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Library of Congress Cataloging-in-Publication Data

Keller, Markus. The science of grapevines : anatomy and physiology/Markus Keller. p. cm. Includes bibliographical references and index. ISBN 978-0-12-374881-2 (hard cover : alk. paper) 1. Grapes–Anatomy. 2. Grapes–Physiology. I. Title. QK495.V55K44 2010 634.8–dc22 2009033159

British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library.

ISBN: 978-0-12-374881-2

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Printed in China 091011987654321 Preface

Grapes were among the first fruit species to stomatal pores may be an exception) are rooted be domesticated and today are the world’s in biochemistry. They are driven or at least most economically important fruit crop. facilitated by enzymes, which in turn are built According to 2009 statistics by the Food and based on blueprints provided by genes. I have Agriculture Organization of the United therefore taken it for granted that it is Nations, grapevines were planted on almost understood that a developmental process 7.3 million hectares producing more than 67 or change in chemical composition implies a million metric tons of fruit in 2007. This makes change in enzyme activity, which implies grapes the number 25 food crop in terms of a change in the activity of one or more genes. planted area and number 16 in terms of ton- This does not imply, as used to be thought, that nage. More than 70% of this crop was used to “one gene makes one enzyme,” nor that “one make wine, 27% consumed as fresh fruit (table enzyme makes one chemical,” but it merely grapes), 2% as dried fruit (raisins), and less means that all enzymatic processes have a than 1% was processed to grape juice or dis- genetic basis. tilled to brandy. Many biochemical and biophysical processes This book is an introduction to the physical apply to many or even all plants. Perhaps no structure of the grapevine, its various organs process is truly unique to grapevines. Chances and tissues, their functions, their interactions are that if grapes do it, some or many other with one another, and their responses to the species employ the same solution to a survival environment. It focuses essentially on the phys- issue because they share a common ancestor ical and biological functions of whole plants that invented the trick a long time ago. For rather than the metabolism and molecular example, microbes hit upon photosynthesis biology of individual cells. It is nonetheless and respiration long before these discoveries necessary to review some fundamental enabled some of them to combine forces and processes at the cell, tissue, and organ levels evolve into plants. Consequently, although this in order to build up an appreciation of whole- book is about grapevines, and primarily about plant function. The book covers those elements the wine grape species Vitis vinifera, I have bor- of physiology that will enhance our under- rowed heavily from research done with other standing of grapevine function and how they plant species, both wild and cultivated, peren- relate to grape production. Although of neces- nial and annual, woody and herbaceous, sity the text contains a plethora of technical including the “queen of weeds”—at least in terms and details, I have tried to resist the the fast-paced world of modern molecular biol- temptation to dwell in biochemical and molec- ogy—Arabidopsis thaliana, the otherwise incon- ular biological jargon. Most physiological spicuous thale cress. I have even taken the processes (water movement through the vine’s liberty to borrow insights gained using micro- hydraulic system and evaporation from the organisms, such as the yeast Saccharomyces

ix x PREFACE cerevisiae that gives us wine and beer and The magnitude of the task of reviewing as bread, that enable us to think about these much of the pertinent literature as possible issues. often forced me to rely on review papers, This book aims to be global in scale. It covers where they were available. I apologize to those physiological aspects of tropical viticulture all friends and colleagues whose work I did not the way to those that pertain to the production cite or cited incompletely or incorrectly. of ice wine at the temperate northern margins Science—and scientists—can only ever hope to of grape growing. It moves from vineyards at approximate the truth. This and the simple fact sea level to vineyards at high altitude. It con- of “errare humanum est” will guarantee a number siders the humid conditions of cool, marine of errors throughout the text. These are entirely climates, the moist winters and dry summers my responsibility, and I would be grateful for of Mediterranean climates, as well as the arid any feedback that might help improve this book environment typical of continental climates in and further our understanding of the world’s the rain shadow of massive mountain ranges. most important and arguably most malleable Yet a book of this nature is necessarily incom- fruit crop. After all, the full quote from Seneca plete, and so is the selection of published infor- the Younger, who was a contemporary of mation included in the text. No one can read Columella, the Roman author of agriculture everything that has been and is being pub- and viticulture textbooks, reads “errare humanum lished, even in the admittedly relatively narrow est, sed in perseverare diabolicum” (“to err is field of grapevine anatomy and physiology. human, to persevere is devilish”). About the Author

Markus Keller is the Chateau Ste. Michelle University in Wagga Wagga, Australia, before Distinguished Professor of Viticulture at coming to eastern Washington. Dr. Keller is Washington State University’s Irrigated Agri- the author of numerous scientific and technical culture Research and Extension Center in papers and industry articles in addition to Prosser, Washington. He received his master’s being a frequent speaker at scientific confer- degree in agronomy (plant science) in 1989 ences and industry meetings and workshops. and a doctorate in natural sciences in 1995 from He also has extensive practical experience in the Swiss Federal Institute of Technology in both the vineyard and the winery as a result Zu¨ rich. He has taught and conducted research of work in the family enterprise, and he was in viticulture and grapevine physiology on awarded the Swiss AgroPrize for innovative three continents, beginning at the Swiss Federal contributions to Switzerland’s agriculture Research Station for Fruit-Growing, Viticulture industry. His current research focuses on and Horticulture in Wa¨denswil (now Agro- environmental factors and management prac- scope Changins-Wa¨denswil), Switzerland, and tices as they influence crop physiology and then moving to Cornell University in Geneva, production of wine and juice grapes. New York, and from there to Charles Sturt

vii Acknowledgments

Completing this book would have been and encouragement from Nancy Maragioglio impossible without the help and support of and Carrie Bolger at Elsevier are greatly many individuals to whom I express my deep acknowledged. They provided numerous sug- gratitude. Many of the illustrations were gestions that improved this book and were skillfully drawn by Adrienne Mills, and Lynn always quick to answer my countless ques- Mills helped with data collection and assisted tions. Special thanks to my wife, Sandra Wran, with some of the most recalcitrant illustra- for scanning my entire slide collection and tions. I thank Gregory Gasic for reviewing supporting this project in many ways from the entire manuscript, identifying errors, and beginning to end. offering many insightful suggestions. The help

xi Elsevier: The Science of Grapevines: Keller Página 1 de 2

The Science of Grapevines

Keller

Table of Contents

Chapter 1 Botany and Anatomy

1.1 Botanical classification and geographical distribution

1.2 Cultivars, clones and rootstocks

1.3 Morphology and anatomy

Chapter 2 Phenology and Growth Cycle

2.1 Seasons and daylength

2.2 Vegetative cycle

2.3 Reproductive cycle

Chapter 3 Water Relations and Nutrient Uptake

3.1 Osmosis, water potential and cell expansion

3.2 Transpiration and stomatal action

3.3 Water and nutrient uptake and transport

Chapter 4 Photosynthesis and Respiration

4.1 Light absorption and energy capture

4.2 Carbon uptake and assimilation

4.3 Photorespiration

4.4 Respiration

Chapter 5 Partitioning of Assimilates

5.1 Photosynthate translocation and distribution

5.2 Canopy-environment interactions

5.3 Nitrogen assimilation and interaction with carbon metabolism

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Chapter 6 Developmental Physiology

6.1 Yield formation

6.2 Grape composition and fruit quality

Chapter 7 Environmental Constraints and Stress Physiology

7.1 Responses to stress

7.2 Water: too much or too little

7.3 Nutrients: deficiency and excess

7.4 Temperature: too cold or too warm

7.5 Living with other organisms: defense and damage

Glossary

References

Internet Resources

http://www.elsevierdirect.com/toc.jsp?isbn=9780123748812 5/4/2011 CHAPTER 1

Botany and Anatomy

OUTLINE

1.1. Botanical Classification and Geographical 1.3. Morphology and Anatomy 20 Distribution 1 1.3.1. Root 21 1.3.2. Trunk and Shoots 27 1.2. Cultivars, Clones, and Rootstocks 9 1.3.3. Nodes and Buds 33 1.2.1. Variety versus Cultivar 9 1.3.4. Leaves 36 1.2.2. Cultivar Classification 13 1.3.5. Tendrils and Clusters 41 1.2.3. Clones 15 1.3.6. Flowers and Grape Berries 43 1.2.4. Rootstocks 16

1.1. BOTANICAL CLASSIFICATION species is a closed gene pool, an assemblage AND GEOGRAPHICAL of organisms that does not normally exchange DISTRIBUTION genes with other species. Their genes compel the individuals belonging to a species to per- The basic unit of biological classification is petuate themselves over many generations. the species. According to the “biological species Yet all life forms on Earth are interrelated; they concept,” a species is defined as a community all ultimately descended from a common of individuals—that is, a population or group ancestor and “dance” to the same genetic code, of populations, whose members can interbreed whereby different combinations of three con- freely with one another under natural condi- secutive nucleotides of each organism’s deoxy- tions but not with members of other popula- ribonucleic acid (DNA) specify different tions (Mayr, 2001; Soltis and Soltis, 2009). In amino acids that can be assembled into pro- other words, such communities are reproduc- teins. Because they are thus interrelated, organ- tively isolated. Although each individual of a isms can be grouped according to the degree of sexual population is genetically unique, each their genetic similarity, external appearance,

The Science of Grapevines 1 Copyright # 2010 Markus Keller. Published by Elsevier Inc. All rights reserved. 2 1. BOTANY AND ANATOMY and behavior. In the classification hierarchy, expressed it clearly: "Wherever many closely closely related species are grouped into a allied yet distinct species occur, many doubtful genus, related genera into a family, allied forms and varieties of the same species likewise families into an order, associated orders into a occur" and, furthermore, "there is no funda- class, similar classes into a division (plants) or mental distinction between species and vari- a phylum (animals), related divisions or phyla eties," and, finally, "varieties are species in the into a kingdom, and, finally, allied kingdoms process of formation" (Darwin, 2004). Indeed, not into an empire but a domain. The “evolu- modern genetic evidence indicates that the tionary species concept” recognizes this ances- various Vitis species evolved relatively recently tor–offspring connection among populations from a common ancestor so that they have not that may follow distinct evolutionary paths to yet had time to develop the complete repro- occupy separate ecological niches but may con- ductive isolation that normally characterizes tinue to interbreed for some time (Soltis and biological species. Thus, Vitis species are Soltis, 2009). defined as populations of vines that can be As is the case with many plants, the species easily distinguished by morphological traits, of the genus Vitis are not very well-defined such as the anatomy of their leaves, flowers, because of the extreme morphological variation and berries, and that are isolated from one among and within populations of wild vines another by geographical, ecological, or pheno- (Currle et al., 1983; Hardie, 2000; Mullins et al., logical barriers; such species are termed ecospe- 1992). This implies the following: (1) All Vitis cies (Hardie, 2000; Levadoux, 1956; Mullins species are close relatives that share a relatively et al., 1992). The following is a brief overview recent common ancestor, and (2) evolution is of the botanical classification of grapevines, still at work, throwing up new variants all the starting with the domain at the top of the time (see Chapter 2.3). Many vine species are hierarchy and finishing with a selection of actually semispecies—that is, populations that species at the base. partially interbreed and form hybrids under natural conditions, which is in fact common Domain Eukarya All living beings, making among plants and may be an important avenue up the earth’s biological diversity or biodiver- for the evolution of new species (Soltis and sity, are currently divided into the three great Soltis, 2009). Despite some hybridization where domains of life: the Bacteria, the Archaea, and their natural habitats overlap, however, the the Eukarya. The Eukarya (eukaryotes; Greek eu various Vitis gene pools usually stay apart so ¼ true, karyon ¼ nucleus) include all terrestrial, that the populations remain recognizably dif- sexually reproducing “higher” organisms with ferent. Grapevines are a good example of the relatively large cells (10–100 mm) containing a limits of taxonomic systems, demonstrating true cell nucleus, in which the DNA-carrying that there is a continuum of differentiation chromosomes are enclosed in a nuclear mem- rather than a set of discrete, sexually incompat- brane, and cell organelles such as mitochondria ible units. As early as 1822, the Rev. William and plastids (Mayr, 2001). They evolved fol- Herbert asserted that "botanical species are lowing injections of oxygen into the atmo- only a higher and more permanent class of sphere caused by abiotic (i.e., nonbiological) varieties," and in 1825 the geologist Leopold factors such as plate tectonics and glaciation von Buch postulated that "varieties slowly (Lane, 2002). The vast majority of life and become changed into permanent species, which the bulk of the world’s biomass—the small are no longer capable of intercrossing" (both (1–10 mm), single-celled prokaryotes (Greek cited in Darwin, 2004). Charles Darwin later pro ¼ before) with cell walls composed of 1.1. BOTANICAL CLASSIFICATION AND GEOGRAPHICAL DISTRIBUTION 3 peptidoglycans (protein–polysaccharides)—is fermentation, other fungi cause diseases of grouped into the other two domains. However, grapevines (see Chapter 7.5); for example, gray both the photosynthetic organelles (chloro- rot is caused by Botrytis cinerea Pers.:Fr. and pow- plasts, from cyanobacteria) and the “power dery mildew by Erysiphe necator [aka Uncinula plants” (mitochondria, from proteobacteria) of necator (Schwein.) Burr.]. Animals can also be eukaryotic cells have descended from (symbi- important pests of grapevines, especially certain otic) bacteria that “infected” other single-celled insects (e.g., phylloxera, Daktulosphaira vitifoliae organisms (or were “swallowed” by them) over Fitch), mites, and nematodes. 1 billion years ago. These organelles still retain some of their own DNA (i.e., genes), although Division (Phylum) Angiospermae (Synonym more than 95% of their original genes have Magnoliophyta) The angiosperms or, in new since been and are still being lost or transferred terminology, the magnoliophytes are the flow- (donated) to their host’s nucleus (Timmis et al., ering plants, which include approximately 2004). Some bacteria cause diseases of grape- 270,000 species. They are believed to have vines; for example, crown gall is caused evolved from a common ancestor that lived by Agrobacterium vitis and Pierce’s disease by approximately 160 million years ago during Xylella fastidiosa (see Chapter 7.5). the late Jurassic period, and they make up the most evolutionarily successful group of plants. Kingdom Plantae The Eukarya comprise at Angiosperms are the plants with the most least four kingdoms; the number changes as the complex reproductive system: They grow their relationships among organisms become better seeds inside an ovary (Greek angeion ¼ pot, known. The Animalia comprise the multicellular vessel) that is itself embedded in a flower. After animals with two sets of chromosomes, cells the flower is fertilized, the other flower parts without cell walls, except in arthropods (insects, fall away and the ovary swells to become a spiders, and the like), which have chitin cell fruit, such as a grape berry. Indeed, the produc- walls, and are the domain of zoology. The Plantae tion of fruits is what defines the angiosperms or plants have a haplo-diploid life cycle and cell and sets them apart from the gymnosperms, walls composed of cellulose, and they are studied with whom they are classed in the Superdivi- in the field of botany. The Fungi include the hap- sion Spermatophyta or seed plants. loid mushrooms, molds, and other fungi, with cell walls composed of glucans and chitin; they Class Dicotyledoneae (Synonym Magno- are studied in mycology. The Protista or Proto- liopsida) This class is large and very diverse, ctista are a catch-all group of all other “higher and its members are often called dicot plants. order” organisms from single-celled microbes, The vast majority of plants (200,000 species), including unicellular fungi, plants (green algae), including most trees, shrubs, vines, and flowers, and animals (protozoans), to large, multicellular and most fruits, vegetables, and legumes belong seaweeds (algae and kelp). There are approxi- to this group. Like all members of the Dicotyledo- mately 500,000 plant species, which are classified neae, grapevines start their life cycle with two into 12 phyla or divisions based largely on repro- cotyledons (seed leaves) preformed in the seed. ductive characteristics. The vascular or higher plants, to which grapevines belong due to their Order Rhamnales (Vitales According to the water conduits, form the Subkingdom Tracheo- Angiosperm Phylogeny Web) Grapevines bionta. Whereas one group of fungi (singular fun- belong to the order Rhamnales, which gets its gus), the yeasts (especially Saccharomyces name from the genus Rhamnus, the buckthorns. cerevisiae), turns grapes into wine through The order has three families: Rhamnaceae 4 1. BOTANY AND ANATOMY

(e.g., Ziziphus jujuba Mill., jujube tree), Leeaceae • Fused flower petals that separate at the base, (the oleasters), and Vitaceae. Plants of the family forming a “calyptra” or cap Leeaceae are more recognizable as being related • Soft and pulpy berry fruits to grapevines than those belonging to the Rham- naceae, being shrubs or trees with flowers aggre- Genus Muscadinia Members of the genus gated in inflorescences, black berries, and seeds Muscadinia usually have glabrous (hairless) that resemble grape seeds, also named pips. leaves, simple tendrils, nonshredding bark, Some taxonomists have now separated the Vita- nodes without diaphragms, and hard wood ceae (Jansen et al., 2006) and Leeaceae from the (Currle et al., 1983; Mullins et al., 1992). Because Rhamnales and placed them in the order Vitales. they do not root from dormant cuttings, they are usually propagated by layering, although Family Vitaceae The members of this family they do root from green cuttings. The “home- are collectively termed grapevines. The family land” of this genus extends from the southeast- contains approximately 1000 species assigned ern United States to Mexico. The genus has to 17 genera that are typically shrubs or woody only three species, which are all very similar lianas that climb by means of their leaf-opposed and may not even deserve to be classed as tendrils—hence the name Vitaceae (Latin viere ¼ separate species (Currle et al., 1983; Mullins to attach). Although most species of this family et al., 1992; Olien, 1990). reside in the tropics or subtropics, a single spe- • Muscadinia rotundifolia Small (formerly Vitis cies from the temperate zones has become the rotundifolia Michaux): A dioecious plant, world’s leading fruit crop grown in almost 90 although breeding has yielded perfect- countries for wine and juice production or as flowered and female cultivars, such as fresh table grapes or dried grapes (raisins). Vita- Noble, Carlos, or Magnolia, known as ceae roots are generally fibrous and well “muscadines” that are grown as table, jelly, branched, and they can grow to several meters or wine grapes. The species is native of the in length. The leaves are alternate, except dur- southeastern United States. The musky ing the juvenile stage in plants grown from flavor and thick skins of the fruit can be seeds, and can be simple or composite. The unattractive. The species has co-evolved fruits are usually fleshy berries with one to four with and therefore resists or tolerates the seeds. All cultivated grapes belong to either grapevine diseases and pests native to North the genus Muscadinia (2n ¼ 40 chromosomes) America, including the fungi powdery or the genus Vitis (2n ¼ 38 chromosomes). The mildew and black rot, the slime mold downy former classification of Muscadinia and Euvitis mildew, the bacterium causing Pierce’s as either subgenera or sections of the genus disease, the aphid phylloxera (Daktulosphaira Vitis has fallen out of favor among taxonomists vitifoliae), and the nematode Xiphinema index (Mullins et al., 1992). Because of the different (which transmits the grapevine fanleaf numbers of chromosomes, crosses between virus), but is sensitive to winter frost and these two genera rarely produce fertile hybrids. lime-induced chlorosis (Alleweldt and Key morphological characteristics of the two Possingham, 1988). Although usually genera include the following: incompatible in both flowering and grafting • Simple leaves with Vitis species, it does produce fertile • Simple or forked tendrils hybrids with V. rupestris, which allows it to • Generally unisexual flowers—that is, either be used in modern (rootstock) breeding male (staminate) or female (pistillate) programs. 1.1. BOTANICAL CLASSIFICATION AND GEOGRAPHICAL DISTRIBUTION 5

• Muscadinia munsoniana Small (Simpson): dioecious (Greek dis ¼ double, oikos ¼ house), Native to Florida and the Bahamas, with containing imperfect male (i.e., female sterile better flavor and skin characteristics than or staminate) or female (i.e., male sterile or pis- M. rotundifolia, but not cultivated. tillate) flowers on different individual plants, • Muscadinia popenoei Fennell: Native to whereas the cultivated varieties of V. vinifera southern Mexico (“Totoloche grape”), have perfect or, in a few cases, physiologically relatively unknown. female flowers (Negrul, 1936; Pratt, 1971; see also Figure 1.1). Members of this genus are very Genus Vitis The genus Vitis occurs predomi- diverse in both habitat and form. Nevertheless, nantly in the temperate and subtropical climate all species within the genus can readily inter- zones of the Northern Hemisphere (Mullins breed to form fertile interspecific crosses called et al., 1992; Wan et al., 2008a). All members of hybrids, which implies that they had a rela- this genus are perennial vines or shrubs with tively recent common ancestor. Moreover, all tendril-bearing shoots. This genus probably species can be grafted onto each other. The comprises 60–70 species (plus up to 30 fossil genus is often divided into two major groups: species and 15 doubtful species) spread mostly the American group and the Eurasian group. throughout Asia (40 species) and North The dominant species of the two groups differ America (20 species) (Alleweldt and Possing- greatly in their useful agronomic traits ham, 1988; Wan et al., 2008b,c). The Eurasian (Table 1.1), which makes them attractive breed- species Vitis vinifera L. gave rise to the over- ing partners (Alleweldt and Possingham, 1988; whelming majority of grape varieties cultivated This et al., 2006). Unfortunately, none of the today. Plants that belong to this genus have many attempts and thousands of crosses that hairy leaves with five main veins, forked ten- have been tested to date has truly fulfilled the drils, bark that shreds when mature, nodes breeders’ hopes to combine the positive attri- with diaphragms, and soft secondary wood. butes while eliminating the negative ones They all can form adventitious roots, which contained in the natural genetic variation of permits propagation by cuttings, yet only V. the two groups. Perhaps the genes conferring vinifera, V. riparia, and V. rupestris root easily disease resistance are coupled to those respon- from dormant cuttings. Although the ancestor sible for undesirable fruit composition. Indeed, of all Vitis species may have had perfect (i.e., hybrids have often been banned in European bisexual or hermaphroditic) flowers (McGo- wine-producing countries because of their per- vern, 2003), the extant wild species are ceived poor fruit (and resulting wine) quality.

FIGURE 1.1 Flower types in the genus Vitis: perfect flower (left), female flower (center), and male flower (right). Illus- trations by A. Mills. 6 1. BOTANY AND ANATOMY

TABLE 1.1 Broad Viticultural Traits of American grow near a permanent source of water, such as and Eurasian Grapevine Species a river, stream, or spring (Morano and Walker, 1995; Figure 1.2). Following is an incomplete list Trait Eurasian Species American Species of some of the more important species: Fruitfulness Good Poor or highly • Vitis labrusca L.: Vigorous climber (“northern variable fox grape”) native to the eastern United Fruit quality Good Poor States from Georgia to southeastern Canada, Usefulness Highly diverse Niche products with Indiana as its western limit. This products species differs from all others in that it Propagation Good Variable usually has continuous tendrils (a tendril at capacity every node). Some of its cultivars (e.g., Lime Good Highly variable Concord and Niagara) are commercially tolerance grown in the United States for juice, jam, Phylloxera Poor Good or variable jelly, and wine production. The classification tolerance of these cultivars, however, is still debated; Disease Poor Good or variable Concord (which has perfect flowers) resistance probably is a natural hybrid of V. labrusca and V. vinifera, and thus it has been classed as Vitis labruscana L. Bailey (Mitani et al., The only unequivocal success story thus far has 2009; Mullins et al., 1992). The distinct foxy been the grafting of phylloxera-susceptible flavor (caused by methyl anthranilate) European wine grape cultivars to rootstocks unique to this species is popular in the that are usually hybrids of tolerant American United States but strange to Europeans. The Vitis species (see Chapter 1.2). species is cold tolerant and resistant to powdery mildew and crown gall, but it is American Group Depending on the taxono- susceptible to phylloxera, downy mildew, mist, this group contains between 8 and 34 spe- black rot, and Pierce’s disease and has poor cies, of which several have become economically lime tolerance (i.e., prefers acid soils). important as wine or juice grapes. Because of Hybrids of V. labrusca were exported to their varying resistance to the North American Europe at the beginning of the 19th century. grapevine diseases and pests, members of this Some of these plants carried powdery group are also being used as rootstocks (see mildew, downy mildew, black rot, and Chapter 1.2) or crossing partners in breeding pro- phylloxera, which drove most populations of grams (Alleweldt and Possingham, 1988; This wild vines extinct and brought the European et al., 2006). As an aside, crosses are always listed wine industry to the verge of destruction. as maternal parent paternal parent (i.e., the • Vitis aestivalis Michaux: Vigorous climber mother’s name comes first). The species of this native to eastern North America, growing in group generally have thinner shoots with longer dry upland forests and bluffs. It is very cold internodes and less prominent nodes than the hardy (to approximately 30 C), drought Eurasian species. They also have small buds, tolerant and also tolerates wet and humid and the leaves have very shallow sinuses and summers (“summer grape”), and is resistant often a glossy surface. All grape species native to powdery and downy mildew and Pierce’s to North America are strictly dioecious (i.e., none disease. The species is very difficult to of them has perfect flowers), and most of them propagate from cuttings. Its fruits are used 1.1. BOTANICAL CLASSIFICATION AND GEOGRAPHICAL DISTRIBUTION 7

FIGURE 1.2 The “bank grape,” V. riparia, growing in a forest in upstate New York (left) and the “canyon grape,” Vitis arizonica Engelmann, growing up a riverside tree in Utah’s Zion National Park (right). Notice the large size of the wild vines and the long “trailing trunks” at the bottom right. Photos by M. Keller.

to make grape jelly, and the cultivars Norton phylloxera, and is resistant to fungal and Cynthiana are commercially grown as diseases but susceptible to Pierce’s disease. wine grapes in the southern and midwestern • Vitis rupestris Scheele: Native to the United States (Tarara and Hellman, 1991). It southwestern United States from Texas to is possible that the two names are synonyms Tennessee, the species is now almost extinct. for the same cultivar and/or that they are It is found in rocky creek beds (“rock hybrids between V. aestivalis and V. labrusca grape”) with permanent water, and it is or even V. vinifera. vigorous, shrubby, and rarely climbs. It has • Vitis riparia Michaux: Widespread in North deep roots for anchorage but is not very America from Canada to Texas and from the drought tolerant on shallow soils, and its Atlantic Ocean to the Rocky Mountains. This lime tolerance is variable. The species species climbs in trees and shrubs along tolerates phylloxera and is resistant to riverbanks (“bank grape”) and prefers deep powdery mildew and downy mildew, but it alluvial soils, but does not do well in is susceptible to anthracnose. calcareous soils (i.e., prefers acid soils), and • Vitis berlandieri Planchon: Native to central its shallow roots make it susceptible to Texas and eastern Mexico, this species drought (a trait it also confers to the climbs on trees on deeper limestone soils rootstocks derived from its crosses with between ridges. It is one of very few other species). It is the earliest to break buds American Vitis species that have good lime and ripen of all the American species, tolerance. Its deep root system makes it matures its canes early, is very cold hardy relatively drought tolerant, but it is very (to approximately 36C), is tolerant of susceptible to waterlogging. The species 8 1. BOTANY AND ANATOMY

breaks buds and flowers much later than and Hopf, 2001). This is the most well- other species and is the latest ripening of the known species of the Eurasian group American group with very late cane because it gave rise to most of the cultivated maturation. It is somewhat tolerant of grapes grown today. Because the cultivated phylloxera and resistant to fungal grapevines have hermaphroditic or, rarely, diseases and Pierce’s disease, but it is very physiologically female flowers, they are difficult to propagate and to graft often grouped into the subspecies V. vinifera (Mullins et al., 1992). ssp. sativa (also termed V. vinifera ssp. • Vitis candicans Engelmann: Very vigorous vinifera). However, the concept of subspecies climber native to the southern United States (or geographical races) as taxonomically and northern Mexico. The species is drought recognizable populations below the species tolerant, relatively tolerant of phylloxera, and level is biologically virtually worthless and, resistant to powdery and downy mildew and according to many taxonomists, V. vinifera Pierce’s disease, but it is difficult to sativa is merely the domesticated form of propagate. Other southern species, such as V. vinifera (ssp.) sylvestris. According to this V. champinii Planchon and V. longii Prince, are view, the differences between the two forms probably natural hybrids of V. candicans, are the result of the domestication process V. rupestris, and other native species. They are (This et al., 2006)—that is, they arose through highly resistant to nematodes. human rather than natural selection. The species is highly tolerant of lime, even more Eurasian Group There are approximately 40 so than V. berlandieri, and drought. known species in this group, most of them • Vitis sylvestris (or silvestris) (Gmelin) Hegi: confined to eastern Asia. Chinese species are Native to an area spanning central Asia to particularly diverse, growing in the dry south- the Mediterranean region, this group west, the northern and southern foothills of contains the dioecious wild vines (now also the Himalayas, the very cold northeast, and termed Lambrusca vines) of Asia and the hot and humid southeast. Although some Europe, growing mainly in damp of them are resistant to fungal diseases and woodlands (“forest grape”) on alluvial soils may tolerate high humidity (Li et al., 2008; of riverbanks and hillsides. Taxonomists Wan et al., 2007, 2008a), most of these species debate whether this group of vines deserves are little known, and there may be several addi- species status or whether it is a subspecies of tional species that have not yet been described. V. vinifera (ssp. sylvestris) because, apart Most Eurasian species are not resistant to the from their flowers, the two look very similar North American grapevine diseases, and yet and interbreed readily (Mullins et al., 1992). It one of them has come to dominate the grape has almost disappeared in Europe and is and wine industries throughout the world. considered an endangered species in some countries, mainly because of the destruction • Vitis vinifera L.: Native to western Asia and of its natural habitats and its susceptibility to Europe between 30 and 50 N, but phylloxera and the mildews introduced from temporarily confined to the humid and North America (Arnold et al., 1998). The high forested to arid, volcanic mountain ranges of humidity and periodic flooding in wooded the southern Caucasus between the Black river valleys may have protected the Sea and the Caspian Sea, and to the remaining populations from destruction by Mediterranean region during the ice ages phylloxera. These wild grapes are thought to (Hardie, 2000; Mullins et al., 1992; Zohary be more cold tolerant than their cultivated 1.2. CULTIVARS, CLONES, AND ROOTSTOCKS 9

siblings and to be resistant to leafroll and Cultivated grapevines of sufficiently similar fanleaf viruses (Arnold et al., 1998). vegetative and reproductive appearance are • Vitis amurensis Ruprecht: The genetically usually called “grape varieties” by growers diverse native of northeastern China and and “cultivars” by botanists. Botanically Russian Siberia (“Amur grape”) is said to be speaking, a variety includes individuals of a the cold-hardiest of all Vitis species (wild) population that can interbreed freely, (Alleweldt and Possingham, 1988; Wan et al., whereas, according to the International Code 2008a). However, budbreak occurs of Nomenclature for Cultivated Plants (Interna- approximately 1 month earlier than in tional Society for Horticultural Science, 2004), a V. vinifera, and bloom and fruit maturity are cultivar is "an assemblage of plants that has also advanced, which seems to be true for been selected for a particular attribute or com- many other Chinese species (Wan et al., bination of attributes, and that is clearly dis- 2008c). It is resistant to downy mildew and tinct, uniform, and stable in its characteristics Botrytis but susceptible to phylloxera (Du and that, when propagated by appropriate et al., 2009). Some cultivars with female or, means, retains those characteristics." A cultivar rarely, hermaphroditic flowers and small can be produced both sexually (i.e., from a clusters are grown in northeastern China. seedling) and asexually (i.e., as a clone), but • Vitis coignetiae Pulliat: Native to Japan, and only the latter method will give grapevine des- used locally for jam production, the species cendents that are genetically identical (i.e., strongly resembles the American V. labrusca “true to type”). Seedlings of so-called grape (Mullins et al., 1992). varieties are not identical copies of their mother plant. Because grapevines are heterozygous across a large number of chromosome positions 1.2. CULTIVARS, CLONES, AND or loci (singular locus) in their genomes, each ROOTSTOCKS seed may give rise to a cultivar with distinct characteristics (Mullins et al., 1992, Thomas 1.2.1. Variety versus Cultivar and Scott, 1993; also see Chapter 2.3). Grapes were among the first fruit crops to be The study of the botanical description, iden- domesticated, along with olives, figs, and dates tification, and classification of plants belonging (Zohary and Hopf, 2001; Zohary and Spiegel- to the genus Vitis and of their usefulness for Roy, 1975). Although all four wild fruits had viticulture is termed ampelography (Greek ampe- ranges stretching far beyond the eastern Medi- los ¼ vine, graphos ¼ description). The descrip- terranean region, the deliberate cultivation of tors have traditionally included such visual grapes for winemaking as well as fresh fruit traits as shoot tips (shape, hairs, and pigmen- and raisin production probably started about tation), leaves (shape of blade with lobes, 7000 to 8000 years ago in the eastern part of sinuses, and serrations), fruit clusters (size the Fertile Crescent that spans the modern- and shape), and berries (size, shape, and day countries of the Near East: Egypt, Israel, pigmentation) (Galet, 1985, 1998; Viala and Lebanon, Jordan, Syria, eastern Turkey, Iraq, Vermorel, 1901). The advent of DNA finger- and western Iran. This is approximately the printing has led to its adoption for the identifi- same time that farmers invented irrigation cation of selections in grape collections and and precedes by approximately 2000 years the is increasingly being used to uncover the his- earliest civilization of Sumer in southern Meso- torical origins and genetic relationships of potamia (McGovern et al., 1996). As an interest- grapevines. ing aside, the Sumerian symbol for “life” is a 10 1. BOTANY AND ANATOMY grape leaf, and the “tree of life” of many in a region comprising the Iberian Peninsula, ancient civilizations is the grapevine. These Central Europe, and Northern Africa (Arroyo- early societies regarded wine as “nectar of the Garcı´a et al., 2006). Whereas almost all wine gods,” and wine drinking was considered to and juice grapes contain seeds, many raisin be a hallmark of civilization: “Savages” or “bar- and most table grape cultivars are seedless, a barians” drank no wine. The biblical legend, selected trait that seems to originate from a modeled on the earlier Mesopotamian Epic of very narrow genetic base, mainly Sultanina Gilgamesh (McGovern, 2003), of Noah’s ark (synonyms Sultana, Thompson Seedless, and stranding near volcanic Mount Ararat on the Kishmish), an ancient Middle Eastern grape Turkish/Iranian/Armenian border and his variety (Adam-Blondon et al., 2001; Iba´n˜ez subsequent planting of the first vineyard also et al., 2009). Most current cultivars are not pro- reflects precisely the probable origin of viticul- ducts of deliberate breeding efforts but are ture. Nevertheless, new archaeological discov- the results of continuous selection over many eries show that wine might have been made centuries of groups of grapevines that were from wild grapes in China as far back as 9000 spontaneously generated by mutation and years ago (McGovern et al., 2004). In Europe, intraspecific crosses via sexual reproduction grapes were grown at least 4000 years ago (Riv- (see Chapter 2.3) and via somatic (Greek soma era Nu´ n˜ez and Walker, 1989). Clearly, humans ¼ body) mutations—that is, mutations occur- quickly learned how to use fermentation as ring in the dividing cells of the shoot apical one of the most important food preservation meristem (see Chapter 1.3), typically during technologies; they also learned to preserve bud formation (so-called bud sports). grapes as raisins by drying them. Domestica- Mutations (Latin mutatio ¼ change) in the tion of V. vinifera was also accompanied by a grape genome arise through rare, chance mis- change from dioecious to hermaphroditic takes in DNA replication during the process reproduction and an increase in seed, berry, of cell division. Whenever a cell divides, it first and cluster size (This et al., 2006); perhaps early has to double (i.e., copy) its DNA so that each viticulturists selected not only more fruitful daughter cell gets a complete set of chromo- vines but also plants that were self-pollinated, somes. Millions of nucleotides are regularly thus eliminating the need for fruitless pollen copied with amazing fidelity, and sophisticated donors. repair mechanisms attempt to fix most mis- Today, an estimated 10,000 or so grape culti- takes that do occur. However, every so often, vars are being grown commercially, although a nucleotide is switched with another one, or DNA fingerprinting suggests that a more accu- a whole group of nucleotides is inserted, rate figure may be approximately 5000 (This repeated, omitted, or moved to a different et al., 2006). Many cultivated grapes are closely location on the chromosome. Many DNA related to one another, and many are known by sequences, called “jumping genes” or transpo- several or even many synonyms (different sons, even move on their own, some of them names for the same cultivar) or homonyms employing a “cut-and-paste” strategy, and (identical name for different cultivars). The may cause mutations if they cannot be silenced vast majority of cultivars belong to the species by the grape genome (Benjak et al., 2008; Lisch, V. vinifera, and comparisons of the DNA 2009). New genes quite often seem to arise from contained in chloroplasts suggest that they duplication of an existing gene and subsequent might have originated from (at least) two geo- mutation of one of the two copies (Dı´az- graphically distinct populations of V. sylvestris: Riquelme et al., 2009; Firn and Jones, 2009). one in the Near and Middle East and the other Many mutations are also caused by damage to 1.2. CULTIVARS, CLONES, AND ROOTSTOCKS 11 the DNA by so-called reactive oxygen species and because they accumulate over time, the that are produced during oxidative stress variation also increases. Although ancient culti- (Halliwell, 2006; Mller et al., 2007; see also vars (e.g., Pinot noir) can be quite heterogeneous Chapter 7.1). Mutations, if they are not lethal, and are planted as many different clones, the can give rise to slightly different gene variants genetic similarity of these clones typically is termed alleles (Greek allelos ¼ each other). The still on the order of 95–99% (Bessis, 2007; genome of the Vitis species is approximately 500 Wegscheider et al., 2009). Similarly, almost all of million DNA base pairs long and comprises an the approximately 20 cultivars with “muscat” estimated 30,000 genes (Jaillon et al., 2007; Velasco aroma, most of them used as raisin or table grapes et al., 2007) that form both a vine’s “building and many of them with a number of synonyms, plan,” including its flexible architectural and are direct descendants of Muscat a` petits grains engineering design, and its “operating manual” (synonym Muscat blanc) or Muscat of Alexandria by encoding (i.e., prescribing) the amino acid (Crespan and Milani, 2001), which is also one makeup of proteins. In comparison, humans are parent of the Argentinian Torronte´s cultivars thought to have fewer than 35,000 genes. Because (Agu¨ero et al., 2003). there are genes that influence growth habit, leaf Hybridization, on the other hand, occurs by shape, disease resistance, cluster architecture, cross-pollination and fertilization (see Chapter berry color, and other quality attributes, some of 2.3) of flowers from different plants, which are the mutations also affect these traits (Bessis, genetically distinct, and thus give rise to a 2007). For instance, dark-skinned (i.e., anthocya- genetically novel individual. Both deliberate nin-accumulating) fruit is the “default” version interbreeding and natural hybridization have in the Vitaceae, and it appears that virtually all occurred many times in the history of viticul- V. vinifera cultivars with green-yellow fruit (so- ture. Many cultivars were originally selected called white cultivars) have a single common from domesticated local wild grapes (V. sylves- ancestor that arose from mutations of two neigh- tris or V. vinifera ssp. sylvestris) and further boring genes of an original dark-fruited grape- developed by interbreeding with other wild vine (Cadle-Davidson and Owens, 2008; This grapes or by the introduction of exotic varieties et al., 2007; Walker et al., 2007). These mutations (Arroyo-Garcı´a et al., 2006). Intriguingly, were probably caused by the insertion of a jump- although the majority of the French wine grape ing gene that renders grapes unable to “switch cultivars are genetically closely related to each on” the anthocyanin assembly line (Kobayashi other and to the wild grapes of western Europe, et al., 2004). The chances of both mutations most Italian and Iberian cultivars are different occurring together are extremely small; this from these and also more distinct among them- might have been a one-time event in the history selves, and most table grapes are altogether in a of grapevines, perhaps as recently as approxi- completely different “genetic league.” Never- mately 200,000 years ago (Mitani et al., 2009). theless, many “noble” grape lineages are as Although this implies that white-fruited cultivars interwoven as those of their human selectors. are likely to be genetically more closely related to For instance, it is thought that Cabernet franc each other than are cultivars with dark fruit, (which is closely related to Petit Verdot) and roughly half of all current grape cultivars have Traminer (synonym Savagnin) may have been fruit with “white” skin. Instability of at least selected from wild grapes, whereas Cabernet one of these mutations may account for the occa- Sauvignon resulted from a natural cross sional appearance of dark-skinned variants of between Cabernet franc and Sauvignon blanc white cultivars (Lijavetzky et al., 2006). Mutations (Bowers and Meredith, 1997; Levadoux, 1956). also add to the genetic variation within a cultivar, Cabernet franc also fathered Merlot and 12 1. BOTANY AND ANATOMY

Carmene`re (Boursiquot et al., 2009). Syrah (syn- genetically similar to many southeastern Euro- onym Shiraz), is derived from a cross between pean wine grapes and, surprisingly, also to the Dureza and Mondeuse blanche, whereas Syrah red-fleshed “teinturier” cultivar Alicante crossed with Peloursin gave rise to Durif (syno- Bouschet, may have been the result of a Gouais nym Petite Sirah) (Meredith et al., 1999; Vouilla- blanc cross with an unknown partner. In fact, moz and Grando, 2006). Durif and Peloursin, based on genetic evidence the ancient eastern on the other hand, are closely related to Malbec European (Croatian) cultivar Gouais blanc has and Marsanne, which are distinct from the thus far been proposed to be involved in more (other) Bordeaux and Burgundy cultivars. than 75 European cultivars. This prominent posi- Chardonnay, Gamay noir, Aligote´,Auxerrois, tion as a parent of many premium wine grape Melon, and other French cultivars all originated varieties is somewhat ironic because its fruit from the same two parents, Pinot and Gouais quality has long been considered inferior. Yet its blanc (synonym White Heunisch), which means vigorous growth and high fertility made it a they are full siblings (Bowers et al., 1999). Pinot favorite with growers, who grew it alongside blanc and Pinot gris are somatic berry-pigment selections from wild vines (e.g., Traminer with mutants of Pinot noir (Figure 1.3); indeed, highly variable yields) for centuries so that it variation in berry color seems to be a fairly com- became widespread throughout Europe in the mon outcome of somatic mutation (Bessis, 2007; Middle Ages. Moreover, because of their sour Furiya et al., 2009; Mu¨ller-Stoll, 1950; Regner berries, Gouais vines were often planted as buf- et al., 2000). The Pinot family in turn is thought fers around vineyards to deter potential grape to be derived from a spontaneous cross between thieves. Another old cultivar from the Adriatic Traminer and Pinot Meunier (synonym Schwarz- coast has also attained some popularity in riesling). Whereas Pinot also appears to be one of both the Old and the New World, albeit under the more distant ancestors of Syrah, Traminer fea- different names: Primitivo in southern Italy, tures as a parent of Sauvignon blanc, Sylvaner, Crljenak kasˇtelanski in Croatia, and Zinfandel in and other cultivars (Sefc et al., 1998). Gouais California (Fanizza et al., 2005; Maletic´ et al., 2004). blanc, crossed with a Traminer variety, probably As novel DNA fingerprinting tests show, gave rise to Riesling, and with Chenin blanc it what breeders get is not always what they set yielded Colombard and other cultivars. Even out to develop, despite their best efforts to Lemberger (synonym Blaufra¨nkisch), which is exclude nondesired pollen from their crosses (Bautista et al., 2008; Iba´n˜ez et al., 2009). The most well-known example is the German wine grape cultivar Mu¨ller-Thurgau, which has long been disseminated as a deliberate cross of Riesling flowers with Sylvaner pollen (hence the synonym Riesling Sylvaner) (Becker, 1976). More than 100 years after its introduction, however, genetic analysis exposed Madeleine Royale, a likely progeny of Pinot noir, as the illegitimate father (Dettweiler et al., 2000). Similarly, Cardinal, a table grape cultivar with worldwide distribution, may be derived from Alphonse Lavalle´e (a possi- ble progeny of Gros Colman and Muscat FIGURE 1.3 Pinot noir (left) growing next to Pinot gris/ Hamburg) and Ko¨nigin der Weinga¨rten (proba- Pinot blanc (right; photo by M. Keller). bly a descendant of Pearl of Csaba) and not, as 1.2. CULTIVARS, CLONES, AND ROOTSTOCKS 13 has been assumed, from Flame Tokay and cultivar name Traminer was first mentioned Alphonse Lavalle´e(Iba´n˜ez et al., 2009). Natural in 1349, Pinot gris (synonym Rula¨nder) in hybridization can also cross species boundaries, 1375, Pinot in 1394, Riesling in 1435, Chasselas as exemplified by the North American juice (synonym Gutedel) in 1523, and Sangiovese in grape cultivar Concord, which was selected from 1590, although it is not certain that the same awildV. labrusca seedling that may have been name was consistently applied to the same cul- pollinated by an unknown V. vinifera cultivar or tivar (von Bassermann-Jordan, 1923). Tradi- a seedling with partial V. vinifera parentage tional European vineyards were—and in some (Mitani et al., 2009; Mullins et al., 1992). Misidenti- areas still are—composed of a population of fication of planting material imported to new heterogeneous vines, and sometimes several grape growing regions is another common cultivars were planted together in the same problem. For instance, the so-called Bonarda in block so that many cultivated varieties have Argentina and Charbono in California are not no defined origin. The identification and culti- related to the various Italian cultivars of the same vation of “pure” cultivars is quite recent. names but are in fact both identical to the old French cultivar Corbeau (Martı´nez et al., 2008). 1.2.2. Cultivar Classification Although each cultivar originally began as a Following several millennia of cultivation and single vine that grew from a seedling, most repeated selection of spontaneous mutants and major cultivars grown today have been propa- natural as well as man-made intra- and interspe- gated vegetatively for a long time, some for cific crosses in many different regions, there is a many centuries and perhaps even millennia vast range of cultivated forms and types of grape- (Bessis, 2007; Hardie, 2000). Propagation by vines. Because thousands of grape cultivars are cuttings was undoubtedly in use by Roman being grown commercially, various attempts times 2000 years ago (Columella, 4–ca. 70 AD). have been made to group them into families or For instance, Gouais blanc is thought to have tribes. Unlike botanical classifications, these been brought from Croatia (then Dalmatia) to groupings are not always based on phenotypic France (then Gallia) along with Viognier (the or genotypic differences, whereby the genotype Croatian cultivar Vugava bijela seems to be is the sum of the genetic material of an organism, identical with Viognier and a close relative of and the phenotype arises from the interaction of the Italian Barbera and the Swiss Arvine) the genotype with the environment during around 280 AD by the Roman Emperor Probus, development (Mayr, 2001). The most common who encouraged vineyard development to methods involve classification on the basis of ensure economic stability. In fact, if a map of place or climate of origin, viticultural charac- the distribution of viticulture in Europe, the teristics, final use, or winemaking characteristics. Near East, and North Africa were to be over- Arguably the first cultivar classification was laid with a map of the Roman Empire at its attempted by Plinius (ca. AD 50) in Rome, who greatest extent, there would be an almost exact created three groups according to berry color geographic overlap. It is believed that the rare and yield: extant Swiss variety Re`ze is, if not identical with, at least a direct descendant of Raetica, • Anemic cultivars: Varieties with small, white which was considered by Roman writers to be grapes one of the two best wine grape varieties of the • Nomentanic cultivars: Varieties with red Empire (Vouillamoz et al., 2007). A long gap grapes, low yielding in cultivar description and identification fol- • Apianic cultivars: High-yielding varieties, lowed the demise of the Roman Empire. The poor quality 14 1. BOTANY AND ANATOMY

During the Middle Ages, European wine from the fact that grape cuttings have forever grapes were simply divided into two groups been carried to distant locations and used according to perceived wine quality (von for (deliberate or natural) interbreeding with Bassermann-Jordan, 1923): locally domesticated and selected variants. Therefore, cultivars have also been grouped • Vinum francicum:“Frentsch”grapes,low- according to their viticultural characteristics, yielding varieties of high quality (e.g., Traminer) although these can be modified by local soil • Vinum hunicum: “Huntsch” grapes, high- and climatic conditions and cultural practices: yielding varieties of poor quality (e.g., Heunisch) • Time of maturity (Table 1.2): The early The division proposed by the Russian bota- maturing Chasselas is used as a basis or nist Negrul (cited in Levadoux, 1956, and Mul- standard. lins et al., 1992) distinguishes three main • Vigor: Rate of shoot growth. ecological or ecogeographical groups of vari- • Productivity: Yielding ability. proles ¼ eties, called “proles” (Latin offspring), Classification is also possible in terms of based on their region of origin: what the grapes will be used for (Galet, 2000), • Proles pontica: Fruitful varieties with although some cultivars are used for various medium-sized, round berries that originated purposes: from the banks of the Aegean and Black Seas • Table grapes: Large, fleshy or juicy grapes, and spread throughout eastern and southern often seedless, some of them with muscat or Europe (e.g., Furmint, Clairette, Black foxy aromas. Examples include Cardinal, Corinth, and Rkatziteli) Cinsaut, Chasselas, and Muscat of Proles occidentalis • : Wine grape varieties of Alexandria. western Europe with small clusters and • Raisin grapes: Predominantly seedless small berries (e.g., Riesling, Chardonnay, grapes. Examples include Thompson Se´millon, Sauvignon blanc, Gewu¨ rztraminer, Seedless, Flame Seedless, Black Corinth the Pinots, and the Cabernets) (synonym Zante Currant), and Delight. Proles orientalis • : Mostly table grape varieties • Juice grapes: Some highly aromatic grapes, with large clusters and large, elongate berries especially in the United States. Examples originating from the Near East, Iran, include Concord and Niagara. Afghanistan, and central Asia (e.g., Thompson • Wine grapes: Very sweet, juicy grapes, often Seedless, the Muscats, and Cinsaut) low yielding. Examples include Riesling, Chardonnay, Se´millon, Sauvignon blanc, Modern genetic research has mostly borne Gewu¨ rztraminer, the Pinots and out Negrul’s grouping (Aradhya et al., 2003). Cabernets, Merlot, Tempranillo (synonyms Moreover, it also confirmed that many cultivars Aragonez, Tinta Roriz), Nebbiolo, and many of the proles occidentalis are closely related to the others. wild V. sylvestris (or V. vinifera ssp. sylvestris), • Brandy (distillation) grapes: Generally white that almost all of the proles pontica varieties are grapes producing bland, acidic wines. closely related to each other, and that the proles Examples include Ugni blanc (synonym orientalis are genetically distinct from the other Trebbiano), Colombard, and Folle blanche. groups. Yet Negrul’s classification is not clear- cut and contains some ampelographic errors. Of course, divisions can also be made on the In addition, all of these classifications suffer basis of winemaking characteristics, but this 1.2. CULTIVARS, CLONES, AND ROOTSTOCKS 15

TABLE 1.2 Classification of Some Grape Cultivars Based on Their Relative Heat Requirement to Reach Acceptable Fruit Maturity, Subjectively Defined as 18–20 Brix

Group Red Wine Cultivars White Wine Cultivars

1 (early) Madeleine Angevine 2 Blue Portuguese Chasselas, Mu¨ ller-Thurgau 3 Gamay, Dolcetto Pinot gris, Pinot blanc, Aligote´, Gewu¨ rztraminer 4 Pinot noir Chardonnay, Sauvignon blanc, Sylvaner 5 Cabernet franc, Lemberger Riesling 6 Merlot, Malbec, Zinfandel, Tempranillo, Cinsaut, Se´millon, Muscadelle, Chenin blanc, Marsanne, Barbera, Sangiovese Roussanne, Viognier 7 Cabernet Sauvignon, Syrah, Nebbiolo Colombard, Palomino 8 Petit Verdot, Aramon, Carignan, Grenache, Muscat of Alexandria, Ugni blanc Mourve`dre 9 (late) Tarrango, Terret noir Clairette, Grenache blanc

Modified from Viala and Vermorel (1909), Gladstones (1992), and Huglin and Schneider (1998).

method is strongly influenced by the market intense fruit pigmentation or other perceived environment and changing consumer demand: quality attributes, and resistance to a particular disease. Due to vegetative propagation, such • Grape composition: Basic characteristics clonal traits arise from somatic mutations (see (sugar, acid, pH, tannins, flavors, and Chapter 1.3) rather than during sexual repro- aroma) important for winemaking. duction (i.e., in germ cells, see Chapter 2.3). • Varietal aroma: White grapes can be Nonetheless, unlike in animals that keep aromatic (e.g., Riesling, Gewu¨ rztraminer, somatic and germ cells separate, somatic muta- and Muscats) or nonaromatic (e.g., tions may ultimately be propagated both vege- Chardonnay, Se´millon, and Sylvaner). tatively and by seeds and result in individual • Production costs: Value for winemaking plants of the same cultivar having slightly dif- reflected in wine and grape price structure ferent genotypes and sometimes phenotypes and appellation systems. (Franks et al., 2002; This et al., 2006). This geno- typic diversity, which accumulates over time, is 1.2.3. Clones termed clonal variation (Mullins et al., 1992; Riaz et al., 2002). If the change is sufficiently distinct In viticultural parlance, a clone (Greek klon (e.g., major change over a short time or many ¼ twig) is a group of grapevines of a uniform small changes over a long period), such clones type that have been vegetatively propagated may come to be called cultivars, as exemplified (usually by cuttings) from an original mother by the example of the fruit skin color mutants vine that would normally have been selected of Pinot: Pinot noir, Pinot gris, and Pinot blanc for a particular desired trait. Such traits include (Bessis, 2007; Regner et al., 2000). In fact, many low vigor, high yield, loose clusters, large or clones of the Pinot family are chimeras, which small berries, seedlessness, different or more are defined as plants with more than one 16 1. BOTANY AND ANATOMY genetically different cell population that arose cultivar are typically very small. For example, from a mutation in only one of the two function- all 39 Sangiovese clones analyzed using a ally distinct cell layers or lineages of the shoot DNA assay were found to have been derived apical meristem (Riaz et al., 2002; Thompson and from the same mother vine (Filippetti et al., Olmo, 1963). This includes another old Pinot 2005). (gris) clone, Pinot Meunier (synonym Schwarz- It is currently fashionable to plant clonal riesling), whose leaves are densely coated with selections in vineyards, although choices are white hair (Franks et al., 2002; Hocquigny et al., often based on the “fallacy of the perfect 2004). Chimeras also exist among clones of Char- clone”—that is, on the false assumption that donnay and Cabernet Sauvignon (Moncada et al., the “best” clone found on one site will also per- 2006), which suggests that layer-specific muta- form best in a new location. Yet the perfor- tions in the apical meristem may be an important mance or suitability of a clone on a particular source of clonal and varietal variation in grape- site is strongly modified by the site’s environ- vines. Therefore, as in the case of species dis- ment (soil, climatic conditions, cultural prac- cussed in Chapter 1.1, there is no clear-cut tices, etc.) and also depends on the desired distinction between clones and cultivars, a fact end use of the grapes produced on that site recognized by Charles Darwin while he devel- and even regulatory circumstances (e.g., yield oped his revolutionary theory of evolution by restrictions). This makes it nearly impossible (random) mutation and (directional) natural to predict clonal performance in new environ- selection (Darwin, 2004). ments. Planting several clones of a cultivar in Although clonal vines were selected and the same vineyard block not only enables vegetatively propagated for fruitfulness, fruit growers to conduct their own evaluation at a flavor, and wine quality in Roman times (Colu- particular site but also provides some insur- mella, 4–ca. 70 AD), organized and methodical ance against fluctuations in yield, fruit quality, clonal selection only began in Germany in the and disease susceptibility that come with the second half of the 19th century and did not absence of genetic variation of a single clone. begin in France until the 1950s. Selection is usu- ally based on the absence of symptoms of virus 1.2.4. Rootstocks diseases (e.g., leafroll and fanleaf), healthy growth, and good performance such as consis- Rootstocks are specialized stock material to tent yields and high wine quality, but criteria which grape cultivars with desirable fruit can also include specific viticultural traits such properties are grafted; the shoot portion of the as fruit set, disease resistance, or drought toler- two grafting partners is termed the scion, ance. Nevertheless, many so-called clones are whereas the rootstock provides the root system actually phenotypes caused by various combi- to the fused combination of genotypes. For the nations and degrees of virus infections rather grafting operation to be successful, the vascular than genuine genetic differences among the cambium responsible for cell division (see Chap- plants themselves. Today, clones exist for most ter 1.3) of the two grafting partners must make major grape cultivars, and their success is contact with each other so that they can “build” based on the ability of some clones to perform a connection between their separate “plumbing” differently in diverse environments. Differ- systems for water and nutrient supply. Grafting ences among clones, and thus within a cultivar, of grapevines was common in ancient times; in offer viticulturally relevant diversity and his De Re Rustica, the Roman writer Columella potentially better planting material. Nonethe- described techniques known to his ancestors. less, the genetic differences among clones of a The use of rootstocks derived from American 1.2. CULTIVARS, CLONES, AND ROOTSTOCKS 17

Vitis species saved grapegrowing in Europe diseases. Infested propagation material can from extinction due to the introduction, on thus be a potential source of inoculum (infec- imported planting material, of the aphid-like tious material) for new vineyards and new viti- insect phylloxera (Daktulosphaira vitifoliae)in cultural regions. the late 19th century (see Chapter 7.5). On the The technique of grafting combines the downside, rootstocks or their progeny that tolerance of soil-borne pests of American Vitis escaped, for example, from abandoned vine- species with the winemaking quality (i.e., yards have also become feral across Europe, consumer acceptance) of V. vinifera. Today, and their interbreeding populations are in some grape rootstock varieties are used not only for areas behaving as invasive species (Arrigo and their tolerance or resistance to root parasites, Arnold, 2007; Laguna, 2003). such as phylloxera and nematodes, but also for Commonly used rootstocks are either indi- their ability to influence crop maturity or their vidual Vitis species or crosses of two or more tolerance of adverse soil conditions such as species (Table 1.3), and due to the dioecious drought, waterlogging, lime, and acid or saline nature of their parents, they are either male or soils (May, 1994; Pongra´cz, 1983; see also female plants. Examples of male rootstocks Table 1.3). Rootstocks can also contribute to the include Teleki 5C and SO4 and Riparia gloire management of vine vigor and of grape maturity de Montpellier, whereas female rootstocks and composition (Currle et al., 1983; Dry and include Kober 5BB, 101-14 Millardet et de Coombe, 2004; Galet, 1998). Although the influ- Grasset, and Fercal (Meneghetti et al., 2006). ence on scion vigor is an important consider- The majority of rootstocks in use today are ation in rootstock choice, there are no truly hybrids of three species: V. riparia, V. rupestris, dwarfing rootstocks, unlike in the case of, for and V. berlandieri (Galet, 1998). Nevertheless, instance, apple. The vertical and horizontal the genetic basis of the world’s rootstocks is distribution of the root system of various root- extremely narrow because as many as 90% of stocks, it seems, is mostly a function of soil all V. vinifera vines are grafted to fewer than texture, composition, and water availability 10 different rootstock varieties. As discussed rather than an inherent trait of the rootstock geno- previously for scion cultivars, this strategy puts type (Smart et al., 2006). Grafting does not directly vineyards at risk from mutant strains of soil affect the color or flavor profile of the grapes pro- pests, including phylloxera, with potentially duced on the scion variety because the chemicals devastating effects if a resistance breaks responsible for these quality-relevant traits are through. An example is the failure in the produced inside the berry (see Chapter 6.2) and 1980s after many years of widespread use of are therefore determined by the genotype of the V. vinifera V. rupestris rootstock AxR1 in the scion. However, an indirect effect on fruit California. Moreover, with the exception of composition, especially on acidity, is possible those derived from M. rotundifolia, the so-called due to the potential influence of the rootstock on “resistant” rootstocks are not immune to phyl- scion vigor, canopy configuration, yield forma- loxera or nematodes (microscopic round- tion, and, possibly, nutrient uptake (Keller et al., worms); they just tolerate them better than do 2001a; Ruhl et al., 1988; Schumann, 1974). V. vinifera cultivars and therefore suffer less Some of the lesser known eastern Asian species, from infestation (Mullins et al., 1992). Tolerant such as V. amurensis, are used increasingly in species or cultivars may grow quite well in modern rootstock breeding programs for their the presence of these pests, but they do not pro- cold tolerance (Alleweldt and Possingham, hibit pest numbers from building up over time. 1988), although this diminishes the phylloxera They also can be symptomless carriers of virus resistance of the resulting crosses. 18

TABLE 1.3 Agronomic Characteristics of Important Grapevine Rootstocks ucpiiiyt gDeficiency Mg to Susceptibility ucpiiiyt Deficiency K to Susceptibility htptoaResistance Phytophthora aeo ec Grafting Bench of Ease rw alResistance Gall Crown co ri Maturation Fruit Scion hloeaResistance Phylloxera eaoeResistance Nematode ad olTolerance Soil Sandy rfe co Vigor Scion Grafted cdSi Tolerance Soil Acid lySi Tolerance Soil Clay loigTolerance Flooding ruh Tolerance Drought aiiyTolerance Salinity aeo Rooting of Ease ieTolerance Lime .BTN N ANATOMY AND BOTANY 1.

Rootstock Parent Species

Riparia Gloire V. riparia NA Rupestris V. rupestris ND St. George Rupestris du Lot V. rupestris D 420A Millardet V. berlandieri V. riparia YD et de Grasset 5BB Kober V. berlandieri V. riparia YYD SO4 V. berlandieri V. riparia YN 8B V. berlandieri V. riparia 5C Teleki V. berlandieri V. riparia A 161-49 Couderc V. berlandieri V. riparia 99 Richter V. berlandieri V. rupestris YYD 110 Richter V. berlandieri V. rupestris YYD 1103 Paulsen V. berlandieri V. rupestris NY D 140 Ruggeri V. berlandieri V. rupestris NY D 44–53 Male`gue V. riparia V. rupestris YNA 3309 Couderc V. riparia V. rupestris NA 101-14 Millardet V. riparia V. rupestris A et de Grasset Schwarzmann V. riparia V. rupestris Gravesac V. berlandieri V. riparia V. rupestris 1616 Couderc V. solonis V. riparia A Salt Creek V. champinii (Ramsey) Dogridge V. champinii Harmony V. champinii V. solonis

V. riparia ROOTSTOCKS AND CLONES, CULTIVARS, 1.2. Freedom V. champinii V. solonis V. riparia

Excellent/high...... Poor/low A: Advanced; D: Delayed N: No; Y: Yes Modified from Currle et al. (1983), Pongra´cz (1983), Galet (1998), and Dry and Coombe (2004). 19 20 1. BOTANY AND ANATOMY

1.3. MORPHOLOGY AND 50–75% of the biomass (dry matter) of ANATOMY cultivated grapevines. This proportion is much lower in young plants and increases with age and plant size; it is less in heavily cropped Grapevines are very vigorous, woody clim- vines and is also lower in humid climates than bers named lianas that are perennial (i.e., they in dry climates. The entire plant body is rigid live more than 2 years), polycarpic (i.e., they and strong yet flexible and adaptive enough flower many times during their life), and decid- to explore soil resources, intercept sunlight, uous (i.e., they shed their leaves each year). and develop seeds for propagation. This combi- With the aid of their tendrils and flexible nation of strength and flexibility is possible trunks, wild vines climb on trees to a height because plants employ a “bricks and mortar” of 30 m or more and spread out their foliage strategy for growth. The vine’s basic building over the tree canopy. Using the trees for sup- blocks, the cells (bricks), have the ability to port (i.e., seizing them as a natural trellis sys- divide and expand, and they are joined tem), they can grow to tremendous size; the together by an adhesive matrix, the cell walls canopy of a single vine may cover a surface (mortar). More precisely, the two cell walls of area of dozens of square meters. Grapevines adjoining cells are “glued” together by the so- can live to several hundred years of age: The called middle lamella, which prevents sliding famous Great Vine in London’s Hampton between cells. Growing cells secrete a primary Court Palace is thought to have been planted wall, which is reinforced in mature cells that before 1770 and continues to produce 200–300 have ceased expanding with a lignified second- kg of fruit each year. Vegetative propagation, ary wall for extra mechanical strength. The cell whether naturally by layering or artificially walls partly take on the function of bones in from cuttings, bud grafts, tissue culture, and animals: They give the plant body form and other means, extends a vine’s lifespan virtually rigidity, and like bones, cell walls are rich in indefinitely. In fact, both rooted cuttings and calcium. The cells differentiate into various grafted plants show clear signs of rejuvenation types, each “designed” to carry out specific compared to their “mother” plants (Munne´- physiological tasks under the direction of dif- Bosch, 2008). For instance, growth rates, leaf ferent sets of genes; although each cell of the gas-exchange rates, and fruiting of recently same plant contains the same complement of propagated vines are those of young plants and genes, different genes can be active or inac- are independent of the age of the vines from tive—that is, switched “on” or “off.” Such sim- which the propagation material was taken. ilar groups of cells that form functional units Like other higher plants, grapevines com- are termed tissues, and different tissues, such prise vegetative organs (roots, trunk, shoots, as epidermis, parenchyma, phloem, and xylem, leaves, and tendrils) and generative or repro- in turn make up the plant’s organs, which are ductive organs or fruiting structures (clusters described next. with flowers or berries). The vegetative part The evolution of such complex multicellular can be divided into a belowground portion structures from single-celled ancestors required (roots) and an aboveground portion (trunk the simultaneous development of innovative and shoots). The aboveground portion together solutions to solve the problem of communication with the reproductive organs is termed the between the cells forming a tissue, between vine’s canopy. The roots, trunk, and its trained different tissues within an organ, between extension, the cordon, together form the per- the diverse organs of the plant body, and among manent structure of the vine, which makes up plants of a community. As will become apparent 1.3. MORPHOLOGY AND ANATOMY 21 throughout this book, plants master the art of True roots develop from the hypocotyl intercell communication by cell-to-cell channels (Greek hypo ¼ below, kotyledon ¼ depression) named plasmodesmata, whereas a vascular of the embryo (Greek embryon ¼ unborn child). system composed of the xylem and phloem In vines grown from seeds, seed germination ensures interorgan communication (Lough and begins with water absorption, called imbibi- Lucas, 2006). The roots and shoots “converse” tion, that enables the embryonic root, or radicle with each other, at least in part, via the produc- (Latin radicula ¼ little root), to grow and rup- tion and release of different hormones (Greek ture the seed coat, forming a primary root or hormaein ¼ to stimulate); cytokinin and abscisic tap root, from which multiple secondary roots, acid are the main root hormones, whereas auxin branch roots or lateral roots, grow. This type and gibberellin are the main shoot hormones. of root morphology is typical of dicotyledons Hormones are defined as extracellular signaling and is called an allorhizic root system (Osmont molecules that act on target cells distant et al., 2007). In vegetatively propagated vines, from their site of production. Perhaps even more roots originate from the cambium layer (Latin fascinating, information exchange between cambiare ¼ to exchange) of woody cuttings neighboring plants is achieved by emitting and (Pratt, 1974). Most of these so-called adventi- receiving volatile chemicals transmitted through tious roots form near the nodes, but they also the air and by releasing soluble chemicals grow on the internodes. These roots are often into the soil and recruiting symbiotic micro- termed main roots, and they branch off into organisms that infect and thereby interconnect secondary, tertiary, etc. lateral roots to form a the roots of different plants. Of course, successful root system with a complicated architecture, communication requires not only the production which resembles the secondary homorhizic and export of appropriate signaling molecules root system typical of monocotyledons from transmitting cells (output signals) but (Osmont et al., 2007). In other words, main also the presence of a sophisticated network of roots develop directly from a cutting, whereas signal perception, decoding, and transformation lateral roots develop from other roots. Unlike in the receiving cells (input signals). Such the arrangement of shoot lateral organs, the signals integrate genetic programs that perceive number and placement of lateral roots are not and respond to both developmental and envi- predetermined (and hence inflexible) but ronmental cues to direct cell, tissue, organ, depend on the availability of water and nutri- and plant growth and physiology (Lough and ents in the soil (Malamy and Benfey, 1997). Lucas, 2006). Because the grapevine is unable to move away from poor soils, it relies on the ability to detect 1.3.1. Root these environmental cues and extend lateral roots to take advantage of favorable soil The root system is the interface between the regions. Moreover, in contrast to many other grapevine and the soil. It provides physical plant species, lateral root initiation in grape- support for the vine in the soil and is responsi- vines is not restricted to the unbranched apical ble for water and nutrient uptake. The roots zone. Grapevines are also able to grow new lat- also serve as storage organs for carbohydrates eral roots on older parts of the roots that have and other nutrients, which support the initial already developed a vascular cambium. Under growth of shoots and roots in spring, and for conditions of very high humidity and high tem- water. In addition, they are a source of plant perature, trunks and other aboveground parts hormones (cytokinins and abscisic acid), which of the vine can also form aerial roots. Whereas modify shoot physiology. this phenomenon is mostly restricted in the 22 1. BOTANY AND ANATOMY genus Vitis to greenhouse conditions (and rare mucilage secreted by the cap covers the surface even there), it is quite common in the natural of the maturing root. The mucigel harbors habitat of the Muscadinia (Viala and Vermorel, microorganisms and probably aids in the estab- 1909). lishment of symbiotic mycorrhiza (Greek mykes The root system of established grapevines ¼ fungus, rhı´za ¼ root) in the root tips, which comprises a far-reaching, highly branched form a network of fungal mycelium that con- structure with a surface area far exceeding that nects plants belowground. This network helps of the leaf canopy it supports. A mature, with the uptake of nutrients and water in cultivated grapevine can have more than 100 exchange for carbohydrates supplied to the km of total root length with a surface area fungi by the plants (Bais et al., 2006; Marschner, greater than 100 m2, whereas its leaf area is 1995; Smith et al., 2001). The site of active cell usually less than 10 m2. The woody roots, division (the apical meristem in the center) whose diameter rarely exceeds 3 or 4 cm, serve and differentiation (the first phloem cells to anchor the vine and transport and store soil- appear to the outside of the meristem) lies just derived nutrients, whereas the small absorbing behind the cap and is followed (0.7–1.5 mm roots (“fine roots” 0.1–1 mm in diameter) are behind the tip) by the elongation zone, a short responsible for acquisition of resources such region of cell expansion and further differentia- as water and nutrients. The woody roots of tion (the first xylem cells appear; see Figure 1.4). mature vines are widely distributed, with hori- Cell division is stimulated by the hormone zontal roots exploring the soil for distances of auxin (indole-3-acetic acid and related com- up to approximately 10 m from the trunk pounds) that is delivered from the shoot tips (Smart et al., 2006; Figure 1.4). Although the via the phloem and via active cell-to-cell trans- majority of roots, especially the fine roots, are port down the vascular parenchyma and is normally concentrated in the top 0.5–1 m, roots then also recycled, or refluxed, within the root can grow to a depth of more than 30 m when tip (Friml, 2003; Kramer and Bennett, 2006). they encounter no impermeable barriers (Galet, Cell elongation and differentiation, on the other 2000; Lehnart et al., 2008; Morlat and Jacquet, hand, are promoted by a group of growth hor- 1993; Pourtchev, 2003; Viala and Vermorel, mones termed gibberellins that are produced, 1909). Indeed, grapevines are among the most upon stimulation by auxin, close to or at their deeply rooted plants, and their root biomass site of action—that is, in tissues with rapidly can range from 5 to 40 t ha 1, which may be a expanding cells (Yamaguchi, 2008). The activity reflection of the competition for water and of the meristem also requires high concentra- nutrients during the vines’ co-evolution with tions of the “cell-division hormone” cytokinin their “trellis” trees (Smart et al., 2006). (trans-zeatin and related compounds), pro- The growing root tip or apex is covered by a duced in the root tips, and low concentrations slimy root cap whose starch-containing central of the “cell-expansion hormone” gibberellin, portion is named the columella. The cap pro- whereas cell elongation requires the opposite tects the root meristem from abrasion, facili- (Wang and Li, 2008; Weiss and Ori, 2007). tates penetration of the soil, and contains Because the phloem and xylem cells differenti- gravity sensors, which guide the root down- ate behind the root tip, hormones as well as ward through the soil. As the root advances the water and nutrients feeding the dividing and encounters new regions of moisture and cells must move across the cells after leaving nutrients, the cap is continuously sloughed off the vascular tissues. and replenished from the inside, whereas the The cells produced in the meristem and polysaccharide-rich slime called mucigel or elongation zone form a central ring called the 1.3. MORPHOLOGY AND ANATOMY 23

FIGURE 1.4 Distribution of Merlot root system in a drip-irrigated vineyard (left; photo by M. Keller) and diagrammatic longitudinal section of the apical region of the root (right; reproduced from Taiz and Zeiger, 2006). endodermis, which divides the root into two primary phloem, and primary xylem and is regions—the cortex toward the outside and thus also called the vascular cylinder, whereas the stele toward the inside. The cortex is the cortex develops the hypodermis and epi- responsible for nutrient uptake from the soil dermis, which form the dermal (“skin”) tissues and storage of starch and other nutrients, (Evert, 2006; Galet, 2000). This epidermis, how- whereas the stele is responsible for nutrient ever, is short-lived, and the underlying hypo- transport up and down the plant (see Chapter dermis forms the boundary between root and 3.3). The stele differentiates into the pericycle, soil even a few millimeters behind the root tip 24 1. BOTANY AND ANATOMY

(Storey et al., 2003). The endodermis consists of consisting of interactions between auxin, a single layer of cells with thickened radial and ethylene, cytokinin, brassinosteroid, and ABA transverse cell walls, named Casparian strips or regulates root architecture in response to Casparian band, impregnated with suberin and environmental (mainly water and nutrient lignin (see Chapter 3.3). The pericycle, rather availability) and developmental influences than the apical meristem as in the shoot, (Osmont et al., 2007). beneath the endodermis gives rise to the lateral Several millimeters behind this region fol- roots by renewed cell division, following dedif- lows the absorption zone, which is densely ferentiation of pericycle founder cells, where covered by root hairs. The colorless root hairs the meristem transitions into the elongation are long protrusions of epidermal cells (epider- zone (Mapfumo et al., 1994a; Nibau et al., mis cells that form root hairs are termed tricho- 2008; Osmont et al., 2007; Viala and Vermorel, blasts) and are mostly responsible for water 1909). Lateral root growth seems to be initiated and nutrient uptake (Gilroy and Jones, 2000; by shoot-derived auxin and aided by locally Pratt, 1974). They are very thin (10–15 mmin produced growth hormones termed brassinos- diameter but can flatten to squeeze through soil teroids (which resemble animal steroid hor- pores <2 mm) and can make up more than 60% mones) and small amounts of gaseous of the root’s surface area, which greatly ethylene (C2H4), which is released from the dif- increases the contact surface between root and ferentiating xylem. Apparently, the incoming soil, or the plant–soil interface, and the auxin induces this ethylene release, which in exploited soil volume (Clarkson, 1985; Sonder- turn blocks further auxin movement, and the gaard et al., 2004; Watt et al., 2006). As the root resulting local auxin accumulation then grows, new root hairs constantly grow behind induces lateral root growth adjacent to the pri- the elongation zone while the older ones are mary xylem (Aloni et al., 2006b). On the other worn away along with the epidermis, the origi- hand, higher concentrations of ethylene and nal cortex, and the endodermis. Hence, the cytokinin moving up from the root tips inhibit absorption zone advances along with the root lateral root formation (Ivanchenko et al., 2008; tip, leaving behind the conducting zone that Osmont et al., 2007). The fact that cytokinin con- continues into the trunk and all aboveground centration is highest closest to the site of its organs of the vine. production ensures that lateral roots are not Procambial cells generate both the primary formed too close to the tip, which would inter- phloem and the primary xylem and then fere with its continued growth (Aloni et al., develop into the vascular cambium, which 2006b). Moreover, the “dormancy hormone” serves as a lateral meristem and forms a contin- abscisic acid (ABA) seems to control the activa- uous sleeve of one to several cell layers within tion of the new lateral root meristem after the root–shoot axis. Once elongation growth emergence of the lateral root from its parent has ceased, the cambium forms new, secondary root: A high ABA concentration (e.g., due to phloem cells toward the outside and secondary water stress or high nitrogen availability) inhi- xylem cells, which collectively form the wood, bits meristem activation and keeps the lateral toward the inside (Viala and Vermorel, 1909). root in a dormant state (De Smet et al., 2006; The cambium is therefore responsible for the Malamy, 2005). Once activated, auxin usually radial growth of a root and moves outward as maintains the cell-producing activity of the the root grows thicker. Its cells can be regarded meristem. Thus, the meristem and elongation as “stem cells,” and the transcellular flow (i.e., zones together form the zone of active root cell-to-cell movement across the cell mem- growth in length, and a signaling network branes and cell walls) of auxin from the shoot 1.3. MORPHOLOGY AND ANATOMY 25 tip toward the roots in addition to cytokinin along with the phloem flow; van Bel, 2003) are produced in the root tips and physical pressure the conduits for assimilates, whereas the stimulate cambial activity and subsequent companion cells (which are crammed with cell differentiation (Aloni, 2001; Aloni et al., mitochondria) carry out the metabolic func- 2006b; Dengler, 2001; Ye, 2002). The cambium tions abandoned by the sieve elements and act becomes dormant in late summer and is reacti- as transfer cells loading sugar and other materi- vated in spring by root-derived cytokinin in als from and to the surrounding parenchyma response to auxin released by the swelling buds cells into and out of the sieve elements. (Aloni, 2001). Simultaneously, a cork or bark To this end, pairs of companion cells and cambium named phellogen forms in the pericy- sieve elements (which arise from a common cle, which later generates cork (phellem) mother cell; see Viala and Vermorel, 1909) are toward the outside and secondary cortex (phel- interconnected through numerous “lifelines” loderm) toward the inside (Mullins et al., 1992; termed pore/plasmodesma units, and the com- Pratt, 1974). Because of the sloughing off of panion cells and parenchyma cells are connected the outermost cell layers, the mature root con- through plasmodesmata (membrane-lined chan- sists of cork on the outside, cork cambium, sec- nels connecting plant cells). The sieve elements, ondary cortex, phloem, vascular cambium, and with a diameter up to approximately 35 mm (Viala xylem at the center (Viala and Vermorel, 1909). and Vermorel, 1909), have thicker cell walls than The deposition of the waxy, hydrophobic the adjacent parenchyma cells and are stacked (water-repellent) biopolyester suberin in the end-to-end and interconnected by large (up to cork cell walls and the oxidation of phenolic 1 mm) sieve pores in the end walls (named sieve compounds from dying cells give these roots a plates) to form the sieve tubes (van Bel, 2003). In brownish color. contrast to the xylem cells, they retain The phloem, vascular cambium, and xylem their plasma membrane, which is necessary to together are called the vascular tissue, or vascu- generate osmotic gradients for assimilate lar system, which forms discrete vertical strands transport in a direction opposite to transpiration named vascular bundles, separated by rays con- (see Chapter 5.1). sisting of parenchyma cells that are rich in nutri- The xylem (Greek xylos ¼ wood) consists of ent reserves (e.g., starch and proteins) and vessel elements, tracheids, fibers, and xylem tannins (Currle et al., 1983; Stafford, 1988; Viala parenchyma. The vessel elements and tracheids and Vermorel, 1909). The phloem and xylem form strong secondary cell walls that become are placed in parallel within each vascular bun- heavily lignified by the incorporation of lignin dle (a pattern termed collateral vascular bundle) (Latin lignum ¼ wood) and together are called and are interconnected by the parenchyma rays, the tracheary elements. These are arranged which allows for the transfer of nutrients longitudinally and embedded in the fibers and between the two “circulatory systems.” The parenchyma, the latter forming radial rays phloem (Greek phloios ¼ bark) is composed of that separate bundles of tracheary elements (living) sieve elements, companion cells, and (Figure 1.5). The tracheary elements are the phloem parenchyma. In a unique division of actual xylem conduits or water pipes of the vine. labor, the hollow and almost “clinically” dead Before they can assume their function, however, sieve elements (before they become functional, these cells must die; their organelles and cyto- they resorb their nucleus and vacuole and only plasm are disassembled, digested, and removed retain their cell membrane, a thin layer of cyto- in an orderly manner termed programmed cell plasm, plastids, and a few enlarged mitochon- death, and the dead cells become the wood (Jones dria, tied together so they are not dragged and Dangl, 1996; Plomion et al., 2001; Turner et al., 26 1. BOTANY AND ANATOMY

FIGURE 1.5 Cross sections of 1-year-old (left), 2-year-old (center), and 3-year-old V. vinifera root (right). Reproduced from Viala and Vermorel, 1909).

2007). Their thickened secondary cell walls perforation plates of the large vessel elements provide mechanical strength to prevent the pipes formed early in the growing season are simple from collapsing under the negative pressure (i.e., have a single hole), whereas those of the generated by transpiration (Ye, 2002; see also vessels formed later (latewood) are sometimes Chapter 3.3). The lignification also renders the scalariform (i.e., have several elongated holes that cell walls relatively waterproof, although lignin make the remaining cell wall appear like rungs of is not completely impermeable to water. The fiber a ladder). The much rarer tracheids, which look cells also commit suicide in a tightly controlled much like vessels without perforation plates, manner after the death of the tracheary elements occur in the latewood and, because they are single (Courtois-Moreau et al., 2009) so that the only cells, are much shorter (1–10 mm) than the live cells in the mature xylem are those of the vessels; many vessels in grapevines are more than parenchyma, and even these cells die after a few 100 mm long, and some exceed 1 m. Both vessels years (Plomion et al., 2001). These cells serve as and tracheids are radially interconnected storage sites for water and nutrients such as car- (although not with each other) through pairs of bohydrates and proteins or amino acids (Zapata porous (water-permeable) cell wall depressions et al., 2004), contain raphide crystals to deter named pits. Whereas the connections between herbivores, and are involved in the defense individual vessel elements within a vessel are against pathogen penetration. The thick (4 mm) provided by either pits or perforation plates, cell walls of the very long (0.5 mm) and thin those between one vessel and another or between (20 mm) fibers give the root mechanical support. neighboring tracheids are provided by pits (De Multiple vessel elements are stacked end-to- Boer and Volkov, 2003; Sun et al., 2006). The pits end and interconnected by perforation plates are generally scalariform and bordered; bordered (holes created by targeted removal of the cell pits are pores with overarching secondary cell walls and membranes) in the end walls to walls with a narrower aperture or pit channel form long hollow columns termed vessels. The opening into a wider pit chamber. The two 1.3. MORPHOLOGY AND ANATOMY 27 adjoining vessels or tracheids are separated (so-called spur pruning, which generally inside the chamber by a permeable “membrane,” retains one to three buds per spur) or longer which consists of the middle lamella and primary canes (so-called cane pruning, which typically cell walls. Although not a true cell membrane retains eight or more buds per cane). Both spur because it does not contain a lipid bilayer, the fine and cane pruning are usually done manually, pore size (5 nm) of pit membranes tends to slow which permits maximum control over both water flow but also limits the spread of air bub- node number and position on the vine. bles (see Chapter 3.3) and pathogens. Because of Cordon-trained grapevines may also be pruned their more open construction, vessels can conduct by machine, leaving a range of shorter and lon- water more efficiently than can tracheids. Water ger spurs centered on the cordon; this is also also moves laterally through pits (more accu- called a box cut. Machine pruning is sometimes rately through pit pairs because the pits of the done as a prepruning operation only, followed two cells line up to connect the cells), both by manual touch-up to control bud numbers. between parallel vessels or tracheids, and from Alternatively, very light mechanical pruning, and to the surrounding parenchyma cells, but which trims off only the ends of the shoots or not to fibers. Due to their length (0.5 mm) and canes, may be done in summer and/or winter, large diameter (10–150 mm), individual vessel ele- a method termed minimal pruning (Clingeleffer, ments can be surrounded by and connected with 1984; Possingham, 1994). This practice relies on many dozens of the much smaller parenchyma the vines’ self-pruning ability, in which the cells (Sun et al., 2006; Viala and Vermorel, 1909). immature apical portion of the shoots generally dies back and falls off in winter (see Chapter 1.3.2. Trunk and Shoots 5.1). An assortment of combinations of training systems, pruning methods, and other cultural The aboveground or aerial axis of the grape- practices are employed in commercial viticul- vine, which comprises the shoots, arms, and ture. They all have as their common goals to trunk, is called the stem by botanists. It pro- constrain the natural vigor of the vines and vides support for the growing vine and is maintain them at a small, manageable size; to responsible for water, nutrient, and assimilate sustain their shape and productivity over transport. The flexible stem (in addition to the the several decades of a vineyard’s life; to opti- roots) also serves as storage organ for carbohy- mize fruit production and quality depending drates and other nutrients, which support early on the intended end use of the grapes; and growth in spring and during stress periods, to facilitate labor and permit various degrees and for water. Because of their liana natures, of mechanization. cultivated grapevines typically require a trellis Phyllotaxy (Greek phyllon ¼ leaf, taxis ¼ order) system for support, unless they are trained describes the regular arrangement of leaves in very close to the ground. The trunk is often space or the pattern of lateral organs on the extended along a horizontal wire to form one shoot, whereby the points at which the lateral or more permanent arms or cordons that sup- organs connect to the shoot are called nodes. In port the 1-year-old wood, which in turn gives vines grown from seeds, the epicotyl (embryonic rise to the fruiting shoots. Such vines are said shoot) emerges from the embryo, forming a to be cordon trained, whereas vines without primary shoot without tendrils, and leaves cordons are said to be head trained. The shoots, arranged in a spiral fashion, with leaves which are called canes after they have matured offset by 137.5 (the so-called golden angle), and the leaves have fallen off, are generally displaying 2/5 (sometimes 3/7) phyllotaxy pruned back in winter to either short spurs (Galet, 2000; Viala and Vermorel, 1909). This 28 1. BOTANY AND ANATOMY means that two revolutions around the shoot The apical meristem is the site of cell division, must be made to find a leaf that is located where all shoot organs are initiated (in contrast directly above the reference leaf, which is five to the root apical meristem that does not form leaves below. This juvenile phase generally ends lateral roots) and where the pattern of the when 6–10 leaves have developed and the vine shoot system is established. It consists of three enters adult development (Mullins et al., 1992). subpopulations of cells: the central zone at the All new leaves are now being produced tip and the rib and peripheral zones below it alternately—that is, on two opposite sides of the (Clark, 1997; Evert, 2006; Kerstetter and Hake, shoot with a single leaf at each node (distichous 1997). The central zone with very small, rela- or 1/2 phyllotaxy with 180 angles)—and the first tively slowly dividing cells with small vacuoles, tendrils are formed. In vegetatively propagated located at the apex of the apical meristem, serves vines or adult plants, primary shoots usually as a source of cells (“stem cells”) for the originate from the buds at nodes of woody canes, other two zones, which are involved in cell dif- and their leaves always display 1/2 phyllotaxy. ferentiation. The rib zone at the base of the apical Shoot growth from buds is sometimes divided meristem forms the vascular and other tissues in into “fixed” growth and “free” growth (Mullins the central part of the shoot. The peripheral et al., 1992). Fixed growth occurs from leaf pri- (or morphogenetic) zone with small, rapidly mordia and compressed internodes preformed dividing cells with small vacuoles surrounds during the previous growing season that the central zone on the flanks of the apical overwinter in the dormant bud. It is responsible meristem and produces the outer shoot tissues for the rapid growth of the first 6–12 leaves in and the lateral meristems that give rise to the spring; the number increases with increasing primordia, which in turn develop into the lateral temperature during bud development (Buttrose, organs (Figure 1.6). In the great majority of 1970a; Morrison, 1991). Free growth, on the species of the family Vitaceae (including all other hand, occurs later in the season from the species of economic importance), there are two production of new leaf primordia and internodes types of lateral meristems: One is responsible in the shoot’s apical meristem. for leaf formation and the other for inflorescence

FIGURE 1.6 Diagrammatic longitudinal section of Concord shoot tip (left; reproduced from Pratt, 1971, reprinted by permission of AJEV); lateral organs arising from various positions in the dormant bud, illustrating the repeating three-node pattern unit of the shoots of many Vitis species (center; reproduced from Carmona et al., 2002); and chimeric Cabernet Sau- vignon shoot (right; photo by M. Keller). 1.3. MORPHOLOGY AND ANATOMY 29 and tendril formation (Carmona et al., 2007; the soil surface and leads to the formation of Gerrath and Posluszny, 2007). The various zones colorless, etiolated shoots that elongate rapidly overlap with another structural feature of the under the influence of gibberellin in search of apical meristem—the horizontal layering of cells light (Hartweck, 2008). The initial set of cells divided into the tunica (which usually consists from which all subsequent cells of a lateral of two parallel cell layers, the outermost of organ are derived are termed “founder cells” which generates the epidermis) on the outside or “anlagen” (German Anlage ¼ conception, and the corpus on the inside (Evert, 2006; layout, investment) that result in a primor- Thompson and Olmo, 1963). Because the cells dium. The lateral organs include leaves, ten- of the tunica divide in an anticlinal plane drils, clusters (inflorescences), and axillary (perpendicular to the surface) and those of the buds. Axillary buds contain meristem primor- corpus in different directions, the two layers dia that are called secondary, axillary, or lateral perpetuate separate cell lineages (Clark, 1997; meristems and form several compressed phyto- Kerstetter and Hake, 1997). Mutations occurring mers before becoming dormant; they can be in only one of the two layers can therefore give reactivated later to produce lateral shoots, rise to chimeric plants (see Figure 1.6) and to whose leaves form at right angles to those of clonal variation within grape cultivars (see the main shoot (Gerrath and Posluszny, 1988a; Chapter 1.2). McSteen and Leyser, 2005, Pratt, 1974; In contrast to the mature cells of the pith, Figure 1.7). Likewise, the inflorescence primor- rays, and cortical parenchyma (Kriedemann dia contain their own flower meristems that and Buttrose, 1971), the meristem cells do not later produce the grape flowers and their contain chlorophyll, so they cannot perform different floral organs, which are themselves photosynthesis and must import all carbon modified leaves—a concept originally pro- and other nutrients. The “installation” of the posed by the German writer and philosopher photosynthetic machinery begins as soon as Johann Wolfgang von Goethe (1790). Contrary the cells are released from the meristem, unless to their influence in the root tip, cytokinins those cells remain in darkness. This occurs, for stimulate cell division in the shoot apical meri- example, in a trunk sucker arising from below stem and are necessary for the transition from

FIGURE 1.7 Repeating three-node pattern of a Syrah shoot (left); mistakes do happen in nature—three consecutive ten- drils on V. vinifera shoot (center); and dormant bud and lateral shoot in a leaf axil (juncture between petiole and shoot) of a Malbec main shoot (right). Photos by M. Keller. 30 1. BOTANY AND ANATOMY undifferentiated cells to differentiated pri- the other members of the family leave a mordia (Dewitte et al., 1999; Mok and Mok, “blank” at every third node and how they keep 2001; Werner et al., 2003). count is still mysterious. The unusual position Just how the vine “knows” when and where of clusters and tendrils opposite leaves is a to initiate the various lateral organs is still result of the lack of elongation of the internodes somewhat of a mystery. Local accumulation of separating nodes that bear only rudimentary auxin and perhaps cytokinin seems to be the leaves termed bracts from those that bear true signal for organ formation by activating the cell leaves (Morrison, 1991; Tucker and Hoefert, wall-loosening protein expansin (see Chapter 1968). The first (i.e., lowest) two or three nodes 3.1), and the identity of each lateral organ usually carry only leaves, the next two nodes may be determined by hormone gradients are generally the ones with clusters, followed (Reinhardt et al., 2000; Wang and Li, 2008). by a node with a leaf only and then a succes- Once initiated, the primorida become sinks for sion of repeating mirror-images of three-node auxin, draining auxin from surrounding cells units with the clusters replaced by tendrils and thereby preventing their growth, which (see Figure 1.6). Some cells do not become pri- ensures the regular, phyllotactic positioning of mordia and will instead develop into the inter- lateral organs (Benjamins and Scheres, 2008; nodes of the shoot axis. These cells differentiate Berleth et al., 2007; Kuhlemeier, 2007). Meris- into the epidermis, cortex, endodermis, cam- tems and leaves are in constant communication bium, phloem, and xylem (Galet, 2000; Viala and clearly influence each other; preexisting and Vermorel, 1909). The repeating units are leaves are required for the correct positioning called phytomers, and each phytomer consists of a new leaf, possibly by defining the routes of a node, an internode, a leaf, and an axillary of auxin transport to the meristem (Piazza bud containing an axillary meristem for et al., 2005; Reinhardt et al., 2000). The older branching (McSteen and Leyser, 2005). leaves even influence specific traits of newly The epidermis develops a waxy cuticle developing leaves, such as leaf size and the (Latin cutis ¼ skin) on its outer cell walls as a density and size of stomata, so that the unfold- protective layer of all aboveground organs. ing leaves are adapted to the environment into The epidermis also has stomata, various types which they are “born” (Lough and Lucas, of hair, or trichomes (Greek trichos ¼ hair), 2006). Moreover, the axillary buds are initially and, on young organs, often small and short- not connected to the vascular bundles of the lived translucent structures variously named shoot; this connection is established at the time pearls, pearl glands, pearl bodies, or sap balls of bud outgrowth. It appears that the buds (Pratt, 1974; Figure 1.8). Pearls also develop on release auxin just before they break, and this tendrils, inflorescences, petioles, and the abax- auxin induces differentiation of vascular tissues ial surface of leaves, and they are especially that connect the buds (and hence the growing frequent under warm, humid conditions and lateral organs) to the preexisting vascular net- high nutrient availability promoting vigorous work (Aloni, 2001). growth (Paiva et al., 2009; Viala and Vermorel, The production of leaf-opposed tendrils and 1909). Although they remain covered by a thin clusters appears to be unique to the Vitaceae epidermis that sometimes develops a stoma, family and is typically discontinuous; that is, they are formed of highly vacuolated, thin- two of every three nodes bear a tendril (Gerrath walled subepidermal cells accumulating and Posluszny, 2007; Pratt, 1974). One notable sugars, oils and, perhaps, proteins. Pearls have exception is V. labrusca, which has a continuous no glandular activity but instead are also pattern—that is, a tendril at every node. Why termed food bodies because they ostensibly 1.3. MORPHOLOGY AND ANATOMY 31

bundles is probably induced by acropetal (toward the tip) polar, active transcellular movement of auxin (at a rate of 1cmh 1), perhaps in the epidermis, from developing leaf primordia and young leaves (Benjamins and Scheres, 2008; Berleth et al., 2007; Woodward and Bartel, 2005). In addition, gibberellin imported via the phloem and/or produced by the growing xylem cells may regulate xylem cell differentiation and elongation (Israelsson et al., 2005). As in the root tip, the first phloem cells appear before the first xylem cells, providing a conduit for the import of carbohy- drates and other material at the earliest stages of leaf development. Each new leaf generated by the apical meristem is connected with pre- existing vascular bundles in the shoot by, normally, five divergent bundles called leaf FIGURE 1.8 Ant feeding on pearls on a Chardonnay traces (Figure 1.9). Most leaf traces arise three shoot. Photo by M. Keller. nodes below their point of departure to a leaf, whereas tendril and axillary bud traces usually begin one node below their point of departure attract and feed ants, which in turn help defend (Chatelet et al., 2006; Gerrath et al., 2001). The the vulnerable young plant organs against shoot vascular bundles that continue their herbivorous insects (Paiva et al., 2009). course through the next internode and ensure The cortex consists mostly of parenchyma the continuity of the vine’s vascular system cells that contain chloroplasts, starch, calcium are termed sympodial bundles. Their xylem oxalate crystals, and phenolic compounds vessels are connected with the vessels of the (including anthocyanins on the sun-exposed leaf traces via intervessel pits, whereas the leaf side of the shoots of many varieties). Some traces ensure continuous vessels between the parenchyma cells below the epidermis differen- shoot and the leaf blade. The vessels terminate tiate into strands of collenchyma with thick cell in the leaf at approximately 50–60% of the leaf walls of cellulose, strengthening the growing length; the xylem conduits beyond this limit shoot. The endodermis is also called the starch are tracheids rather than vessels (Chatelet sheath because of its high concentration of et al., 2006). amyloplasts. These are specialized plastids res- The procambium develops into the vascular ponsible for starch production from imported cambium, which is divided into intrafascicular sucrose and its storage in starch grains or gran- cambium and interfascicular cambium. The for- ules (Emes and Neuhaus, 1997; Martin and mer produces secondary phloem toward the Smith, 1995; Pratt, 1974). In addition, paren- outside and secondary xylem toward the inside, chyma cells also accumulate and store protein and the latter forms parenchyma cells that reserves inside specialized protein storage become the rays separating the conducting tis- vacuoles, which are disassembled into trans- sues or vascular bundles (see Figure 1.9). As in portable amino acids during remobilization the root, the cambium is responsible for radial in spring. The differentiation of the vascular growth, but in contrast to its action in the root, 32 1. BOTANY AND ANATOMY

FIGURE 1.9 Cross section of V. vinifera shoot prior to periderm formation (left) and of cane after two layers of phloem have been discarded (center), and longitudinal section through Vitis shoot (right: A, pith; B, stele; C, cortex; D, diaphragm). Reproduced from Viala and Vermorel (1909). cytokinin stimulates this cambial activity in the time the grapes begin to ripen). Annual rings shoot (Werner et al., 2003). Cytokinins are pro- are composed entirely of xylem cells because duced in the root tips and unfolding leaves and the vascular cambium moves outward with the are transported upwards in the xylem and growing trunk, leaving only one live ring of downwards in the phloem (Aloni et al., 2005; phloem at the end of each growing season, Nordstro¨m et al., 2004). Cambium activity which is reactivated in spring (Davis and Evert, results in annual rings of secondary xylem; 1970; Esau, 1948). The xylem remains functional when we count annual rings, we are counting for several years; older, dysfunctional xylem rings of xylem. The annual rings of grapevines then becomes the heartwood. are called diffuse-porous (Mullins et al., 1992; Some parenchyma cells form outgrowths Sun et al., 2006) because the size of the xylem into the lumen of tracheary elements, entering cells produced in spring is similar to that of via the interconnecting pits; such outgrowths those produced in summer, before the cambium are termed tyloses (Greek tylos ¼ knob, knot) activity ceases (usually at approximately the and serve mainly to seal injured xylem (e.g., 1.3. MORPHOLOGY AND ANATOMY 33 due to summer or winter pruning or to freez- and is accompanied by deposition of starch in ing), perhaps to prevent entry of pathogens the xylem and phloem parenchyma cells (Eifert (Evert, 2006; Sun et al., 2006; Viala and et al., 1961; Plank and Wolkinger, 1976). It ends Vermorel, 1909). Within the ring of vascular with the death and abscission (Latin abscissio ¼ bundles there is a core of dead, unsclerified separation) of the apical meristem and sealing parenchyma cells called the pith. Some second- of the sieve pores and plasmodesmata inside ary phloem cells also form a cork cambium the sieve tubes by callose for overwintering (phellogen), which later produces cork (phel- (Davis and Evert, 1970; Esau, 1948; Viala and lem) toward the outside and secondary cortex Vermorel, 1909). Like cellulose (b1!4-glucan) (phelloderm) toward the inside (Galet, and starch (a1!4- and a1!6-glucan), callose 2000; Pratt, 1974). Phellem, phellogen, and (b1!3-glucan) consists of long chains of glu- phelloderm together form the outer bark cose molecules. The callose is degraded in the or periderm, whose multilayer combination of following spring, restoring the transport func- dead but elastic and heavily suberized cork tion of the phloem for another growing season cells and waxes, by protecting the shoot (and (Pratt, 1974). The reactivated phloem from the later the cane, cordon, and trunk) from wound- previous season is important during the early ing as well as water and nutrient loss, not only growth period, before it is replaced with newly takes over the function of the epidermis/cuticle formed phloem and discarded around mid- it replaces but also provides some insulation season, concurrent with periderm formation against temperature fluctuations (Franke and (see Figure 1.9). Depending on the species, the Schreiber, 2007; Lendzian, 2006). The cork layer vascular cambium resumes its function from is interspersed with pores called lenticels approximately 2 weeks (V. vinifera) to 2 months that, it seems, assume the role of the vanished (V. riparia) after the phloem has been reacti- stomata, permitting continued gas exchange vated, and a new phellogen is formed (Davis between the interior tissues and the atmo- and Evert, 1970; Esau, 1948). This cambium sphere. Additional protection, especially reactivation (early cell divisions), which is against fungal infection, arises from the incor- induced by auxin, is extremely fast and occurs poration of phenolic compounds termed stil- almost concurrently in the canes and trunk, benes (mainly resveratrol and ¼ E-viniferin), before any bud swelling can be observed which is one of the reasons woody tissues (Aloni, 2001). The new phellogen isolates most decay only very slowly (see Chapter 7.5). of the secondary phloem produced in the pre- All tissues outside the vascular cambium are vious growing season, and the dead phloem, collectively referred to as the bark; these together with the old periderm, is eventually include epidermis, cortex, and phloem. The sloughed off as strips of dead bark (Davis and periderm, along with the outermost layers of Evert, 1970; Esau, 1948). The tissue layers that secondary phloem, dies and turns brown (from are eliminated annually (i.e., the dead parts oxidation of cellular components) starting at of the bark) are collectively termed the rhyti- the base and moving up toward the tip as the dome (Greek rhytidu`ma ¼ wrinkle) (Viala and shoot becomes a cane. Thus, browning is Vermorel, 1909). caused by the death of the green cortex rather than by lignification, which occurs in all sec- 1.3.3. Nodes and Buds ondary cell walls but especially in those of the xylem cells, independently of and long before The lateral organs of the shoot are attached browning. Browning is also called shoot “matu- at nodes, which can be distinguished from the ration” (French aouˆtement from aouˆte´ ¼ mature) rest of the shoot or cane by their characteristic 34 1. BOTANY AND ANATOMY swelling due to a thicker pith and cortex arise in the axil (upper angle) between a shoot (Figure 1.10). In all Vitis species, a diaphragm and a leaf, directly above the petiole insertion consisting of hard, thickened pith cells with point (Galet, 2000; Viala and Vermorel, 1909). sclerified (Greek skleros ¼ dry, hard) cell walls All buds are formed by shoot apical meristems. divides the pith at the node. It is thought that There are three types of buds: prompt buds, the diaphragm aids in directing water and dormant buds, and latent buds. Prompt buds nutrients into the leaves. The leaf and bud are are the only true axillary buds of the primary inserted just above the node, and each leaf is or main shoot and are also called lateral buds connected to the shoot via its own vascular (Gerrath and Posluszny, 2007; Pratt, 1974). bundles inside a petiole. When the petiole falls They develop from the earliest formed (lateral) off, it leaves a leaf scar on the node. meristem and can break in the current growing Buds are young, compressed shoots (i.e., the season and give rise to secondary, so-called lat- internodes have not yet elongated; see Fig- eral shoots. Species such as V. rupestris, V. arizo- ure 1.6) enclosed in scales or bracts that are nica,orV. monticola often grow many long green in summer and turn brown during shoot laterals, which in turn can give rise to tertiary maturation. The bracts (leaflike structures with- shoots (Viala and Vermorel, 1909). By contrast, out petiole or lamina) protect the bud from des- tertiary shoots are rare in V. vinifera, and lat- iccation and freezing. In addition, long, woolly erals form mainly if the apical dominance of epidermal hairs growing on the inside of the the primary shoot is broken (Alleweldt and bracts form a down that cushions the bud and Istar, 1969), for example, by removal of the gives it a woolly appearance when it opens shoot tip (see Chapter 2.2). Although prompt (breaks). Buds are always axillary; that is, they buds are usually not fruitful, some clusters,

FIGURE 1.10 Location of the main features of a Vitis cane (left; illustration by A. Mills) and one-node Concord spur with one count node and three basal buds (right: A, spur with buds; B, cross section of basal bud; C, longitudinal section of basal bud; D, cross section of compound bud; E, longitudinal section of compound bud; reproduced from Pool et al., 1978, reprinted by permission of AJEV). 1.3. MORPHOLOGY AND ANATOMY 35 referred to as “second crop,” can grow on lat- Initially, 3 or 4 leaf primordia are produced, eral shoots, especially on those growing from each of them flanked by a pair of scales and the preformed part of the main shoot. Remov- wrapped in abundant hair (Morrison, 1991; ing the shoot tip, a cultural practice variously Snyder, 1933; Srinivasan and Mullins, 1981). named shoot tipping, topping, or hedging in The bud’s apical meristem then starts to pro- viticultural parlance, appears to promote the duce lateral meristems opposite to leaf primor- development of such second-crop clusters. dia. The first two or three of these will These clusters are typically small and ripen normally give rise to inflorescences, and the well after the clusters on the main shoot, if at remainder will become tendrils. Intriguingly, all. Many lateral shoots fail to form a proper in the majority of cultivars only the buds pro- periderm and are abscised in late fall or winter. duced from fixed growth (i.e., at the first 6–10 Dormant buds are formed later than prompt nodes), it seems, are able to initiate inflores- buds and arise from their own shoot apical cences; all buds formed during the shoot’s meristems in the axil of the first leaf (reduced subsequent free-growth phase only initiate ten- to a small, scale-like leaf called a prophyll) of drils (Morrison, 1991; Sa´nchez and Dokoozlian, lateral shoots, whether or not these shoots 2005). The inflorescence meristem produces emerge from the prompt buds (Gerrath and several branch meristems in spiral phyllotaxy, Posluszny, 1988a, 2007; Pratt, 1974; Viala and each of them subtended by a bract (Gerrath Vermorel, 1909). Some of these dormant buds and Posluszny, 1988b; May, 2004; Srinivasan will give rise to next year’s crop, but they and Mullins, 1981). The secondary buds gener- remain dormant until the following spring, ally remain small and are less fruitful. They which is why they are also called “winter often grow out when the primary bud has been buds.” A dormant bud is in fact an overwinter- damaged, for example, by spring frost or ing compound bud (also called an “eye”) insects, but also when vines have been pruned because it generally contains three separate severely so that their shoot system is out of bal- buds (see Figure 1.10)—a larger primary bud ance with the capacity of the root system. in the center flanked by two smaller secondary Latent buds are buds that remain dormant buds. The latter are also termed accessory buds or hidden for several years and often become and are often separately named a secondary part of the permanent structure of the vine. and a tertiary bud because they develop in the Even decades-old cordons and trunks still carry axils of the first and second prophyll, respec- viable latent buds that can give rise to water tively, of the shoot primordium that forms the shoots or suckers, especially following frost primary bud (Morrison, 1991; Pratt, 1974). events or on vines that have been pruned too Higher order buds have been reported, but severely (Galet, 2000; Lavee and May, 1997; these usually remain rudimentary (Zelleke Sartorius, 1968). Such shoots are usually not and Du¨ ring, 1994). Just like the shoot apical fruitful, although exceptions do occur. meristem, the apical meristem of the primary Basal buds, located at the base of a shoot or bud generates two types of lateral meristems: cane (see Figure 1.10), are formed in the shoot’s One is responsible for leaf production and the prophyll axils and thus can be prompt buds, other for inflorescence and tendril production dormant buds, or latent buds. Because the (Carmona et al., 2007). These form as many as basal internodes elongate only insignificantly, 12 leaf primordia (8–10 on average) and their these buds appear to be situated in a whorl associated prompt bud, up to several inflores- (“crown”) around the shoot (cane) base (Pratt, cence primordia, and several tendril primordia 1974; Viala and Vermorel, 1909). They are before they become dormant (see Chapter 2.2). therefore also called “crown buds” and can be 36 1. BOTANY AND ANATOMY quite numerous, although typically inconspicu- primordia is termed the plastochron (Greek ous. Although basal buds are usually less fruit- plastos ¼ formed, chronos ¼ time). We distin- ful than the other buds, this varies by cultivar: guish four types of leaves according to their Zinfandel, Sangiovese, Gamay, and Muscat of position on the shoot: cotyledons, scales, bracts, Alexandria have particularly fruitful basal and foliage leaves. Cotyledons (embryonic buds, whereas fruitfulness is very low in Viog- leaves) are preformed in the embryo and are nier, Gewu¨ rztraminer, and Thompson Seedless. the first two leaves to emerge from the embryo Of course, this has implications for the type of during seed germination. They are short-lived winter pruning; cultivars with low basal bud and fall off soon after germination. The scales fruitfulness are better suited for cane pruning, form around buds and protect them from water whereas those with high fruitfulness can be loss and mechanical injury. Bracts are small, pruned to short spurs. scalelike leaves at branch points on the stem of inflorescences and tendrils. The first two 1.3.4. Leaves leaves of a shoot grown from a bud ordinarily also develop as bracts and are separated by A leaf differentiates along with a very short internodes. Subsequent leaves corresponding node from a leaf primordium develop into mature leaves termed foliage (Latin primus ¼ first, ordiri ¼ to begin to weave) leaves, which are separated by elongated inter- produced by the apical meristem in the shoot nodes. Foliage leaves consist of lamina (blade), tip (Figure 1.11). The production of leaf primor- petiole (stalk) and, at the base of the petiole, a dia is induced by the “cell-expansion” protein pair of stipules, which are short-lived leaflike expansin, which in turn is induced by the plant structures or sheaths nearly surrounding the hormone cytokinin produced either locally near shoot’s node (Viala and Vermorel, 1909). the apical meristem or in the root tips, from The petiole (Latin petiolus ¼ little foot) is the where it is imported via the xylem (Mok and leaf stem that connects the lamina to the shoot Mok, 2001, Pien et al., 2001). The (thermal) time and contains multiple (typically 12–14) vascu- between the formation of two successive leaf lar bundles (Viala and Vermorel, 1909). Several vascular bundles are often derived from the same shoot vascular bundle (i.e., leaf trace); this branching is called anastomosis. Depending on species and cultivar, the petiole can be between 2 and 12 cm long and grows toward the light to position the leaf for optimal sunlight inter- ception (Figure 1.12). Where the petiole joins the blade, it divides to form the five main veins of the lamina. An abscission layer forms at each end of the petiole toward the end of the grow- ing season and leads to leaf fall. The basic function of the lamina (Latin lam- ina ¼ leaf) is to capture sunlight for energy FIGURE 1.11 Longitudinal section of dormant Cabernet (ATP) production and carbon dioxide (CO2) Sauvignonbud.A,shootapicalmeristem;B,lateralbudpri- for carbohydrate production to support the mordium; C, inflorescence primordium; L, leaf primordium; U, uncommitted primordium. Reproduced from Morrison, vine’s metabolism and growth (see Chapter 4). J.C. (1991). Bud Development in Vitis vinifera L. International To maximize light absorption, leaves must be Journal of Plant Sciences, The University of Chicago Press. as wide as possible, whereas to maximize gas 1.3. MORPHOLOGY AND ANATOMY 37

FIGURE 1.12 Cross section of a V. vinifera leaf with prominent main vein (left; reproduced from Viala and Vermorel, 1909), Zinfandel petiole bending toward the light (center), and pattern of Concord leaf venation (right; photos by M. Keller).

exchange, they must be as flat as possible; both evenly to and from all over the lamina. The of these properties, however, make leaves veins are enclosed by a set of thickened paren- inherently vulnerable to overheating and dehy- chyma cells termed the bundle sheaths, which dration (Tsukaya, 2006). Different species and are visible as almost transparent, projecting cultivars of grapevine differ in their leaf shape, “ribs,” especially on the abaxial side of the leaf and leaf morphology forms the main basis of (Pratt, 1974; see Figure 1.12). The bundle ampelography (see Chapter 1.1). The predomi- sheaths function to conduct water away from nant leaf form of the genus Vitis is palmate, in the xylem to the mesophyll cells and serve as which the five main vascular bundles (major “windows” transferring light to the photosyn- veins; see Figure 1.12) serve the leaf’s five lobes thetic mesophyll cells in addition to providing that in most V. vinifera cultivars are partly sepa- mechanical support to protect the leaf blade rated from each other by more-or-less deep against collapse during severe dehydration gaps termed sinuses (Mullins et al., 1992; Viala and other stresses. and Vermorel, 1909; see Figure 1.12). The main The leaf margins are serrated (toothed), and vascular bundles divide into an ever finer, elab- both the number of teeth and their sharpness orate network whose branches (minor veins) or sinus depth vary greatly among Vitis species are interconnected or eventually (at the fifth and cultivars (Figure 1.13). It is thought that the order) end blindly between the leaf mesophyll sinus depth, but not the teeth number, is cells. Interconnected veins form enclosed areas inversely related to the rate and/or duration or islets termed areoles, approximately 1 or of cell division during leaf formation so that 2 mm across, and 10 mm of minor veins services leaves with greater cell numbers should have up to 10 mesophyll cells (Wardlaw, 1990). The shallower sinuses (Tsukaya, 2006), but this vein network acts as an “irrigation/collection association has not been investigated in grape- system,” with the major veins acting as a rapid vines. The teeth “funnel” the tracheids of the supply network and the minor veins forming a terminal veins into water pores called slow distribution and collection network hydathodes, which can discharge drops of (Canny, 1993). This system delivers water and xylem sap (Evert, 2006; Pratt, 1974). This pro- nutrients, and it collects assimilates relatively cess is called guttation (Latin gutta ¼ drop) 38 1. BOTANY AND ANATOMY

which in turn determines the extent and direction of expansion of these cells by means of sugar and water import (see Chapter 3.1). As a side note, the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) is a synthetic, nontransportable auxin that stimu- lates cell division but inhibits cell expansion and differentiation, and interferes strongly with pat- terning, leading to severely distorted leaves. Thus, the leaf’s shape and the basic architecture of its vascular system are determined by genetic and hormonal controls during the initial stages FIGURE 1.13 Chardonnay leaf (left) and Merlot leaf of leaf formation. Newly formed leaf cells require (right). Illustrations by A. Mills. approximately 2 weeks to expand to their full size (in contrast to root cells, which finish expanding and should not be confused with dew, which is within a few hours), which gives leaves sufficient the formation of droplets of atmospheric water developmental flexibility to adjust their final that condenses on cool surfaces. Guttation is size to environmental conditions prevailing especially pronounced during warm nights during leaf formation. Leaf size and shape can with high relative humidity (i.e., when transpira- be markedly altered by patterns (gradients) of tion is minimal and root pressure may force water water availability across the leaf during the up the vine, see Chapters 2.2 and 3.3). So-called period of leaf expansion. The final size of a leaf scavenging cells surrounding the xylem vessels is usually closely tied to its cell number, although near their endings actively and selectively collect greater cell expansion often partially compen- xylem solutes (e.g., nutrient ions and hormones sates for lower cell number (Tsukaya, 2006). such as cytokinin) and recycle them back to the Because both cell division and cell expansion are phloem for re-export (Sakakibara, 2006), whereas dependent on sugar supply, leaves developing undesirable solutes (e.g., excess calcium) may be when a vine is photosynthetically “challenged” excreted along with the water. Thus, the will produce few and small cells (Van Volken- hydathodes may function as safety (overflow) burgh, 1999). Therefore, leaves can grow to a valves that can rid the leaves of excess water. In large size if nutrient availability is unlimited leaf primordia and unfolding leaves, the during their expansion, but they will remain hydathodes are also thought to be primary sites small if they form while environmental condi- of auxin production, which is, at least in part, tions are unfavorable. responsible for the formation of the characteristic The lamina grows by cell division and leaf shapes (patterning) and differentiation of expansion in basal and intercalar (Latin interca- vascular bundles, whereby cells that experience lare ¼ to insert) meristems and consists mainly rapid auxin flow will differentiate into veins of primary tissue; there is no secondary growth (Aloni, 2001; Aloni et al., 2003, 2006b; Benjamins to speak of (Evert, 2006). Cell division generally and Scheres, 2008; Berleth et al., 2007; Dengler, stops when the leaf is only half or less of its 2001). Auxin stimulates cell division at high con- final size so that the remainder of leaf growth centration and cell expansion at low concentra- is caused solely by the expansion of preformed tion. It may exert its growth-promoting cells, which occurs mainly due to enlargement influence via differences in its content among of their vacuoles (see Chapter 3.1). In addition cells (i.e., auxin gradients arising from active to the influence of auxin, both cell division cell-to-cell transport away from the source), and cell expansion are stimulated by cytokinins 1.3. MORPHOLOGY AND ANATOMY 39 produced by the dividing leaf cells as well as with a thin, continuous, extracellular mem- by cytokinins imported via the xylem from the brane termed the cuticular membrane or cuticle root tips. Moreover, cell expansion is also pro- (Bargel et al., 2006; Kerstiens, 1996). The cuticle moted by gibberellins. Gibberellin production consists of cutin, polysaccharides, phenolics, by expanding cells just behind the shoot apical and soluble lipids that make up the intracuticu- meristem and by the young, rapidly expanding lar wax embedded in the outer parts of the leaves and petioles near elongating internodes polymer matrix. It is covered by a layer of over- is induced by auxin (Hartweck, 2008). lapping solid, semicrystalline lipid platelets— At the same time as the leaf cells expand and the epicuticular wax. The intra- and epicuticu- the rate of cell division decreases, they also lar waxes are typically referred to as cuticular build up their photosynthetic machinery, wax, which seals the plant surface. Although namely the chlorophyll-rich chloroplasts (see waxes are complex mixtures of different lipids, Chapter 4.1). By the time the various tissues of they are essentially (>90%) made up of chains the leaf blade begin to differentiate, the leaf is of more than 20 methylene (CH2) groups that generally only six cell layers thick (Pratt, are assembled within expanding epidermis 1974). The outer surface layers differentiate into cells and then secreted to the surface along the adaxial (upper, top, and sun-exposed) and with cutin as the leaf unfolds and expands abaxial (lower, bottom, and shaded) epidermis. (Samuels et al., 2008; Scho¨nherr, 2006). The The epidermis does not contain chloroplasts self-cleaning properties of this water-repellent but can have various types of hairs called tri- or hydrophobic (Greek hydros ¼ water, phobos chomes, particularly along the veins on the ¼ fear) surface film keep the leaves clean by abaxial side (Viala and Vermorel, 1909). The greatly reducing the adhesion of water and par- number and length of trichomes varies with ticles (Bargel et al., 2006). Together with the species and cultivar and gives the underside thick outer cell walls of the epidermis, the cuti- of some leaves (e.g., Vitis candicans; Clairette, cle provides mechanical support that maintains Meunier) a mat whitish appearance, whereas the integrity of leaves and other plant organs others (e.g., Vitis monticola; Grenache) are gla- and determines their growth rate and extent brous (Latin glaber ¼ hairless, bald). Trichome of growth. This epidermis/cuticle skin also density is especially high in young leaves, but forms the first mechanical barrier to invading it decreases after unfolding due to gradual pathogens as well as physical or chemical shedding of trichomes, especially on the adax- injury by repelling fungal spores and dust par- ial side (Karabourniotis et al., 1999). Trichomes ticles, and it reduces nutrient leaching (Bargel probably help reduce water loss (by regulating et al., 2006; Kerstiens, 1996). Its most important the leaf surface temperature and increasing the function, however, is the protection of the plant boundary layer resistance; see Chapter 3.2), from desiccation. The waxy cuticle restricts deter insects (as mechanical barriers), provide gas diffusion so that most water vapor and protection from damaging ultraviolet radiation other gases must pass through openings called (due to their phenolic compounds absorbing stomata (Greek stoma ¼ mouth; Figure 1.14). and scattering radiation; see Chapter 5.2), and These small pores (15–40 mm in length) are may even afford some insulation. The outside formed by degradation of the cell wall joining walls of epidermis cells, which are much two specialized cells, the kidney-shaped guard thicker than the cell walls of interior tissues cells, which, in contrast to the surrounding (Kutschera, 2008a,b), contain cutin—a strong, epidermis cells from which they are derived, elastic biopolyester composed of highly poly- contain a few chloroplasts (Lawson, 2008). Vitis merized hydroxy fatty acids—and are covered leaves are said to be hypostomatous because 40 1. BOTANY AND ANATOMY

FIGURE 1.14 Cross section of a leaf (left; illustration by A. Mills and M. Keller) and stoma on a Chardonnay leaf, surrounded by crystalline platelets of epicuticular wax (right; photo by M. Keller). the stomata are located almost exclusively in cannot increase in length and therefore bend the abaxial epidermis. outward. The aperture (pore width) of open Grapevine leaves typically have between 50 stomata is approximately 8 mm, whereas it is and 400 stomata/mm2, with V. vinifera and almost zero when the stomata are closed, for most rootstocks at the lower end (<250 mm 2) example, at night or during severe water stress. and V. labrusca and V. cinerea at the higher The tissue between the two epidermal layers end (>300 mm 2) of this range (Du¨ ring, 1980; is called the mesophyll (see Figure 1.14). As the Liu et al., 1978; Scienza and Boselli, 1981). Inter- leaf expands, the mesophyll cells stop growing estingly, mature leaves seem to be able to before the epidermis cells do. This pulls the detect the conditions around them and send a mesophyll cells apart and leads to the forma- (still unknown) signal to the newly developing tion of a large system of intercellular spaces leaves that adjusts the number of stomata that that facilitate the diffusion of gases such as will form on those leaves. Stomata are responsi- water vapor and CO2 (Van Volkenburgh, ble for regulating the gas exchange (mainly 1999). The mesophyll consists of a single layer CO2 for photosynthesis, oxygen for respiration, of elongated cells termed palisade parenchyma and water vapor from transpiration) between and four to six layers of irregularly shaped, the leaf and the atmosphere. Their major func- loosely packed cells termed spongy paren- tion is to balance the uptake of CO2 with the chyma (Galet, 2000; Pratt, 1974; Viala and loss of water (see Chapter 3.2). Stomata open Vermorel, 1909). Both cell types contain large when the guard cells take up water and swell numbers of chloroplasts (Greek chloros ¼ light (their volume more than doubles); because they green, yellow), which are the actual centers of are attached to each other at both ends, they photosynthesis and assimilation. 1.3. MORPHOLOGY AND ANATOMY 41

The palisade parenchyma lies below the Mullins, 1978, 1980, 1981). Accordingly, the adaxial epidermis and has small intercellular absence of gibberellin production or supp- spaces. It also contains crystals of calcium ression of its activity at the site of organ initiation oxalate, which are arranged inside large cells gives rise to an inflorescence (flower cluster), (typically 50–100 mm long and 20–25 mm wide) whereas the presence of gibberellin results in a in bundles of needle-like crystals called raphides tendril. The cell-division hormone cytokinin, in and, in older leaves inside very small cells (10 contrast, promotes inflorescence formation mm in diameter) lining the veins, in aggregates over tendril formation (Srinivasan and Mullins, of octahedral (star-shaped) crystals called 1978, 1980, 1981). It seems likely that the druses (Evert, 2006; Viala and Vermorel, 1909). response to these hormones is quantitative The spongy parenchyma follows toward the because there is a continuum of transitional or abaxial side and has large intercellular spaces. intermediary structures that are partly tendril- Many of the mesophyll cell walls are thus like and partly inflorescence-like. Moreover, exposed to the air inside the leaf (internal the degree of differentiation depends largely on leaf atmosphere) so that the internal leaf surface the cultivar and environmental conditions is approximately 10–40 times the surface area during the time of cluster initiation and diffe- of the leaf. Water evaporates from the meso- rentiation (see Chapter 2.3). Both tendrils and phyll cell walls into these intercellular spaces, inflorescences, in turn, are modified shoots which are continuous with the outside air when (lateral branches), and there is also a variety of the stomata are open so that water vapor is dis- intermediary forms between inflorescence and charged (transpired) into the atmosphere (see shoot (Figure 1.15). The individual flowers on Figure 1.14; see also Chapter 3.2). The CO2 an inflorescence, again, are modified shoots, follows the reverse diffusion route into the leaf with the separate floral organs having evolved (see Chapter 4.2). from highly modified leaves. Similar to leaves, tendrils grow in intercalary 1.3.5. Tendrils and Clusters fashion in addition to growth in their own api- cal meristem (Tucker and Hoefert, 1968). They Tendrils and fruiting clusters of the grapevine also differentiate an epidermis with numerous are generally considered homologous on the stomata, a spongy parenchyma, and a large basis of anatomical, morphological, and physio- but short-lived hydathode at each of their tips, logical similarities. Darwin (1875) concluded which soon become suberized (Gerrath and from observations of grapevines growing in his Posluszny, 1988a; Tucker and Hoefert, 1968). backyard that "there can be no doubt that the Although two tips are most common, vigorous tendril is a modified flower-peduncle." Indeed, vines often form tendrils with three or more studies of gene expression—that is, of the manu- tips (Figure 1.16). Tendrils enable wild vines facture of RNA and protein from the segment of to access sunlight at the top of tree canopies DNA making up a gene—also suggest that ten- with a relatively small investment in shoot bio- drils are modified reproductive organs that have mass per unit height gain. The tendril’s tips been recruited (i.e., adapted during evolution) search for surrounding objects by making as climbing organs (Calonje et al., 2004; Dı´az- sweeping, rotating movements during growth Riquelme et al., 2009). As sterile reproductive (Darwin, 1875; Galet, 2000; Viala and Vermorel, structures, they are prevented from completing 1909); this oscillatory growth pattern is termed floral development by the cell-elongation circumnutation. When one of the tips detects a hormone gibberellin (Alleweldt, 1961; Boss and support (via contact-sensitive epidermis cells), Thomas, 2002; Boss et al., 2003; Srinivasan and the arms rapidly coil around the support in 42 1. BOTANY AND ANATOMY

FIGURE 1.15 Intermediary forms of Syrah inflorescence/tendril/shoot (left; inset: inflorescence that would rather be a tendril; photos by M. Keller) and structure of a grape cluster with berries removed (right; illustration by A. Mills; inset reproduced from Viala and Vermorel, 1909).

FIGURE 1.16 Vitis shoot tip showing tendrils with two and three tips (left) and tips of a tendril coiling around a trellis wire (right). Photos by M. Keller. 1.3. MORPHOLOGY AND ANATOMY 43 two directions (see Figure 1.16). Following this rays and to the inside of the vascular bundles so-called thigmotropic (Greek thigmos ¼ touch, accumulate starch and may serve as a tran- trope ¼ turn) movement, the entire tendril ligni- sient nutrient storage compartment. The epi- fies and stiffens to prevent unwinding (Braam, dermis on the exterior contains numerous 2005). Tendrils that fail to find a support die stomata and is covered with a cuticle and are abscised at the point of attachment to (Theiler, 1970). Clusters vary widely in length the shoot. from 3–5 cm in wild grape species to more The fruiting structures of the grapevine are than 50 cm in some table grape cultivars, and panicles and are known as inflorescences or this is accompanied by varying degrees of flower clusters. They are always inserted branching. The cluster architecture resulting opposite a leaf and are initially protected by from this branching is probably determined bracts covered with trichomes. After fruit set by auxin released from developing flower the inflorescence is called a cluster or bunch. meristems. It is composed of the peduncle (stalk; i.e., the entire branched axis apart from the pedicels 1.3.6. Flowers and Grape Berries of individual flowers) carrying the flowers or, following fruit set, the berries. Each flower or As discussed in Chapter 1.1, wild grapes are berry is attached to the peduncle via a pedicel dioecious, but most V. vinifera cultivars have (final branch or flower stalk) that widens into perfect (hermaphroditic) flowers. Physiologi- thereceptaclethatcarriestheflowerorberry. cally, female flowers are rare among V. vinifera The main stem or axis consists of the hypo- cultivars (e.g., Madeleine Angevine and Picolit) clade (between the shoot and the first branch- but common among M. rotundifolia cultivars. ing) and the rachis (central axis) that is made Many rootstocks have male flowers, whereas up of a main axis (also called inner arm) and some (e.g., Ramsey, 101-14 Mgt, 5BB, and 41B) a lateral wing or shoulder (also called outer have female flowers (Meneghetti et al., 2006). arm). Both arms carry multiple secondary The formation and development of grape flow- branches (and many of these tertiary branches, ers is considered in Chapter 2.3. The flowers some of which in turn carry quaternary (Figure 1.17) are green, inconspicuous, and branches), although the shoulder may be miss- small; their size varies, depending on species, ing or may be a tendril or, occasionally, a from 2 mm (V. berlandieri)to6or7mm(V. lab- shoot. Just as in tendrils, each of the arms rusca). They are made up of a receptacle (some- and branches is subtended by a bract. The times called torus) carrying five sepals (fused flowers usually occur in groups of three (with to form the calyx), five petals (with their bor- two, often slightly smaller, flowers flanking a dering cells interlocked to form a protective central, terminal flower) or five; these basic cap termed calyptra or corolla), five stamens, floral units are termed a triad, a dichasium, and the pistil (May, 2004; Meneghetti et al., or a cyme (Gerrath and Posluszny, 1988b; 2006; Srinivasan and Mullins, 1981; Swanepoel May, 2004; Mullins et al., 1992). The hypo- and Archer, 1988). The stamens are the male clade, which varies in length from 2 to 10 cm reproductive organs with bilobed anthers depending on cultivar, connects the cluster (microsporangia), each lobe carrying two pol- with the shoot, similar to the function of the len sacs containing more than 1000 pollen petiole for the leaf, and therefore also contains grains (each composed of a large vegetative cell multiple (typically approximately 30) vascular and two sperm cells), and their stems termed bundles that are separated by rays (Viala and anther filaments. The pistil is the female repro- Vermorel, 1909). The parenchyma cells of the ductive organ with a superior ovary 44 1. BOTANY AND ANATOMY

FIGURE 1.17 Diagrammatic longitudinal section of a Muscat Hamburg flower (left; from Rafei, 1941) and longitudinal section of a Vitis seed (right; illustration by A. Mills). surmounted by a short style and a papillate with undeveloped style and stigma (Caporali stigma (Considine and Knox, 1979a; Hardie et al., 2003; see also Figure 1.1). et al., 1996b; Pratt, 1971). The set of stamens of A network of vascular bundles, or traces, a flower are collectively termed the androe- supplies each of the floral organs (see Fig- cium, and the female parts together are named ure 1.17). The base of the ovary is encircled by the gynoecium. The gynoecium of grape flow- a whorl of odor glands termed osmophors but ers generally consists of two seed pockets sometimes referred to as nectaries. The ovary called carpels (megasporangia) that are fused comprises two, rarely three, cavities termed to form the pistil; the central region in the locules, each normally containing two seed pri- ovary where the two inner carpel walls meet mordia called ovules (Nitsch et al., 1960) that is termed septum, which divides the ovary, are formed by a placental wall to which they and later the fruit, into two locules. Female remain attached via a funiculus (Latin funiculus flowers are difficult to distinguish from perfect ¼ thin rope), which serves the same function as flowers; they have stamens that look normal the umbilical cord in mammals. Both the calyp- but produce sterile pollen, whereas in male tra and the pistil (after capfall) have a cuticle- flowers the pistil is reduced to a tiny ovary covered epidermis containing chloroplasts 1.3. MORPHOLOGY AND ANATOMY 45 and, through their stomata (10–20 per mm2), epidermis and later forms a cuticle on the out- are capable of their own gas exchange (Blanke side. The protective testa contains inclusions and Leyhe, 1987, 1988, 1989a). The pistil tissues of calcium oxalate crystals and is rich in pheno- are rich in calcium oxalate crystals, starch, and lic compounds such as tannins that protect the phenolic compounds, although the starch dis- seed against premature “consumption” by appears during berry development (Hardie microorganisms or insects (Dixon et al., 2005). et al., 1996a; Sartorius, 1926). The apparent folding of the integuments leaves The ovules (female gametophytes) with their a small opening (pore) termed micropyle fertilized egg (zygote) and remaining embryo through which the pollen tube enters during sac (composed of seven cells including the egg pollination (Sartorius, 1926; see also Chapter cell flanked by two synergid cells and the cen- 2.3), and water will enter and the radicle will tral cell) can develop into heart- or pear-shaped exit during seed germination. The endosperm seeds whose main role is to protect and nurture progressively expands at the expense of the the developing embryo (Roberts et al., 2002). nucellus that may serve as a food supply for Thus, a grape berry with two locules can have the embryo and endosperm early during seed a maximum of four seeds, and one with three development (Nitsch et al., 1960). locules can have six seeds. In practice, the seed The vascular bundles that previously served number is usually one or two in V. vinifera cul- the ovary give rise to a complex network of tivars but two or three in many Chinese Vitis vascular traces that supply the seed (ovular species (Wan et al., 2008c). Depending on culti- bundles) and the pericarp (ventral and dorsal var and seed number, the seeds make up bundles). The dorsal, or peripheral, vascular between 2 and 7% of the fresh weight of mature bundles of most V. vinifera cultivars lie approx- grapes (Viala and Vermorel, 1909). After fertili- imately 200–300 mm beneath a mature berry’s zation, the pistil develops into the fruit, with surface and can give some berries a “chicken- the ovary wall becoming the pericarp—that is, wire” appearance (see Figure 1.18), whereas in the flesh and skin (Figure 1.18); thus, the fruit V. labrusca the bundles are approximately twice can be viewed as a swollen or mature ovary as far from the surface (Alleweldt et al., 1981). (Gillaspy et al., 1993). Grapes are classified as The ventral, or central, vascular bundles and a berry fruit because the thick pericarp encloses their associated parenchyma cells are termed the seeds. the “brush” and remain attached to the pedicel The ovule is made up of the outer and inner when a ripe grape is plucked from the cluster. integument and the nucellus (Pratt, 1971) and Individual tracheary elements (mostly vessel the embryo sac. After fertilization, the integu- elements with some tracheids) in the berry are ments will form the seed coat termed testa, approximately 250–300 mm long with an aver- and the nucellus will surround the developing age diameter of approximately 3.5–5 mm (Cha- endosperm and the embryo (see Figure 1.17) telet et al., 2008a). Although existing tracheary with an epicotyl (embryonic shoot tip), two elements are stretched at the beginning of rip- cotyledons (embryonic or seed leaves), a hypo- ening, most of them remain intact, and new ele- cotyl (embryonic shoot base), and a radicle ments are added throughout berry (embryonic root). The epicotyl contains at its development. The pericarp consists of three tip the shoot apical meristem, and the radicle anatomically distinct tissues: the exocarp (or contains at its tip the root apical meristem, epicarp), mesocarp, and endocarp (Galet, 2000; which defines the apical–basal axis of the Viala and Vermorel, 1909). embryo and future seedling. The outer integu- The exocarp forms the grape’s dermal sys- ment consists of a palisade layer and an tem or “skin,” which, depending on thickness 46 1. BOTANY AND ANATOMY

FIGURE 1.18 Structure of a ripe grape berry (left; reproduced from Coombe, 1987, reprinted by permission of AJEV), scanning electron micrograph detail of ruptured Syrah berry surface with epicuticular wax and raphide crystals of calcium oxalate (top right; photo courtesy of S. Rogiers), and stylar end of Syrah berry covered with epicuticular wax (bottom right; photo by M. Keller).

(which varies by species and cultivar) and structure of the epicuticular wax changes with berry size, makes up between 5 and 18% of age of the berry. Although the wax is normally the fresh weight of mature berries. It is com- crystalline, the crystals appear to degrade posed of the epidermis covered with a waxy slowly over time, fusing into amorphous cuticle and the underlying outer hypodermis masses and eventually into a more-or-less (Bessis, 1972a; Considine and Knox, 1979b; continuous layer on top of the normal, uninter- Fouge`re-Rifot et al., 1995; Hardie et al., 1996b). rupted wax layer (Casado and Heredia, 2001; Epicuticular wax, often referred to as “bloom,” Rogiers et al., 2004a). The wax material is rather covers the surface of the cuticle as overlapping soft and can be altered or removed by the platelets forming a strongly hydrophobic layer impact of rain, by abrasion from wind-blown that protects the berry from water loss and particles, or by contact with other berries, forms the first barrier against pathogen inva- leaves, or other adjacent objects (Shepherd sion (Rosenquist and Morrison, 1988). The and Griffiths, 2006). However, because of its 1.3. MORPHOLOGY AND ANATOMY 47 vital importance as a desiccation protectant, a “boundary” between hypodermis and meso- damaged or removed wax layer is quickly carp, are now situated within the mesocarp regenerated, especially in young berries. The (Chatelet et al., 2008a; Fouge`re-Rifot et al., cuticle is approximately 1.5–4 mm thick, and 1995). The relative thickness of the skin the epidermis consists of a single cell layer that declines from approximately one-eighth to is approximately 6–10 mm thick (Alleweldt one-hundredth of the total berry diameter et al., 1981; Bessis, 1972a; Hardie et al., 1996b). between fruit set and maturity. Unlike the hypodermis and mesocarp cells, The mesocarp, which is commonly called the the cells of the epidermis divide in a strictly “flesh” or “pulp” of the grape berry, forms anticlinal manner (i.e., normal to the surface), soon after fruit set and consists of 25–30 layers thus maintaining their cell “lineage” (Consi- of large (100–400 mm), thin-walled, and highly dine and Knox, 1981). The initially single-lay- vacuolated storage parenchyma cells. The ered outer hypodermis (100–250 mm thick) vacuoles can make up as much as 99% of the increases to 6–10 layers of thick-walled cells cell volume in ripe grape berries (Diakou and after fruit set and then transiently even to Carde, 2001) and serve as an internal reservoir 15–20 layers. These cells contain chloroplasts storing sugars, organic acids, and nutrients and tannin-rich vacuoles, interspersed with (see Chapter 6.2). Mesocarp cells outside the amyloplasts, which form within a few days network of peripheral vascular bundles of the after fruit set but disappear during berry devel- pericarp are termed the outer mesocarp; those opment, and so-called idioblast (Greek idios ¼ toward the inside are the inner mesocarp, unique, blastos ¼ germ, sprout) cells rich in which, at maturity, makes up almost two-thirds bundles of needle-like calcium oxalate crystals of the berry volume. At maturity, the mesocarp called raphides (Evert, 2006; Fouge`re-Rifot cells are approximately 75 times larger than the et al., 1995; see also Figure 1.18). As an aside, skin cells, and their radial width is approxi- a cell may contain more than one kind of vacu- mately 10 times that of the skin cells, whereas ole, so it is possible that different components the cell walls in the skin are almost 20 times may be stored in specialized vacuoles within thicker than those in the mesocarp. It is no the same cell (Marty, 1999; Mu¨ ntz, 2007). The wonder, then, that the skin sets the upper limit outermost hypodermal cells become stretched to which a berry can expand. and radially compressed, and their cell walls The innermost tissue of the pericarp, the thicken approximately 10-fold as the berry endocarp, surrounds the seeds and consists of expands (Considine and Knox, 1979b; Hardie the inner hypodermis with cells rich in star- et al., 1996b). The innermost cells expand and shaped calcium oxalate crystals (druses) and seem to be progressively converted to meso- also the inner epidermis (Fouge`re-Rifot et al., carp cells. Thus, the skin becomes thinner (4 1995; Hardie et al., 1996b). The endocarp is or 5 cell layers) in the ripening berry and the often difficult to distinguish from the mesocarp vascular bundles, which initially marked the in grapes (Mullins et al., 1992). CHAPTER 2

Phenology and Growth Cycle

OUTLINE

2.1. Seasons and Day Length 49 2.3. Reproductive Cycle 68 2.2. Vegetative Cycle 53

2.1. SEASONS AND DAY LENGTH of cultural practices as part of vineyard man- agement (Dry and Coombe, 2004). Phenology (Greek phainesthai ¼ to appear, Grapevines, like other plants, monitor the logos ¼ knowledge, teaching) is the study of seasons by means of an endogenous “clock” natural phenomena that recur periodically in (Greek endon ¼ inside, genes ¼ causing) in plants and animals and of the relationship of order to prevent damage by unfavorable envir- these phenomena to climate and changes in onments. Every plant cell contains a clock (per- season. In other words, it is the study of the haps even several clocks), and the clocks of sequence of plant development. Its aim is to different cells, tissues, and organs act autono- describe causes of variation in timing by seek- mously (McClung, 2001, 2008). The clock is ing correlations between weather indices and driven by a self-sustaining oscillator consisting the dates of particular growth events and the of proteins that oscillate with a 24-h rhythm in intervals between them. Phenology investigates response to light detected by phytochromes a plant’s reaction to the environment and (see Chapter 5.2) and other light-sensitive pro- attempts to predict its behavior in new environ- teins that translate the light signal into a clock ments. In viticulture, phenology is mainly input that uses this time-encoded information concerned with the timing of specific stages of to regulate physiological functions (Fankhauser growth and development in the annual cycle. and Staiger, 2002; Spalding and Folta, 2005). Such knowledge can be used for site and culti- Additional input is provided through feedback var selection, vineyard design, planning of loops that arise from various compounds pro- labor and equipment requirements, and timing duced by the plant’s metabolism (McClung, 2008).

The Science of Grapevines 49 Copyright # 2010 Markus Keller. Published by Elsevier Inc. All rights reserved. 50 2. PHENOLOGY AND GROWTH CYCLE

Just how plants manage to integrate and drought (see Chapter 7.2), nutrient deficiency or transform this environmental and metabolic excess (see Chapter 7.3), or infection by patho- information into daily and seasonal functions gens (see Chapter 7.5). In general, higher and how they avoid being “fooled” by bright temperatures accelerate plant development moonlight is still mysterious, but it could and advance grapevine phenology. Therefore, involve fluctuations in calcium concentration one of the most conspicuous consequences of þ of the cytosol. Cytosolic Ca2 also oscillates climate change associated with the current and with a 24-h period with a peak before dusk, future increase in temperature due to the man- and it participates in the “translation” of many made rise of atmospheric CO2 is a shift of signals, both internal and external (Hotta et al., phenological stages to earlier times during the 2007; McClung, 2001; Salome´ and McClung, season (Chuine et al., 2004; Wolfe et al., 2005). 2005). In contrast to the temperature depen- Because day length during the growing sea- dence of most biochemical processes, the son is longer at higher latitudes, the effects of period of the rhythm is temperature compen- day length and latitude on the internal clock sated so that it remains constant at 24 h over are very similar (Salome´ and McClung, 2005). a wide range of temperatures. The gradual In tropical climates, with little or no change in shift in time of sunrise (actually the change day length (and temperature at lower eleva- between dawn and dusk) as the season pro- tions) during the year, grapevines behave as gresses serves to reset the phase of the clock evergreens with continuous growth and strong every day and enables plants to “know” when apical dominance, and often continuous fruit the sun will rise even before dawn (Fankhauser production, throughout the year (Dry and and Staiger, 2002). This synchronization of the Coombe, 2004; Mullins et al., 1992). With appro- circadian (Latin circa ¼ about, dies ¼ day) clock priate pruning strategies (and sometimes delib- to the external environment is termed entrain- erate defoliation by hand, often in combination ment. It translates day length (i.e., photoperiod) with water stress and sprays of ethephon or into a vine’s internal time as an estimate of both other chemicals to induce near dormancy, fol- the time of day and the time of year, enabling lowed by applications of hydrogen cyanamide it to anticipate and prepare for daily and sea- to induce uniform budbreak), two crops can sonal fluctuations in light and temperature. For often be harvested each year in the tropics. In example, the genes responsible for “building” temperate climates (and at high elevations in enzymes involved in the production of ultravio- the tropics), where winter conditions prevent let (UV)-protecting phenolics are most active the survival of leaves, grapevines have a dis- before dawn, photosynthesis-related genes continuous cycle with alternating periods of peak during the day (and opening and closing growth and dormancy. Under these conditions, of the stomata can anticipate dawn and dusk), the time of active growth generally occurs from whereas genes associated with stress are March/April to October/November (Northern induced in late afternoon (Fankhauser and Hemisphere) or September/October to April/ Staiger, 2002; Hotta et al., 2007). May (Southern Hemisphere). Several distinct On a seasonal scale, flowering, onset of developmental stages or key events have fruit ripening, bud dormancy, leaf senescence, been identified (Figures 2.1 and 2.2) and have and cold acclimation are typical responses to been given a variety of names. These stages day length (so-called photoperiodism), include dormancy, budbreak (budburst), bloom although each of these developmental pro- (anthesis or flowering), fruit set (berry set or cesses is also modulated by temperature, and setting), veraison (French ve´rer ¼ to change: some can be altered by stress factors such as color change or onset of ripening), harvest 2.1. SEASONS AND DAY LENGTH 51

FIGURE 2.1 Grapevine growth stages according to Baillod and Baggiolini (1993). 52 2. PHENOLOGY AND GROWTH CYCLE

FIGURE 2.2 Grapevine growth stages according to Eichhorn and Lorenz. Reproduced from Coombe, B. G. (1995). Adoption of a system for identifying grapevine growth stages. Aust. J. Grape Wine Res. 1, 104-110, with permission by Wiley-Blackwell. 2.2. VEGETATIVE CYCLE 53

(ripeness or maturity), and leaf senescence moisture, and rootstock, but on average it and subsequent fall (abscission). Although by seems to begin when the soil temperature rises definition no visible growth occurs during dor- above approximately 7C (Alleweldt, 1965). mancy, metabolism does not rest completely, Bleeding can last for a few days or several but high concentrations of the dormancy hor- weeks (probably depending on whether or not mone abscisic acid keep it at a minimum neces- air temperatures are conducive to budbreak); sary for survival of the buds and woody it can also be a stop-and-go process that fluctu- tissues. Although the division of meristematic ates with changes in soil (root-zone) tempera- cells in the buds and cambium is blocked, chro- ture (Andersen and Brodbeck, 1989b; Reuther mosome duplication and protein synthesis and Reichardt, 1963). A vine can exude bleed- resume during the later stages of dormancy in ing sap at rates of less than 0.1 L to more than preparation for the activation of growth when 1 L per day, with the highest rates occurring temperature and soil moisture become favor- on warm and moist soils (Alleweldt, 1965; able in spring. Currle et al., 1983). Bleeding is probably caused The annual growth cycle of mature, fruiting by root pressure (Fischer et al., 1997; Priestly grapevines is often divided into a vegetative and Wormall 1925; Sperry et al., 1987), whose and a reproductive cycle. existence in grapevines was first demonstrated by the English cleric Stephen Hales (Hales, 2.2. VEGETATIVE CYCLE 1727). Root pressure arises from the remobiliza- tion of nutrient reserves from starch and pro- teins and pumping of sugars (especially In late winter or early spring, grapevines glucose and fructose) and amino acids (espe- often exude xylem sap from pruning surfaces cially glutamine) into the xylem (Reuther and and other wounds that have not yet been Reichardt, 1963; Roubelakis-Angelakis and suberized (Figure 2.3). Such sap flow or “bleed- Kliewer, 1979). The concentration of sugars ing” marks the transition from dormancy to apparently increases exponentially as the active growth. Initiation of bleeding is related temperature declines close to or below 0 C; to the restoration of metabolic activity in the this response occurs within a few hours and is roots and is influenced by soil temperature, equally rapidly reversible when the temperature

FIGURE 2.3 Bleeding grapevine cane (left); swelling, woolly bud just before budbreak (right). Photos by M. Keller. 54 2. PHENOLOGY AND GROWTH CYCLE rises again (Moreau and Vinet, 1923; Reuther cases root pressure seems to remain active for and Reichardt, 1963). In addition, organic acids more than 1 month after budbreak (Reuther (especially malate, tartrate, and citrate) and min- and Reichardt, 1963). Both bleeding and root eral nutrient ions (especially potassium and respiration, which may serve as a proxy for calcium) are also present in the bleeding sap metabolic activity in the roots, decline as soil (Andersen and Brodbeck, 1989a; Glad et al., moisture decreases, and membrane leakage 1992; Marangoni et al., 1986; Priestley and increases sharply once soil moisture falls below Wormall, 1925). The resulting increase in the approximately 5% (v/v) (Currle et al., 1983; osmotic pressure of the xylem sap provides Huang et al., 2005b). This may be the reason the driving force for osmotic water uptake by why bleeding is minimal or even absent in the roots from the soil, which generates a positive vines growing in dry soil, which can cause hydrostatic pressure in the entire xylem of 0.2–0.4 stunted shoot growth (possibly due to cavita- MPa at the trunk base and declining at a rate of tion) when shoots begin to grow with stored 0.01 MPa m 1 as the sap rises (Scholander et al., water from the trunk and root parenchyma in 1955; Sperry et al., 1987). This pressure in turn the absence of sufficient soil water to supply lifts water up the vine (see Chapter 3.3) and to the unfolding leaves. Nevertheless, the emerging the swelling buds up to several meters of height. shoots quickly become dependent on the Root pressure serves to dissolve and push delivery in the reactivated phloem of sugar out air bubbles that have formed during the and other nutrients remobilized from the winter in the xylem and thus restores xylem permanent vine organs. function (Scholander et al., 1955; Sperry et al., Cell division and auxin production in buds, 1987). This is necessary because when xylem beginning in the proximal leaf primordia of sap freezes, the gas dissolved in the sap is the distal buds, starts 1–3 weeks before bud- forced out of solution because gases are practi- break (see Figure 2.3), which marks the onset cally insoluble in ice. This can lead to cavitation of vegetative growth in spring. At the same (see Chapter 3.3) upon thawing if the bubbles time, abscisic acid (ABA) declines strongly, expand instead of going back into solution. relieving the growth inhibition that keeps the Given that bleeding typically starts before the vine dormant while low temperatures would buds begin to swell, root pressure may also be threaten its survival. The release of auxin and necessary to rehydrate the dormant buds and its basipetal (i.e., from the apex to the base) trans- reactivate them by cytokinin that is delivered port in the cambial region begins suddenly and from the roots (Field et al., 2009; Lilov and rushes like a continuous wave down the vine, Andonova, 1976). In fact, the buds’ water con- stimulating the cambium cells to resume divi- tent quickly rises from approximately 40% to sion and differentiation into new phloem and 80% during the budswell phase (Fuller and xylem cells (Aloni, 2001). The cambium cells Telli, 1999). The delivery of sugars in the become sensitive to auxin when they are absence of phloem flow may enable the buds exposed to cytokinin, which is supplied from to resume growth (i.e., cell division in the api- the roots as a result of root pressure. Although cal meristem), produce the growth hormone it remains to be demonstrated whether the root auxin (or release it from storage pools), and tips are capable of cytokinin production this break. Accordingly, root pressure-driven water early in spring, cytokinins have indeed been ascent may stimulate bud swelling, and the found in the bleeding sap of grapevines and, in maximum rate of bleeding occurs a few days addition to being influenced by the rootstock before budbreak and generally stops 10–14 (Skene and Antcliff, 1972), higher root tempera- days later (Alleweldt, 1965), although in some tures favor cytokinin delivery via the xylem 2.2. VEGETATIVE CYCLE 55

(Field et al., 2009; Zelleke and Kliewer, 1981). As discussed in Chapter 7) but may take 10 times an aside, the same combination of the actions of longer in areas with mild winters. As a conse- bud-derived auxin and root-derived cytokinin quence, budbreak is often erratic in warm and probably also induces the cambium on either (sub)tropical regions, where the majority of the side of new grafts to produce undifferentiated world’stablegrapesaregrown.Thisiswhy cells termed callus and to form a union between hydrogen cyanamide (H2CN2), usually in the form scion and rootstock. The differentiation of cells of calcium cyanamide (CaCN2), is commonly produced in the previous growing season to applied 3 or 4 weeks before the intended time of form the first xylem vessels of the new shoot budbreak to end dormancy (so-called dormancy begins even before the cambium starts produc- release) and promote budbreak of many table ing new cells (i.e., before budbreak), although grapes (Dokoozlian et al., 1995; Lavee and these vessels only mature (i.e., lignify) after bud- May, 1997). The higher winter temperatures in break. Warmer temperatures during vessel dif- such warmer areas lead to higher respiration ferentiation, perhaps by enhancing the supply rates (see Chapter 4), and this might be asso- of remobilized nutrient reserves to the cambium, ciated with an increase in oxidative stress (e.g., result in wider vessels that may be more effec- hydrogen peroxide release by the mitochondria) tive at supplying water to the developing shoots that, under extreme conditions, could result in later in the season (Fonti et al., 2007); this temper- bud damage called bud necrosis (Pe´rez et al., ature effect could be an important driving vari- 2007). Hydrogen cyanamide also leads to hydro- able for seasonal shoot vigor. Because cambium gen peroxide accumulation by inhibiting the reactivation occurs well before the new leaves antioxidant enzyme catalase, but the subsequent þ will begin supplying assimilates, the energy movement of calcium ions (Ca2 )fromthecell and carbon requirements of the dividing cells walls to the cytoplasm, similar to the movement have to be met by the remobilization of stored induced by chilling, then somehow seems to starch and supply of sugar from the permanent initiate cellular mechanisms culminating in structure of the vine. This starch breakdown dormancy release of the buds (Pang et al., 2007). may by induced by gibberellin, whose produc- In cooler, temperate climates, mean daily tion is stimulated by the auxin emanating from temperatures above approximately 8–10 C the buds and shoot tips (Frigerio et al., 2006; induce budbreak and shoot growth, but the Yamaguchi, 2008). precise base temperature requirement depends Although some grape cultivars (e.g., Thompson on the species (e.g., V. berlandieri > V. rupestris > Seedless) are believed not to require chilling (i.e., V. vinifera > V. riparia) and cultivar (e.g., a period of low temperatures) to resume growth Riesling > Cabernet Sauvignon, Viognier > from dormant buds, budbreak generally occurs Cabernet Franc, Syrah, Se´millon > Merlot, earlier, more rapidly, and more uniformly on Pinot, Sauvignon blanc > Chardonnay, Chenin vines that have experienced a period of cool blanc > Gewu¨ rztraminer). Cultivars with a weather (Antcliff and May, 1961; Currle et al., lower base temperature break buds earlier than 1983; Kliewer and Soleimani, 1972). Moreover, those with a higher temperature threshold longer duration of chilling and lower tempera- (Pouget, 1972). The rate of budbreak increases tures typically accelerate the rate of budbreak as the temperature rises above the threshold once warmer temperatures return (Dokoozlian, temperature up to approximately 30 C and then 1999; Lavee and May, 1997). The buds on a declines again. However, it is the temperature single vine usually break within a few days in of the buds themselves, rather than the areas with cold winters (provided the winter is surrounding air temperature, that determines not so cold as to damage or kill the buds, as when and how rapidly those buds break. 56 2. PHENOLOGY AND GROWTH CYCLE

During the day, the temperature of buds can be buds of the cane break early and grow vigorously, several degrees above the ambient temperature, whereas those toward the base of the cane grow especially when they are heated by the sun and weakly or not at all (Figure 2.4). This inhibition when there is little or no wind, yet even at wind of basal bud outgrowth is especially prevalent speeds greater than 5 m s 1 buds may still be in warm climates, where it can lead to erratic approximately 2 C warmer than air (Grace, and nonuniform budbreak. It also varies among 2006). Conversely, at night the bud temperature cultivars; for instance, Carignan and Mourve`dre is often cooler than the air temperature. In con- (synonyms Monastrell and Mataro) show partic- trast to the temperature of the buds, and despite ularly strong correlative inhibition, whereas its impact on the rate of bleeding sap flow, root Syrah and Cinsaut are at the other end of the temperature may have little influence on the spectrum. Like the evolution of tendrils, this time and rate of budbreak (Field et al., 2009; behavior is an adaptation to the habitat of wild May, 2004). vines, which enables them to access sunlight at Shoot growth starts in the distal (topmost or the top of tree canopies with a small investment apical) buds of a cane or vine, proceeding basi- in shoot biomass per unit height gain. This petally to the roots. Yet preferential budbreak adaptive behavior creates both a viticultural pro- of distal buds often inhibits the growth of the blem (mainly in warm climates) and an oppor- proximal (basal) buds (Galet, 2000). This phe- tunity (mainly in cool climates) for cultivated nomenon is often attributed to apical domi- grapevines. The problem arises from the inconsis- nance, although it may actually be a case of tent budbreak of cane-pruned vines. Viticultural correlative inhibition. In correlative inhibition, practices aimed at overcoming correlative inhibi- growing shoots may inhibit each other and tion and promoting uniform budbreak include prevent buds on the same cane from breaking, spur pruning, bending (“arching”) and partial whereas in apical dominance the shoot tip inhi- cracking of canes, and application of hydrogen bits the outgrowth of lateral buds (Beveridge, cyanamide. Spur-pruned vines are typically 2006; Ferguson and Beveridge, 2009). Correlative unaffected by correlative inhibition, and where inhibition is most conspicuous in early spring mixed “populations” of spurs and canes are when, in cane-pruned vines, the most distal retained on the same vine, the buds on the spurs

FIGURE 2.4 Cane-pruned Cabernet Sauvignon vines displaying strong correlative inhibition in spring. Notice that bud- break is not inhibited on spurs adjacent to the canes. Photos by M. Keller. 2.2. VEGETATIVE CYCLE 57 break at the same time as the distal buds of the growth of both shoots and roots is entirely canes (see Figure 2.4; Clingeleffer, 1989). The dependent on nutrient reserves (mainly carbo- opportunity stems from the delay in budbreak hydrates and proteins or amino acids) stored on vines that are spur-pruned only after the in the permanent structure of the vine, until canes’ distal buds have already broken, which new leaves become photosynthetically compe- may enable vines grown in frost-prone locations tent and begin to produce and export carbohy- to escape spring-frost damage (see Chapter 7.4). drates. These reserves are thus depleted and Budbreak is followed by a period of very reach a minimum around bloom time or even rapid shoot growth, with a new leaf appearing later, making vines vulnerable to stress around every few days. In many cultivars, the unfold- the time of bloom (Lebon et al., 2008; Weyand ing leaves, and often the developing inflores- and Schultz, 2006b; Williams, 1996; Zapata cences as well, are reddish before they turn et al., 2004). Carbohydrate reserves are used green (Figure 2.5). This transient accumulation not only as building supplies to construct of anthocyanin pigments in or just below the new plant material in the absence of photosyn- epidermis probably protects the developing thesis but also to generate the energy (by respi- photosynthetic machinery (e.g., protochloro- ration) required to fuel this construction (see phyll) from excess light (Chalker-Scott, 1999; Chapter 4.4). Consequently, grapevines literally Liakopoulos et al., 2006; Rosenheim, 1920). The lose (dry) weight during the initial spring anthocyanins usually disappear by the time growth phase, before the photosynthesizing the leaf has produced roughly half of its maxi- leaves begin to add new biomass from about mum (i.e., mature) chlorophyll and carotenoid the five- to six-leaf stage onward (Eifert et al., content and the palisade cells have expanded 1961; Hale and Weaver, 1962; Koblet, 1969). to occupy approximately 50% of the mesophyll Nitrogen reserves, in addition to carbohy- (Hughes et al., 2007). In addition, the dense drates, are very important for new growth in covering of “hair,” termed pubescence, of the spring; early season growth and especially leaf young leaves also serves the same photoprotec- chlorophyll correlate closely with the amount tive purpose (Liakopoulos et al., 2006). Early of nitrogen reserves stored in the perennial

FIGURE 2.5 Emerging Merlot shoot with pink unfolding leaves and inflorescences days after budbreak (left); Muscat Ottonel shoot with red young leaves (right). Photos by M. Keller. 58 2. PHENOLOGY AND GROWTH CYCLE parts of the vine (Cheng et al., 2004b; Keller and the agricultural Green Revolution in the second Koblet, 1995a; Treeby and Wheatley, 2006). half of the 20th century (Hartweck, 2008). Higher soil temperatures are associated with Meanwhile, the thick, cellulose-reinforced more rapid starch remobilization in the roots, outer cell walls and rigid cuticle of the epider- which may allow the shoots to grow more mis act like a corset, forcing the internode rapidly on vines in warm than in cool soil cells to elongate rather than simply expanding (Field et al., 2009; Skene and Kerridge, 1967; radially in all directions (Kutschera, 2008a,b; Woodham and Alexander, 1966; Zelleke and Schopfer, 2006). The suppression of axillary Kliewer, 1979). bud outgrowth by auxin is indirect because The initial growth phase is characterized by the hormone does not enter these buds, which, strong apical dominance, whereby slow but of course, begin producing and exporting auxin active (i.e., mediated by ATP-consuming pumps) themselves as soon as they begin to grow basipetal flow of auxin released by the leaf (Ongaro and Leyser, 2008). In addition to at primordia and young leaves near the shoot least one unknown mechanism, auxin may apex inhibits lateral shoot growth from prompt exert its remote control by inhibiting cytokinin buds and stimulates extension of internodes production in the roots and in the shoot tissues (Woodward and Bartel, 2005). Intriguingly, adjacent to the axillary buds (Beveridge, 2006; however, if auxin flows too rapidly, such as in Dun et al., 2006; Ferguson and Beveridge, overly vigorous shoots, apical dominance is 2009; McSteen and Leyser, 2005). Damage to compromised and lateral buds are able to grow the shoot tip or its removal (e.g., by summer out (Berleth et al., 2007; Dun et al., 2006; Lazar pruning) releases the repression of cytokinin and Goodman, 2006). Gibberellins produced synthesis and delivery from the roots via the upon stimulation by auxin in or near the shoot xylem (Nordstro¨m et al., 2004; Tanaka et al., tip and brassinosteroids produced in the inter- 2006). Cytokinins that enter the buds stimulate node epidermis promote cell elongation in the growth of lateral shoots, whose leaves are ordi- shoot’s internodes, perhaps by activating the narily smaller than the leaves of the main shoot wall-loosening protein expansin (Cosgrove, and which often stop growing after they have 2000; Frigerio et al., 2006; Olszewski et al., 2002; formed just a few leaves. Incidentally, removal Savaldi-Goldstein et al., 2007; Weiss and Ori, of the shoot tip also depletes auxin in the roots 2007; Yamaguchi, 2008), in addition to promot- and inhibits their growth, at least temporarily ing production of the membrane water channel (Ferguson and Beveridge, 2009; Fu and protein aquaporin (Maurel et al., 2008; see also Harberd, 2003). Growers employ continued Chapter 3.1). Gibberellins act, it seems, by shoot tipping to encourage lateral growth in binding to so-called DELLA proteins in the cell the tropics, where strong apical dominance is nucleus and inducing the degradation of these notorious. In temperate climates, the influence growth-suppressing proteins; however, just of apical dominance decreases once the main how DELLA proteins put the breaks on growth shoot has formed approximately 18–20 leaves remains to be discovered (Achard and Genschik, (around the time of bloom) and its growth 2009; Hartweck, 2008). Mutations that render slows somewhat (Alleweldt and Istar, 1969). DELLA proteins unable to bind gibberellins Cytokinins arriving with the transpiration and are thus immune to degradation result in stream and produced in the nodes then seem dwarf phenotypes that look like bonsai vines to be able to break auxin-dependent apical (Boss and Thomas, 2002; Boss et al., 2003); such dominance, and lateral shoots begin to grow, mutations formed the basis for the short and even when the main apex is intact. Lateral out- high-yielding cereal cultivars that gave rise to growth is especially pronounced (and may 2.2. VEGETATIVE CYCLE 59 occur much earlier than the 18-leaf stage) if summer so that growth can be controlled by warm temperatures stimulate rapid shoot appropriate deficit irrigation strategies (see growth, possibly due to cytokinin production Chapter 7.2). Decreasing day length, perhaps within the lateral buds. In addition, rapid aided by declining temperatures, shortly before transpiration at high temperatures would also the grapes begin to ripen seems to be the trig- stimulate cytokinin delivery from the roots. ger that halts cell division in the apical meri- Abundant nitrogen availability also leads to stem and in the cambium, thus inducing strong lateral shoot growth (Keller and Koblet, dormancy (Davis and Evert, 1970; Esau, 1948; 1995a), possibly because nitrate stimulates Fracheboud et al., 2009; Rohde and Bhalerao, cytokinin production in the roots and export 2007). The cessation of growth and shoot matu- in the xylem (Sakakibara, 2006; Sakakibara ration (i.e., periderm formation) are brought et al., 2006). about by a rapid decline in apex-derived auxin The most distal lateral shoot often gradually in the cambial region and a concomitant assumes the function of the main shoot, and the increase in the root-derived abscisic acid in more basal laterals soon stop growing (Currle the xylem and phloem, where it remains et al., 1983). If water and nutrient availability high during the subsequent dormant period permits, shoot growth often resumes after fruit (Mwange et al., 2005). set and more or less stops in midsummer. The shoot growth cycle is completed with Shortly before or while the grapes begin to leaf senescence associated with the recycling ripen, the shoots begin to form a periderm, of nutrients from the leaves to the canes and hence becoming canes and turning from green permanent parts of the vine (Conradie, 1986; to yellowish-reddish brown. This shoot “matu- Dintscheff et al., 1964; Lo¨hnertz et al., 1989; ration” proceeds acropetally from the base Schaller et al., 1990), abscission (shedding) of toward the tip and is accompanied by replen- leaves, and, finally, dehydration and cold accli- ishment of storage reserves in preparation for mation of all woody parts in fall. Before they the following growing season (Eifert et al., are shed, the senescing leaves develop their 1961; Lo¨hnertz et al., 1989). This process is characteristic and often spectacular yellow delayed by abundant water and nutrient and/or red autumn color. The coloration supply, which also encourages continued or occurs because the green chlorophylls are renewed shoot growth beyond veraison. This degraded more rapidly than the yellow-orange can cause problems in regions with significant carotenoids, which are thus “unmasked” in the summer rainfall, especially on fertile sites that process. The carotenoids also undergo various have high water and nutrient storage capacity, chemical modifications that influence their color. where late-season shoot growth can compete In addition, newly produced red anthocyanins with the ripening clusters and storage reserve are accumulated once a leaf has degraded pools for assimilate supply (Sartorius, 1973). roughly half of its original chlorophyll. Neverthe- Unlike the shoots of many other woody per- less, whereas white-skinned grape cultivars are ennials, grapevine shoots do not form terminal unable to produce anthocyanins, even the leaves buds but continue to grow as long as environ- of most dark-skinned cultivars normally turn mental conditions permit. When this is no lon- yellow (sometimes orange) rather than red. ger possible, the shoot tip with the apical Anthocyanin production is triggered by and meristem dies. The sensitivity of the shoot apex consequently a sign of sugar accumulation to adverse environmental factors (e.g., drought (Gollop et al., 2002; Pirie and Mullins, 1976), or low temperature) may be exploited in areas which can be caused by restricted phloem efflux where the soil dries down sufficiently in early (e.g., due to virus infection, wind-induced injury, 60 2. PHENOLOGY AND GROWTH CYCLE

FIGURE 2.6 Red leaves above the point where a tendril has wrapped itself around a Cabernet franc shoot (left), on undercropped shoots growing from a twisted Cabernet Sauvignon cane (center), and pale yellow leaves on a partly broken Muscat blanc shoot (right). Photos by M. Keller.

or girdling; Figure 2.6) or nutrient deficiency (see interaction with temperature likely vary by Chapter 7.3). The anthocyanins, which seem to species and cultivar. During the carefully be dominated by the glucosides of peonidin orchestrated senescence program, which nor- and cyanidin, and other phenolic components mally starts in the mesophyll cells at the leaf that are accumulated in the leaves may be margins and then progresses inward and released into the soil, where they might be able toward the base, the leaves transition from to suppress the germination of seeds from other photosynthetic carbon assimilation to the plant species the following spring, at least until breakdown (catabolism) of chlorophyll, pro- they are themselves degraded by soil microbes teins, lipids, and nucleic acids and subsequent (Weir et al., 2004), thus acting as preemer- export of valuable nutrients to other plant gence bioherbicides that reduce interspecific parts (Jones and Dangl, 1996; Lim et al., competition. 2007). Before they die, the leaves lose about It is thought that decreasing day length triggers half of their carbon, much of it due to respira- leaf senescence. Temperature, by contrast, has tion to generate the energy required for the no effect on the onset of senescence, although degradation and export metabolism in the face lower temperature may accelerate the rate of of declining photosynthesis (Keskitalo et al., senescence once initiated (Fracheboud et al., 2005; Williams and Smith, 1985). The high 2009). In other words, for a given cultivar respiratory costs of senescence are increasingly grown at a particular latitude, leaf senescence met by the degradation of cell membranes and should start on approximately the same day use of their lipids to provide energy through each year but progress more slowly in warm respiration (Buchanan-Wollaston, 1997). Not than in cool years or on warm than on cool surprisingly, the nucleus with its complement sites. At the other end of the temperature of genes and the mitochondria with their res- spectrum, heat stress (45 C) may also accel- piration machinery are the last cell compo- erate the senescence rate (Thomas and Stod- nents to be dismantled in the process (Lim dart, 1980). Of course, the critical day length et al., 2007). Moreover, chlorophyll is degraded necessary to induce senescence and the not because its products are reusable but, rather, 2.2. VEGETATIVE CYCLE 61 because its light-absorbing properties make it a intermediate (e.g., Cabernet Sauvignon, Syrah, strong phytotoxin in the absence of photosyn- Chardonnay, Teleki 5BB, SO-4, or 3309 Couderc) thesis and to facilitate access to more valuable or weak (e.g., Gamay, 101–14 Mgt, or Riparia materials such as chloroplast proteins and Gloire). Growth typically accelerates with lipids. The nitrogen and much of the carbon of increasing temperature up to an optimum of the chlorophyll molecules, but not their magne- 25–30 C but slows as temperature rises sium, remain in the leaf (Ho¨rtensteiner and further and ceases at approximately 35–38C Feller, 2002), at least until soil microbes decom- (Buttrose, 1969b; Currle et al., 1983). Tempera- pose the leaf once it has fallen to the ground. ture has a strong effect on photosynthesis and Considering that chlorophyll contains only respiration (see Chapter 5.2): Warm (25–30C) approximately 2% of the leaf’s nitrogen, this loss daytime temperatures promote photosynthetic is a rather modest price the plant pays for recy- CO2 fixation, whereas somewhat cooler cling of proteins and lipids (Ho¨rtensteiner, (15–20C) nighttime temperatures limit respi- 2006). Leaves thus manage to remobilize up to ratory CO2 loss. Consequently, a sufficient 50–80% of their nitrogen and phosphorus, 50% (but not excessive) difference between day of their sulfur, and 20% of their iron before their and night temperatures, rather than simply cell membranes collapse, marking the end of a warm daily average temperature, is likely their life (Keskitalo et al., 2005; Niklas, 2006). to promote growth, a phenomenon termed Following this resorption process, the phloem thermoperiodic growth (Went, 1944). Nonethe- is plugged and sealed, and the leaves are shed less, the assumption of a linear increase in at predetermined positions of cell separation at growth rate with rising mean temperature the base of the petiole, termed abscission zones, forms the basis for the calculation of the which are put in place (i.e., differentiated) soon so-called growing degree days (GDD), thermal after the leaves have fully expanded (Roberts time, or heat units. Amerine and Winkler (1944) et al., 2002). The formation of abscission zones proposed that grape-growing regions could is induced by the plant hormone ethylene, be classified by heat summation or cumulative whereas their position is dictated by auxin, GDD for a “standard” growing season (April– which disappears completely during the October in the Northern Hemisphere) as subsequent dormancy period. Each year, grape- follows: vines invest most of their aboveground carbon Xn and nitrogen in leaf production, which cycles GDD ¼ ðTi T Þ i¼1 b as resorbed material in the perennial plant parts and as dead organic material, so-called detritus where Ti is the mean daily air temperature for or humus, through the soil. the period April 1 to October 31 (n ¼ 214 days), T ¼ In viticultural terminology, the rate of shoot and b 10 C is the base temperature. growth or shoot elongation over time is Normally, Ti is calculated as the mean of the referred to as vigor. In addition to genetic daily maximum and minimum temperature. T T > effects (species, cultivar, and rootstock), it is Only daily values of i b 0 are added to strongly influenced by temperature, soil mois- the cumulative GDD, whereas values 0are ture, nutrient availability, vine reserve status, set to zero. Despite some limitations (Dry and pruning level, and even vine age. Some culti- Coombe, 2004) and although variations on this vars (e.g., Thompson Seedless, Cabernet franc, theme have been proposed, including a or Grenache) or rootstocks (e.g., Richter 140, “heliothermal index” (Huglin and Schneider, 1103 Paulsen, or St. George) are considered to 1998), a “latitude-temperature index” (Jackson be vigorous, whereas others are thought to be and Cherry, 1988), and “biologically effective 62 2. PHENOLOGY AND GROWTH CYCLE day degrees” (Gladstones, 1992), the “Winkler also favor high vigor (due to more abundant regions” (Winkler et al., 1974) are still used as a resource availability), as does increasing prun- standard today. Each 1 C increment in tempera- ing severity, because plants in general try to ture averaged over the period April–October reestablish a balanced root:shoot ratio (Buttrose adds 214 GDD to the standard growing and Mullins, 1968; Clingeleffer, 1984; Poorter season so that vineyards at lower latitude or and Nagel, 2000). Nevertheless, the canopy of lower elevation will tend to be classified into lightly pruned vines (e.g., after mechanical or higher (warmer) Winkler regions. The projected minimal pruning) develops earlier and more rise in temperature associated with global rapidly and grows larger than that of heavily climate change (Intergovernmental Panel on pruned vines (e.g., after manual spur or cane Climate Change, 2007) will likely shift many pruning) because a higher number of buds vineyard sites into the next higher Winkler leads to more shoots growing simultaneously region within the next 50 years. The predicted in spring, using a larger pool of reserves increase in average spring and autumn tem- accumulated in the previous growing season peratures will also lead to longer actual (as (Araujo and Williams, 1988; Possingham, 1994; opposed to standard) growing seasons, which Sommer et al., 1995; Weyand and Schultz, are determined by the frost-free period (days 2006a,b; Winkler, 1929). On the other hand, between the last spring and first fall frosts). the presence of fruit tends to depress vegetative Moreover, in addition to its influence on growth of both shoots, especially lateral growth rates, a rise in temperature also typi- shoots, and roots so that the total biomass cally accelerates phenological development, production per vine remains approximately with a consistent trend toward earlier bloom, constant (Eibach and Alleweldt, 1985; Pallas veraison, and harvest (Alleweldt et al. 1984b; et al., 2008; Petrie et al., 2000b). The direction Chuine et al., 2004; Jones and Davis, 2000; of growth also affects growth rate (Currle Webb et al., 2007; Wolfe et al., 2005). et al., 1983; Kliewer et al., 1989); upright- Nevertheless, although the duration of the growing shoots, such as those in a typical phase between budbreak and the time the fruit vertical shoot positioning trellis system, gener- of a given cultivar attains a specific sugar ally grow more vigorously than shoots forced concentrationisshorterinawarmerthanina to grow horizontally, which in turn grow more cooler climate, the cumulative GDD during rapidly than shoots pointing downward, such this period is higher in the warmer climate as those in Geneva double-curtain or Scott– (McIntyre et al., 1987). Henry systems. This effect on growth of the The temperature effect on shoot growth is shoot angle relative to vertical is termed gravi- caused, at least in part, by a stimulation of morphism (Wareing and Nasr, 1961) and is auxin production by high temperature, which associated with variations in xylem vessels, in turn promotes cell elongation. Vigor declines whose number and diameter seem to be tailored with increasing vine size; therefore, shoots of to optimize water and nutrient supply to the small, young vines usually grow more vigor- shoot (Lovisolo and Schubert, 2000; Schubert ously than those of large, old vines. This et al., 1999). Accumulation of auxin near the decline arises from the increased mechanical shoot tip is thought to be responsible for the cost of maintaining an older vine’s permanent production of more but narrower vessels in structure, from the hydraulics of water trans- downward-pointing shoots (Lovisolo et al., 2002b). port, and from the higher number of buds that The time of rapid shoot growth coincides result in more shoots. Increasing soil moisture with secondary growth (increase in diameter) and nutrient (especially nitrogen) availability of the shoots, cordon, trunk, and roots. This 2.2. VEGETATIVE CYCLE 63 secondary growth ends when the decreasing coinciding with shoot browning (Alleweldt, day length after midsummer leads to cessation 1960) and cold acclimation (see Chapter 7.4). of cell division in the cambium, well before The buds’ water content decreases from the temperature becomes too low to limit cell approximately 80% to 50%, and around verai- division (Druart et al., 2007). The shortening son they enter into a phase of endodormancy, day length also seem to decrease gibberellin in which is also called true, inherent, organic, the shoot elongation zone so that growth rates deep, or winter dormancy or rest (Currle et al., decline. The rapid growth period also coincides 1983; Mullins et al., 1992). This transition is with the formation of prompt (lateral) and dor- characterized by a continuous, rapid increase mant (compound) buds in the leaf axils. How- in the temperature and time required for the ever, development of the first compound buds buds to break and by minimal respiration rates. in the basal nodes begins as early as 3 or 4 The shift from paradormancy to endodor- weeks before budbreak or more than 1 year mancy is induced by decreasing day length before these buds will give rise to shoots (i.e., shorter photoperiod) and cooler tempera- (Morrison, 1991). Dormancy in the case of these tures, and it is associated with a strong increase buds means that cell division is blocked, in ABA in the buds and nodes (Currle et al., whereas metabolic processes (e.g., respiration) 1983; Rohde and Bhalerao, 2007). This transi- continue, albeit at a reduced level. Although tion occurs later in cultivars that break buds the buds are under apical dominance from the early (i.e., at lower temperature) the following growing shoot tip, and thus do not normally spring (Pouget, 1972). The genetic programs break, the prompt buds do not become dor- for bud dormancy and cold hardiness are mant and can break relatively readily to form superimposed; shorter photoperiods are suffi- lateral shoots, which contrasts with the com- cient to induce dormancy and leaf senescence pound buds. Nonetheless, following loss of (but not cold hardiness), whereas cooler tem- the shoot tip and removal of lateral shoots or peratures are required to develop cold hardi- severe defoliation (e.g., due to water stress or ness (Fennell and Hoover, 1991). Nonetheless, physical damage caused by pests), even dor- entryintoendodormancyalsoappearstobe mant buds are initially able to resume growth delayed under warm conditions. Endodor- (Alleweldt, 1960). This phase is variously mancy, which is defined as the inhibition of referred to as paradormancy, predormancy, growth by internal bud signals, prevents bud- conditional dormancy, or summer dormancy, break and renewed shoot growth during the defined as the inhibition of growth by distal favorable growth conditions of late summer organs or, specifically, by auxin released by and early autumn, which would lead to cer- the shoot tip and young leaves (Galet, 2000; tain death of the emerging shoots due to fall Horvath et al., 2003; Lang et al., 1987). However, and winter frosts (Horvath et al., 2003; Lang the same buds that are arrested by incoming et al., 1987). (exogenous) auxin will start producing and Approximately 1 week at low but above- releasing auxin as soon as they are released freezing temperatures (chilling) in combination from dormancy and begin to grow. With the with short days appears to break endodor- slowing down of shoot growth (which starts mancy and to stimulate the renewed ability of earlier on less severely pruned vines due to dormant buds to resume growth when favor- their higher shoot number), the buds progres- able (i.e., warm) conditions return (Currle sively (over 2 or 3 weeks) lose the ability to et al., 1983; Galet, 2000; Pouget, 1972). This break even in the absence of apical dominance, period ordinarily coincides approximately with beginning with the most basal buds and leaf fall or soon thereafter in temperate climates 64 2. PHENOLOGY AND GROWTH CYCLE but may extend into midwinter in regions with of a given cultivar is determined by thermal mild winters. The transition seems to be related time (cumulative GDD) during the ecodor- to a decline in ABA in the buds and nodes mancy period. Incidentally, the cambium of (Currle et al., 1983). The upper temperature woody organs also seems to experience similar threshold required to break endodormancy is periods of rest (endodormancy) and quiescence approximately 10 C (mean daily temperature), (ecodormancy) and is reactivated in spring but it is lower for “late” cultivars with a higher when the cambial cells resume cell division base temperature for budbreak and shoot (Begum et al., 2007). growth in spring (and higher for “early” culti- Root growth, especially the formation of lat- vars with lower base temperature). Thus, early eral roots, is stimulated by auxin that has been cultivars enter into dormancy later than late delivered acropetally (from its main place of cultivars, and their dormancy is less intense production in the shoot tips and unfolding and more easily lifted (Pouget, 1972). Although leaves to the root tip) in the phloem (passive chilling is probably not an absolute require- transport, dominant) and parenchyma (active ment to break dormancy in grapevines (at least cell-to-cell transport, which is less important not in all cultivars), and its effect seems to be except during seed germination) and by gibber- quantitative rather than qualitative, no chilling ellins (promoting cell elongation) produced in (especially in combination with an extended the root tips (Friml, 2003; Kramer and Bennett, growing period in late fall) usually leads to 2006). Changes in auxin flow usually lead to delayed, limited, and uneven budbreak. This changes in root growth, and interrupting the can be a problem in warm subtropical and auxin flow (e.g., by cutting off the trunk) will tropical climates, where budbreak is often arti- inhibit root growth completely. Lateral roots ficially induced by application of H2CN2 after grow from the pericycle (and not the apical pruning. Once endodormancy has been lifted, meristem as in the shoot) following the initia- the main factor that prevents budbreak is low tion of renewed cell division by basipetal (from temperature (but lack of water can also inhibit the tip to the base) auxin transport in the cortex budbreak); thus, this last phase is called and epidermis. Following initiation, further ecodormancy (also termed enforced, relative, division of the “founder cells” leads to the for- imposed, or postdormancy or quiescence), mation of a lateral root primordium, which which is defined as the inhibition of growth pushes radially out of the parent root via cell by temporary, adverse environmental condi- expansion, again stimulated by auxin (and tions (Galet, 2000; Horvath et al., 2003; Lang repressed by ABA, e.g., under drought or et al., 1987). In other words, unfavorable grow- osmotic stress from excessive nutrient supply ing conditions simultaneously break endodor- or salinity). After emergence, the apical meri- mancy and impose ecodormancy. The latter is stem is activated and the new lateral root related to cold-induced (or drought-induced) begins to grow autonomously. Note that accumulation of ABA and coincides with maxi- whereas dormant buds seem to inhibit adventi- mal cold hardiness (see Chapter 7). The return tious root formation on hardwood cuttings of warmer temperatures in spring (or unsea- (Smart et al., 2003), the lack of auxin that is nor- sonably warm temperatures during winter) mally produced and released by swelling buds eventually leads to the release from dormancy, may be the reason disbudded cuttings are often which is associated with a rapid (within days) unable or struggle to form roots, unless they gain in the buds’ moisture content back to are dipped in an auxin solution. Similar to 80% and a concomitant loss of cold hardiness shoots, roots also display a kind of apical dom- (Lavee and May, 1997). The date of budbreak inance, whereby the growing tip of a root 2.2. VEGETATIVE CYCLE 65 inhibits the outgrowth of lateral roots from its shortly after budbreak until leaf fall in both pericycle. In contrast to the shoots, however, young and mature vines (Araujo and Williams, the roots use cytokinin rather than auxin to 1988; Williams and Biscay, 1991). However, inhibit lateral root initiation too close to the most of the fine roots (<1 mm in diameter), root tip (Aloni et al., 2006b). This enables the which are the vine’s major water- and nutrient- root to continue growing down (or across) the absorbing structures, are short-lived, so they soil profile in search of water and nutrients. If have to be replaced continuously, probably the root fails to detect nutrients such as nitrate because the soil around them quickly or phosphate, its tip ceases to produce cytoki- becomes exhausted (Comas et al., 2000; Viala nin so that lateral roots can proliferate to and Vermorel, 1909). Only a few of them live explore new territory in the upper soil layers. longer and may eventually become large Although root tips can extend at rates of sev- structural roots. It is estimated that vines allocate eral centimeters per day, it seems that root between 30 and 60% of total net photosynthesis growth generally lags behind shoot growth, products (see Chapter 5.1) to the roots under peaks around the time of bloom and early fruit favorable soil conditions, and this portion is development, and tapers off during fruit ripen- even greater in poor soils or during drought ing (Reimers et al., 1994). Early canopy develop- (Anderson et al., 2003). New roots are initially ment, such as occurs with lightly or minimally white, but they turn brown after approximately pruned grapevines, is associated with more pro- 5 weeks and black after an additional 3–6 nounced early root growth (Comas et al., 2005). weeks. The onset of browning generally indi- The supply of photoassimilates from the leaves cates cessation of root metabolic activity and is evidently critical to sustain root growth, and thus death of the root as a “functional” organ loss of leaf area inhibits root growth far more in terms of water and nutrient absorption and than it does shoot or fruit growth (Buttrose, as a source for new laterals (Comas et al., 1966a). The majority of new roots are pro- 2000). Black roots shrivel and eventually disap- duced while the shoots are growing vigor- pear altogether. Fine roots produced before ously, but root growth can continue as long bloom have an extremely short life span com- as water and nutrient availability permit and pared with those produced later in the growing the soil temperature remains high enough season (Anderson et al., 2003). Fine roots also (Williams and Biscay, 1991). In warm climates, die sooner in dry soil and at shallower soil or in warm growing seasons in cooler cli- depth, and so do roots of heavily pruned vines mates, that permit continued assimilate pro- supporting only few shoots. Such an increase in duction by the leaves, there may be a second root mortality can be considered a response to flush of new root growth around or just after herbivory, whereby plants compensate for the harvest (Conradie, 1980; Lehnart et al., 2008; loss of shoot biomass (i.e., carbon shortage) Mohr, 1996). Surprisingly, root growth has and attempt to restore their root:shoot ratio to even been recorded through the winter season, a balance that is more favorable for growth presumably sustained by nutrient reserves (Bloom et al., 1985). Therefore, mechanical (Bauerle et al., 2008b). Early canopy develop- and, particularly, minimal pruning tend to ment (i.e., larger leaf area) in spring promotes result in more and longer-lived fine roots than early root growth; thus, root growth of lightly spur or cane pruning. Lightly pruned vines pruned vines peaks earlier than that of heavily also produce more fine roots (to balance their pruned vines. The total root biomass increases larger canopy) than heavily pruned vines, even from growth of permanent roots in length and though the former generally support a larger diameter throughout the growing season from crop. In addition, infection with mycorrhizal 66 2. PHENOLOGY AND GROWTH CYCLE fungi greatly prolongs the life span of fine roots, cytokinin, which is then distributed throughout whereas nitrogen addition shortens it. the vine via the transpiration stream in the Grapevines, like other plants, are stationary: xylem, mainly to rapidly transpiring organs Unlike animals, they cannot move from place (Aloni et al., 2005). Depending on soil condi- to place to find better food sources. Because tions, the majority of roots are usually concen- uptake of water and nutrients by the roots trated in the nutrient- and oxygen-rich surface from the soil quickly depletes readily available soil, typically in the top 50 cm, with some roots resources, the roots must keep elongating penetrating to several meters of depth (Celette throughout the growing season and throughout et al., 2008; Morlat and Jacquet, 1993; Smart the vine’s life to maintain the supply of these et al., 2006). Vineyard floor vegetation, such as raw materials. This leads to the formation of a cover crops or permanent swards, tends to shift branched root system that is elaborated with the maximum root density of grapevines to root hairs and symbiotic mycorrhiza (Gebbing 50–100 cm, probably because of competition et al., 1977; Possingham and Groot Obbink, for water and nutrients (Lehnart et al., 2008; 1971; Stahl, 1900). Root density increases as a Reimers et al., 1994). Somewhat paradoxically, vine grows older but appears to settle and however, the average rooting depth tends to remain more or less constant after the vine be much greater in shallow soils (over bedrock) reaches 5–10 years of age. Although 40–50% of than in deep soils because the roots in shallow the volume of a typical soil consists of pores, soils can grow down cracks in the rock, root exploration of the soil ordinarily follows following the path of water infiltration. the path of least resistance; roots predomi- Root growth and distribution is influenced by nantly grow along natural fracture lines of soil species, rootstock, and cultivar. Scion cultivars and reinvade macropores created by old root also alter the root growth of different rootstocks debris forming microniches of high organic (Erlenwein, 1965a). Such genetic influences matter. The decaying dead roots additionally and interactions may be partly responsible for supply nutrients to the growing new roots, the variation in shoot vigor observed with provided these mineralized nutrients are not different scion/rootstock combinations because leached by excessive rainfall or irrigation. Of grapevines try to maintain an equilibrium in course, the general direction of root growth is root:shoot ratio. In fact, total root length nor- downward; that is, the root perceives and mally correlates closely with leaf area, and root responds to gravity (a process termed gravi- dry weight correlates with aboveground dry tropism). The “gravity sensors” are specialized weight in grapevines (Petrie et al., 2000b). amyloplasts (starch-containing plastids) called However, whereas root density may be depen- statoliths that settle at the bottom of the root dent on the genotype, it appears that root cap cells. When a root is forced away from ver- distribution is dictated by soil properties tical (by rocks or other obstacles), the resettling (Mullins et al., 1992). Even genetically identical of these statoliths triggers a redistribution of plants (i.e., clones) can develop vastly different calcium and movement of auxin to the new root systems in terms of both size and archi- lower side of the root back in the elongation tecture, depending on microscale variations in zone so that the cells on that side elongate more soil conditions that the roots encounter during than on the upper side, allowing the root to growth. Such differences result from changes navigate around the obstacle (Kramer and in the number, growth rate, distribution, and Bennett, 2006). Note that the gravity-sensing orientation of lateral roots. Thus, temperature, cells in the root cap are also the ones that pro- soil texture, moisture, and nutrient availability duce most of the plant’s cell division hormone also strongly and rapidly affect root growth 2.2. VEGETATIVE CYCLE 67

FIGURE 2.7 Riesling roots grown at 15C (left) or 25C (center; reproduced with permission from Erlenwein, 1965b), and Cabernet Sauvignon roots growing down fractures in limestone bedrock (right; photo by M. Keller). and distribution. In some cases, the effect of down a moisture gradient in a process called temperature on root growth may be even more hydrotropism, and this ability is so strong that pronounced than its influence on shoot growth it can override the roots’ gravity response (Skene and Kerridge, 1967; Figure 2.7); a mini- (Eapen et al., 2005). How the roots “sense” the mum soil temperature of 6 C seems to be neces- presence of water and how they integrate the sary for roots to grow (Richards, 1983), and a resulting signal with the gravity signal is not 10 C increase in temperature can double or known, but hydrotropism partly seems to result triple the rate of root extension. Although the from the roots’ propensity to grow away from optimum is close to 30 C, temperatures above regions of high osmotic pressure (Takahashi that threshold can kill at least the fine roots et al., 2003). Their growth clearly responds to within days (Huang et al., 2005b). This can result the presence of water tables, the amount and in problems for grapevines grown in pots, where frequency of water application (whether from the soil temperature during the day often rainfall or irrigation), and the type of irrigation exceeds the ambient temperature by several system (e.g., drip vs. furrow). Therefore, grape- degrees. Although under field conditions soil vines can tolerate considerable drought due to temperatures usually fluctuate much less than their ability to selectively grow roots in soil aboveground temperatures due to the large heat patches with available water (Bauerle et al., capacity of wet soils, heavy rainfall or applica- 2008b). This is why the roots of drip-irrigated tion of cold irrigation water can lead to short- vines (especially in sandy soils and dry term changes in soil temperature. Moreover, climates) are often concentrated beneath the dry and sandy soils are not as well buffered drip emitters, whereas in rainfed vineyards or against temperature fluctuations as are wet those irrigated with other methods (e.g., flood- and loamy soils. Mild water deficit has little ing, furrows, and overhead sprinklers) the root effect on lateral root growth, but more severe system is typically much more widespread stress suppresses root growth, although not (Stevens and Douglas, 1994), unless root as severely as it inhibits shoot growth (see growth in the midrows is prevented by soil Chapter 7.2). compaction from heavy machinery. Roots of Roots tend to search out moist soil regions. mature vines also change their growth pattern In other words, they grow toward water or accordingly following conversion from flood 68 2. PHENOLOGY AND GROWTH CYCLE or sprinkler to drip irrigation (Soar and Loveys, growing season under optimal conditions. 2007). Similarly, permanent interrow cover Another trait typical of perennial woody spe- crops tend to lead to a concentration of vine cies is that they require two consecutive grow- roots in the bare, herbicide-treated vine rows ing seasons for flower and fruit production: (Celette et al., 2008; Morlat and Jacquet, 2003). Buds formed in the first year give rise to shoots The roots’ ability to find water even below carrying fruit in the second year. Reproductive the bedrock underlying the “useful” soil hori- growth is very similar in the different Vitis spe- zons (see Figure 2.7) can also greatly expand cies and begins with flower formation, which the apparent rootzone or rooting depth as esti- can be divided into three separate processes: mated from soil pits. This is why grapevines inflorescence initiation (or induction), flower planted on apparently shallow soil are not initiation, and flower differentiation. The first always less vigorous than those planted on step involves the formation of lateral meristems deeper soil, and why their growth is not neces- as uncommitted primordia or initials by the sarily easier to control using deficit irrigation apical meristem inside the shoot’s axial com- strategies. pound buds from spring through early summer In contrast to its effect on shoot growth, (Alleweldt and Ilter, 1969; Carmona et al., 2008; increasing nutrient (especially nitrogen) avail- Gerrath, 1992; Gerrath and Posluszny, 1988b; ability decreases root growth. Soil compaction, Meneghetti et al., 2006; Morrison, 1991; Pratt, leading to a decrease in the soil’s pore volume, 1971; Srinivasan and Mullins, 1981). Primordia also reduces root (and consequently shoot) are first formed in the buds at the shoot base, growth; pressurizing the soil by 1 bar due to and thereafter they also appear progressively compaction may decrease root elongation by in the buds toward the shoot tip. These primor- 90%. Nonetheless, localized soil compaction dia (i.e., meristems) can develop (differentiate) (e.g., due to heavy machinery or plow soles) into inflorescence primordia (i.e., they form reduces shoot growth only when it decreases several inflorescence branch primordia or mer- the total root length (Montagu et al., 2001). When istems), tendril primordia, or sometimes even a single root grows through loose soil into com- shoot primordia. Initiation of uncommitted pri- pact subsoil, compensatory root growth in the mordia occurs after four or five leaf primordia loose soil maintains total root length and thus have been initiated in the buds (i.e., from shoot growth remains unaffected. Such plastic- budbreak to bloom), and subsequent inflo- ity in root system development in response to rescence differentiation begins around anthesis heterogeneous soil conditions enables grape- of the current season’s inflorescences and vines to acquire soil resources with a conserva- may continue until the buds enter dormancy tive investment in root biomass. (Alleweldt and Ilter, 1969; Morrison, 1991; Snyder, 1933; Swanepoel and Archer, 1988). In 2.3. REPRODUCTIVE CYCLE other words, inflorescences are formed during the year that precedes the flowering and fruit- ing year, and the fate of the following year’s Grapevines grown from seeds, like other primordia is determined as early as bloom time woody perennials, have a vegetative phase of of the current year. The number of inflores- several years before they reach their reproduc- cences is lowest at the shoot base and increases tive phase and become able to produce fruit. with higher bud position (Sartorius, 1968). Yet This ensures that the vine has ample resources in most cultivars only the basal six to eight to support fruit production. By contrast, vines buds of a shoot (i.e., those preformed in the grown from cuttings are able to fruit in the first previous growing season) are able to form 2.3. REPRODUCTIVE CYCLE 69 inflorescences (Sa´nchez and Dokoozlian, 2005); unfruitful. Buds that develop inside dense the uncommitted primordia initiated at youn- canopies are less fruitful than the buds at the ger node positions (i.e., those produced by canopy exterior (May et al., 1976; Shaulis et al., the actively growing shoot) always become 1966). However, bud fruitfulness appears to tendrils, never inflorescences (Morrison, 1991). be independent of the light exposure of the Thompson Seedless (synonym Sultana) is an buds but depends strongly on the supply of exception: Fruitful buds are initiated over assimilates to the buds (Keller and Koblet, the entire length of its shoots (Sa´nchez and 1995a; May and Antcliff, 1963; Sa´nchez and Dokoozlian, 2005). The origin of the shoot is Dokoozlian, 2005). These assimilates may come unimportant for the fruitfulness of its buds; from stored reserves or from leaf photosynthesis, shoots arising from 1-year-old spurs or canes but they are mostly supplied by the leaves on produce equally fruitful buds as those growing the same side of the shoot, perhaps especially from older cordons or trunks (Mu¨ ller-Thurgau, from a bud’s subtending leaf (Hale and 1883). Weaver, 1962). This has implications for the The maximum number of future inflores- design of trellis and training systems in viti- cences per bud, the so-called bud fruitfulness, culture. A renewal zone (the zone where buds appears to be determined by approximately are located that produce next year’s shoots 3 months after budbreak—that is, before verai- and crop) with well-exposed leaves, ideally son (Alleweldt and Ilter, 1969). Depending on situated close to the top of the canopy, and a species and cultivar, several inflorescence pri- canopy that is not too dense will maximize mordia may be formed in the primary buds of the potential crop because the number of pri- most Vitis species. Most V. vinifera cultivars mordia determines the vine’s yield potential typically initiate two such primordia, but for for the following year (see Chapter 6.1). In some, such as Se´millon, Muscadelle, Gamais, addition, the growth direction of the shoot or Carignan, three is more common, whereas also appears to influence bud fruitfulness, the fruitful Gouais blanc forms four, V. labrus- at least in some cultivars: Upright-growing cana cultivars form three or four, and V. riparia shoots produce buds with more inflorescence and V. rupestris buds form seven or eight inflo- primordia than horizontally or downward- rescence primordia (Viala and Vermorel, 1909). growing shoots (Alleweldt and Ilter, 1969). Moreover, environmental conditions strongly The commonly accepted view (Carmona modulate the number of primordia. Conditions et al., 2002, 2008; Gerrath, 1992; Gerrath and required for the formation of the maximum Posluszny, 1988b; May, 2004; Meneghetti et al., number of inflorescence primordia include 2006; Morrison, 1991; Mullins et al., 1992; high irradiance, warm temperature (25–30 C, Scholefield and Ward, 1975; Snyder, 1933; especially during the 3 weeks prior to bloom), Srinivasan and Mullins, 1981) of inflorescence and adequate water and nutrient supply and flower differentiation is that the formation (Currle et al., 1983; Meneghetti et al., 2006; of inflorescence meristems and that of flower Mullins et al., 1992; Sartorius, 1968; Sommer meristems are seasonally separated (Figure 2.8). et al., 2000; Srinivasan and Mullins, 1981). According to this view, inflorescence primordia Warm temperatures promote the induction of grow rapidly during the summer, producing inflorescences, whereas cool temperatures several branch meristems before the buds enter (<20 C) promote tendril formation (Buttrose, dormancy in late summer. Further inflores- 1969a, 1970b). Unfavorable conditions (e.g., cence development is arrested with the onset temperatures >35 C) may reduce the primor- of dormancy and does not resume until the dia number to zero, rendering the buds buds begin to swell in late winter or early 70 2. PHENOLOGY AND GROWTH CYCLE

FIGURE 2.8 Cluster initiation (1) and differentiation (2–6: numbers indicate increasing orders and positions of branch- ing) and flower development (8: initiation of sepal lobes; 9: initiation of petal lobes; 10: initiation of stamen primordia; 11: initiation of carpel primordia; 12: appearance of locules; 13: ovule initiation; 14: pollen formation; 15: anthesis) of Concord grapevines (left; reproduced with permission of M. C. Goffinet); inflorescences of the rootstock 3309C emerging after bud- break (right; photo by M. Keller). spring. When the buds are reactivated, the inflo- The pistil with its reproductive structures rescence branch meristems produce additional does not develop until the individual flowers meristems that give rise to clusters of three or become visibly separated on the inflorescence. four flower meristems or floral primordia during Separate genes or groups of genes specify the the period of budswell that precedes budbreak identity of the individual floral organs, although by several days to weeks. This process is termed the mechanism by which they exert their influ- flower initiation, and within each of these clus- ence is not known (Dı´az-Riquelme et al., 2009). ters the floral primordia are initiated “from the The dissenting minority opinion of this top down”; that is, the terminal flower is initiated developmental process differs mainly in the first and the basal flower last. Flower initiation timing of flower initiation (Agaoglu, 1971; happens simultaneously in all parts of the inflo- Alleweldt, 1966; Alleweldt and Ilter, 1969; rescence primordium during the budswell phase Sartorius, 1968). This view holds that a portion and culminates in the formation of a calyx (fused of the calyxes are formed before bud dor- sepals) for each flower. Branching and flower mancy, some during winter dormancy (albeit initiation ostensibly cease once a shoot starts very slowly), and the remainder during the growing out of the bud. The subsequent flower subsequent budswell and budbreak period. differentiation, or the development of the indi- The corolla and stamen also appear before bud- vidual organs of each flower, also called floral break, followed by the pistil, whose formation organogenesis, occurs over approximately 5 is complete within 10–15 days of the emergence weeks during and after budbreak. The corolla of the inflorescence after budbreak. Intrigu- (joined petals) and stamens appear successively ingly, evidence supporting both of these views under the stimulating influence of gibberellin comes from both warm and cool climates, so (Cheng et al., 2004a), while the inflorescences it is not clear what might cause the disparity become visible on the shoot and then separate. in observations. Inflorescences that arise on 2.3. REPRODUCTIVE CYCLE 71 lateral shoots, especially on those laterals on tissues. The anthers produce and release particu- the free-growth portion of a shoot that could larly high amounts of auxin, as well as gibberel- not possibly have been initiated in the previous lin, and thereby synchronize (i.e., delay) the growing season, obviously run through their development of the other flower parts so that entire developmental program from inflores- they become functional just before anthesis cence initiation to fruiting within the same (Aloni et al., 2006a; Olszewski et al., 2002; growing season. Yamaguchi, 2008). Gibberellins help maintain The extent of branching before and after the the inflorescence meristems and favor inflores- dormancy period and the degree to which the cence elongation (Alleweldt, 1959). This is why individual flowers develop in spring determine table grape growers sometimes apply gibberel- the number of flowers that form on each inflo- lin sprays before bloom to lengthen the rachis rescence. The flower number is highly variable, and make clusters less compact (Mullins et al., even on the same shoot: The basal inflorescence 1992). Application during bloom may lead to of a shoot typically produces the most flowers, lower fruit set, which tends to further promote and numbers decline with increasing height of loose clusters. However, gibberellins act anta- insertion on the shoot (May, 2004). Branching gonistically to cytokinins and favor tendril for- of the primordia is induced and controlled by mation by inhibiting the branching process so two groups of plant hormones, namely auxins that bud fruitfulness may decrease. This inhibi- and cytokinins. The production of cytokinins, tory role appears to be somewhat unique to which occurs predominantly in the root tips, grapes because gibberellins are usually involved and their transport to the shoots in the xylem in establishing floral primordia in many plants. are regulated by developmental (genetic) and Approximately 2 weeks after the ovules environmental signals (e.g., water and nitrogen have been formed and 5–10 weeks after bud- availability). Auxin is produced and released break, anthesis marks the beginning of bloom, by the floral meristems (i.e., the tip of each floral exposing the male and female floral organs organ) and controls both formation and differen- (Figures 2.9 and 2.10). Anthesis theoretically tiation (i.e., morphogenesis) of the flowers; it refers to the release of pollen but is commonly also induces development of their vascular regarded as the shedding of the calyptra, or

FIGURE 2.9 Grape flowers immediately before anthesis (left, with cross section center left), during anthesis (center right), and immediately after anthesis (right). Illustrations by A. Mills. 72 2. PHENOLOGY AND GROWTH CYCLE

FIGURE 2.10 Chardonnay inflorescence at the beginning of bloom (left) and in full bloom (right, with insert showing a close-up of a group of flowers). Photos by M. Keller. capfall. Similar to budbreak, anthesis generally flowers are termed star flowers and can appear begins in the shoots growing from distal buds in a range of cultivars (Longbottom et al., 2008; and progresses basipetally toward the trunk. Pratt, 1971). On the other hand, anthesis within an individ- When the anthers burst open, they release ual inflorescence often starts close to its base, their pollen into the air. The production of pollen and all flowers normally open within 5–7 days, is stimulated by the plant hormone cytokinin. although this can be delayed during cold and The transfer of pollen grains (microgameto- rainy conditions. Only few flowers open at phytes) from the anthers to the stigma is called 15 C, and the optimum temperature seems to pollination and is followed by abscission of the be in the range 20–25 C; the rate of cap fall stamens. During anthesis, the stigma releases a slows greatly at 35 C (Galet, 2000; May, 2004). sticky sap that retains and rehydrates the pollen Each day opening typically begins at approxi- for pollination (Considine and Knox, 1979a; mately dawn and ends at approximately noon Meneghetti et al., 2006). This sap is supplied via (Staudt, 1999). Different inflorescences on the the newly formed stylar xylem that has previ- same vine, or on different vines in the same ously been induced by auxin released by the vineyard, follow the same rhythm but may stigma’s papillae. Because the tiny, dry pollen start it on different days so that the entire grains are highly exposed during this process, bloom period in a particular vineyard may last the pollen wall contains high concentrations of 2 or 3 weeks. Capfall occurs when the petals phenolic compounds termed flavonols that act detach from the receptacle and are lifted off as “sunscreen” (Downey et al., 2003b; see also by the anthers. It is thought that it is the rapid Chapter 5.2) protecting the genetic information elongation of the anther filaments that pushes (DNA) contained in the chromosomes from the calyptra away from its base once the basal destruction by UV radiation (Caldwell et al., cells have dehisced, which is a rare mechanism 1998). Nevertheless, excessive UVB radiation, among angiosperms. Occasionally, the petals such as that resulting from sudden exposure to instead open at the top and are spread open sunlight of previously shaded inflorescences, and pushed downward by the anthers. Such especially in conjunction with heat (>33 C), can 2.3. REPRODUCTIVE CYCLE 73 sometimes decrease pollen germination or pol- sperm cells (male gametes; Greek gamete ¼ len tube growth (Torabinejad et al., 1998). In spouse), penetrates the stigma and grows down other words, stress reduces pollen viability and through the intercellular spaces of the style like leads to male sterility. a hydraulic drill, delivering its package of chro- In contrast to their wild relatives and the few mosomes into the egg inside the ovule (Zonia cultivars with female flowers, cultivated grape- and Munnik, 2007). The male and female vines are typically self-pollinated, whereby pol- gametes (sex cells) meet when a pollen tube len originates from the flower’s own anthers enters the embryo sac and releases the sperm. (Mullins et al., 1992; Sartorius, 1926). Wind Their fusion in the ovule is termed fertilization appears to be responsible only for occasional and results in the zygote, which develops into cross-pollination, where pollen originates from the embryo and leads to fruit set. In a typical a flower on a different plant than the polli- grape flower, the pollen tubes cover a distance nated one (Scherz, 1939). In fact, the anthers of approximately 2 mm. Both pollen germina- often burst shortly (1–24 h) before the calyptra tion and pollen tube growth are induced by is shed, releasing the pollen on the flower’s auxin, with which the pollen has previously own stigma, a process termed cleistogamy been “loaded” in the anthers, and stimulated (Heazlewood and Wilson, 2004; May, 2004; by the growth hormone gibberellin that may Staudt, 1999). Despite this self-pollination strat- be produced by the pollen (Gillaspy et al., egy, the osmophors on the ovary may serve to 1993). Growth of the pollen tube represents a attract insects by emitting monoterpenes (simi- more than 1000-fold elongation of the pollen lar to citrus and lavender flowers), although cell and is very rapid; rates of 0.3 mm h 1 are they ostensibly do not offer the insects any nec- not uncommon (Staudt, 1982). Not surpris- tar. An intriguing possibility is that the release ingly, this process is very energy-consuming, of volatiles might be a defense strategy to deter and the growing pollen tubes have high respi- herbivorous insects or fungi that would feed on ration rates. Respiration is fuelled by the break- the highly vulnerable flowers or to attract down of starch reserves that are accumulated enemies of these herbivores (Gang, 2005). Insect in the pollen grains from imported sucrose pollination appears to be much more common, prior to anthesis. and is perhaps required for commercially The speed at which the pollen tubes grow acceptable fruit set, in Muscadinia than in Vitis toward the ovules is critical for fruit set because species (Olien, 1990; Sampson et al., 2001). the ovules are only receptive for a limited However, even in the latter, berries originating period after anthesis, after which they can no from cross-pollinated flowers apparently grow longer be fertilized. The rate of pollen tube larger than those from self-pollinated flowers, growth depends on temperature; at 25–30 C, which suggests that cross-pollination improves fertilization can occur within 12 h, at 20 C after seed formation. In any case, the pollen grains 24 h, and at 15 C after 48 h, whereas even absorb water from the moist stigma to rehy- lower temperatures do not permit fertilization drate (a process that requires approximately because the pollen tubes stop growing before 30 min), germinate on the stigma whose surface they reach the ovules or they simply reach cell layer subsequently suberizes (Considine them too late (Staudt, 1982). Eggs that are not and Knox, 1979a), and form a pollen tube. The fertilized within 3 or 4 days after anthesis will optimum temperature for pollen germination degenerate (Callis, 1995; Kassemeyer and is between 25 and 30 C, whereas temperatures Staudt, 1981). Therefore, slow pollen tube below 10C and above 35C inhibit germina- growth (e.g., due to low pollen “fuel status” tion. The pollen tube, which carries two fused resulting from stress interfering with starch 74 2. PHENOLOGY AND GROWTH CYCLE accumulation prior to pollination) leads to poor Similar to the placenta in mammals, the endo- fruit set (Koblet, 1966). Nonetheless, only one sperm protects the embryo developing from pollen tube from the many pollen grains land- the zygote, controls nutrient transfer from the ing on the stigma needs to reach one of up to berry, and serves as a nutrient store for the four receptive ovules to make fertilization developing embryo and the emerging plant likely. How the pollen tubes find the ovules is organs during seed germination. It seems that not well-known, but it seems that they are double fertilization is an all-or-nothing process: attracted by the synergid cells (which die after Either both the egg cell and the central cell are they have served their purpose) flanking the fertilized or neither is fertilized (Kassemeyer egg cell and follow gradients of g-amino buty- and Staudt, 1981). The resulting diploid embryo rate (GABA) (Berger et al., 2008; Palanivelu and (initially) triploid endosperm together form et al., 2003). GABA is chemically an amino acid, the filial (daughter) generation, whereas all but it is not referred to as such because it is not other berry tissues (including the seed coat and incorporated into proteins. In plant cells, it is the nucellus) are part of the maternal (mother) synthesized from glutamate, and in the human generation or mother plant. Yet the contribution brain GABA released from nerve cells is of the central cell’s maternally derived cyto- the predominant inhibitory neurotransmitter. plasm to the developing endosperm is quite sig- In any case, once the pollen tube reaches the nificant (Berger et al., 2006). Incidentally, the ovule, it stops growing, and its volume and embryo should more accurately be called the pressure rapidly increase. This leads to an zygotic embryo because somatic plant cells can explosive rupture of the tip, which ejects be forced through tissue culture (i.e., in vitro)to- the sperm cells into the ovule (Staudt, 1982). The form so-called somatic embryos, which can four ovules in a flower do not all reach develop into whole plants that are clones of their the same stage of development simultaneously: mother plant (Franks et al., 2002; Martinelli and Some embryo sacs may fail to develop or not Gribaudo, 2001; Mullins and Srinivasan, 1976). mature in time to be fertilized, others are not This somatic embryogenesis is exploited in “found” by a pollen tube, and yet others are vegetative propagation, which is used to help fertilized but are slow to develop (Kassemeyer eliminate viruses and bacteria by so-called and Staudt, 1982; Nitsch et al., 1960; Pratt, micropropagation or meristem culture, whereby 1971). This is why grape berries rarely contain shoot tips less than 0.5 mm in length are excised four seeds. under aseptic conditions and grown into full Fertilization in angiosperms is more com- plants (Barlass et al., 1982; Bass and Vuittenez, plex than in other plants or in animals. During 1977). Although this practice may occasionally the process of double fertilization, one haploid lead to the expression of juvenile leaf characters (i.e., having one set of chromosomes) male in the resulting plant, this is normally confined gamete fuses with the haploid female gamete to the lower portion of the new shoots, and (the egg cell or simply egg) to form the diploid cuttings taken from the distal portion will (i.e., having two sets of chromosomes—one give rise to true-to-type plants (Grenan, 1984). from the mother via her egg and one from the However, such propagation methods are belie- father via his pollen) zygote. The other male ved to encourage the activation of normally gamete (delivered by the same pollen tube) silent jumping genes (see Chapter 1.2), which almost simultaneously fuses with the other could induce occasional somatic mutations female gamete, the diploid central cell of the and thus introduce additional genetic variation embryo sac to give rise to the endosperm (Ber- in some of the tissue-cultured vines (Benjak ger et al., 2008; May, 2004; Raghavan, 2003). et al., 2008). 2.3. REPRODUCTIVE CYCLE 75

In contrast to somatic embryogenesis, two separate sexes and prohibiting them from although they are also derived from somatic mixing prevents this problem (i.e., limits the cells (and hence may carry somatic mutations), damage) while still permitting the (nuclear) but due to genetic recombination in addition to chromosomes to take advantage of the costly occasional mutations, each egg cell is geneti- sexual recombination. cally different from all other egg cells on the As a result of the chromosome mixing, every same vine, and each sperm cell is genetically single seed, even within the same berry, will different from all other sperm cells on the same give rise to a genetically unique plant, slightly vine. The recombination is basically a random different from all others, even though it inher- shuffling of paternal and maternal genes, just ited all its plastids and mitochondria from the like the shuffling of a deck of cards, and occurs mother plant. This production of new geno- approximately 1–3 weeks before anthesis types by mixing of genes (more accurately, (depending on cultivar; Lebon et al., 2008) dur- alleles; i.e., slightly different versions of the ing the production of gametes in a special cell same gene, one of which may or may not be division termed meiosis (Greek meioun ¼ to dominant over the other) in each generation in make small). Just before the cell divides, the addition to occasional mutations (which gener- chromosomes line up alongside each other ate the different alleles in the first place) pro- and randomly exchange pieces of DNA so that vides the endless genetic variety that under the new chromosomes that end up in the natural conditions allows evolution (specifi- gametes constitute a novel and distinctive mix cally, natural selection) to act by “weeding of genes from the mother and the father. This out” those individuals (phenotypes) less well shuffling is the true essence of sex and thus adapted to the prevailing environmental condi- only applies to genes contained in the cell tions (Mayr, 2001). As Charles Darwin (2004) nucleus—that is, on the chromosomes. The stated, "Natural selection acts solely through other cell organelles with their own genes (i.e., the preservation of variations in some way the mitochondria and plastids such as chloro- advantageous." Because “environment” also plasts) are reproduced asexually (by simple comprises other species, including pathogens division), and the male organelles are normally such as fungi, bacteria, and viruses, the major excluded from or left behind in the pollen. benefit of recombination is the decreased risk Only very few of them occasionally “slip” for a plant’s offspring to be wiped out by path- through (Birky, 1995; Timmis et al., 2004), and ogen attack (see Chapter 1). This is the meaning some of their genes may escape destruction of sex: By producing genetic variation (i.e., by and be “absorbed” by the nucleus (Sheppard “not putting all its eggs in the same basket”), et al., 2008). Indeed, this exclusion of the a species greatly increases the chance that at father’s organelle genes is the fundamental rea- least some individuals will resist and thus sur- son why there are two, and only two, sexes in vive pathogen attack (or drought, etc.). Of all sexually reproducing plants (and animals). course, such genetic variation is possible only Building and running an organism as complex in wild grapevines but not in cultivated popu- as a grapevine (or an animal) requires a great lations of vegetatively propagated (i.e., clonal) deal of cooperation among the genes. The plants. Nevertheless, even the various cultivars genes of the plastids and mitochondria have originally arose this way (whether by deliber- never “learned” how to cooperate, and these ate crossing or by natural pollination), and organelles would often attack and kill each recombination accounts for the vast differences other when two cells fuse (which, in fact, they among cultivars in terms of growth, yield for- do in single-celled algae). Assigning them to mation, fruit composition, pest and disease 76 2. PHENOLOGY AND GROWTH CYCLE susceptibility, and reaction to environmental is exploited in table grape and raisin produc- influences. Somatic mutations (bud sports), tion; growers routinely apply gibberellin sprays however, can arise in vegetatively propagated at bloom and fruit set to increase berry size grapevines and may give rise to clonal varia- (Mullins et al., 1992). In contrast to early seed tion that can be subject to selection by humans formation, the seeds later take charge of their (artificial as opposed to natural selection). own maturation by switching on the embryo’s Within days after fertilization, the endo- genes; in other words, there is a change from sperm nucleus begins to divide, whereas the maternal to filial control of seed development zygote (i.e., the future embryo) does not start (Gutierrez et al., 2007). These genes initiate dividing until approximately 2 or 3 weeks and coordinate the import of nutrients and after fertilization (Nitsch et al., 1960; Pratt, accumulation of storage reserves such as starch 1971); by this time, the cells of the nucellus and proteins in the endosperm and the have reached their maximum size, and the subsequent desiccation of the seeds. endosperm, whose nuclei have by now become Although pollination, rather than fertiliza- polyploid, begins to accumulate starch and tion, triggers the initial stages of ovary develop- lipids (Kassemeyer and Staudt, 1983). The first ment, subsequent fruit formation depends on a few divisions of both tissues seem to be mainly supply of seed-derived auxin (Kassemeyer and under the control of maternal genes, which Staudt, 1983; O’Neill, 1997). Therefore, the may give the mother plant some control over development of grape berries is usually depen- which embryos to develop (Raghavan, 2003). dent on pollination, fertilization, and develop- The fertilized ovule develops into the seed, ment of at least one seed (Pratt, 1971). Ovaries whose embryo starts to produce and release that do not contain at least one fertilized ovule auxin to pericarp tissues, where it stimulates or whose seed development is aborted early on gibberellin synthesis (O’Neill, 1997; Pandolfini are abscised from the fruit cluster (Nitsch et al., et al., 2007; Serrani et al., 2008). Gibberellin 1960). Arrested ovule development prior to may also be produced in and released by the anthesis prohibits fertilization and also results seeds upon stimulation by embryo-derived in abortion if all four ovules fail to develop auxin (Dorcey et al., 2009). Auxin reactivates (Kassemeyer and Staudt, 1982). Later degenera- cell division, and gibberellin induces cell tion of the embryo and endosperm can result in expansion (Serrani et al., 2007). Thus, fruit set apparently normal-looking but hollow (and is dependent on both auxin and gibberellin: thus nonviable) seeds termed “floaters” These two hormones induce the pistil to because they float in water, unlike normal develop into the fruit and to differentiate an seeds, which are thus named “sinkers” (Ebadi exocarp (skin) and mesocarp (flesh or pulp). et al., 1996). Although auxin induces gibberellin produc- Nonetheless, some cultivars can produce tion, auxin synthesis might be stimulated by apparently normal seedless berries. This is pollen-derived gibberellins and is necessary called parthenocarpic (Greek parthenos ¼ vir- for embryo formation and fruit tissue produc- gin, karpos ¼ fruit) fruit development, when tion and continues until the embryo is mature. the berries develop without fertilization Therefore, the interaction, also called cross talk, (although typically not without pollination) between auxin and gibberellin plays a key role and lack seeds completely, or stenospermo- in the ovary’s commitment to initiate fruit carpy, when the berries contain at least one fer- development and growth (Gillaspy et al., 1993; tilized but subsequently aborted seed so that in Ozga and Reinecke, 2003). The promoting effect many cases all that remains is a small, soft, white of gibberellin on cell volume in seedless berries rudimentary seed or seed trace (Barritt, 1970; 2.3. REPRODUCTIVE CYCLE 77

Ledbetter and Ramming, 1989; Olmo, 1936; intensity, warm temperature, and adequate soil Pearson, 1932; Roytchev, 1998, 2000; Staudt moisture and nutrient availability. This is the and Kassemeyer, 1984). Stenospermocarpy is a likely reason why years in which the shoots quantitative trait: The seed traces vary in size carry more clusters than usual often follow and hardness over a wide range (Cabezas et al., years with above-average fruit set. Adverse 2006; Ledbetter and Shonnard, 1991). Cultivars environmental conditions (e.g., cloudy, wet, with parthenocarpic fruit (e.g., Black Corinth) cool, or hot bloom period and water or nutrient are often used to produce seedless raisin stress), insufficient or inefficient leaf area (e.g., grapes, whereas most so-called seedless table due to hail or insect or disease attack), shade grapes (e.g., Sultana; synonym Thompson created by dense canopies, or excessively vigor- Seedless) are stenospermocarpic. Intriguingly, ous shoot growth (competing with inflores- the embryos of stenospermocarpic grapes cences for assimilates) often result in poor remain alive and can be rescued and grown fruit set and loose clusters (see Chapter 6.1). into full plants using in vitro techniques, which Because, as a perennial species, the grapevine permits crossing of seedless table grapes to does not depend on producing seeds each year, breed new cultivars (Cain et al., 1983; Emershad such “self-thinning” enables it to adjust its and Ramming, 1984; Emershad et al., 1989). reproductive output to available resources Because the lack of growth hormone production without jeopardizing survival of the plant. By in stenospermocarpic fruit limits cell expan- culling a portion of its reproductive structures, sion and hence berry size (Coombe, 1960; the vine may enable the remaining seeds to Nitsch et al., 1960), gibberellin sprays are often attain adequate size and maturity. applied after fruit set to increase the size of Poor fruit set due to excessive abortion of table grapes with the added benefit of decreased flowers and ovaries is also termed shatter, cluster compactness due to the elongation of shedding, or “coulure” (French couler ¼ to leak, the rachis. to fall off). Loss of such ovaries can occur for The proportion of flowers that develop into up to 4 weeks after anthesis (Kassemeyer and berries following anthesis is typically in the Staudt, 1983; Staudt and Kassrawi, 1973). Fertil- range of 20–50% and is inversely related to ization of at least one of up to four ovules is the number of flowers per inflorescence. Fruit required for the ovary to develop into a berry; set is highly variable between cultivars and is hence, the abscised ovaries typically do not strongly modulated by environmental condi- contain any fertilized ovules, although the tions and rootstocks (Alleweldt and Hofa¨cker, vast majority of them have been pollinated 1975; Keller et al., 2001a; Schneider and Staudt, (Kassemeyer and Staudt, 1982; Staudt and 1978). Cultivars that are susceptible to environ- Kassemeyer, 1984). Normal fruit set despite mental conditions leading to poor fruit set inadequate seed development in a portion of include Merlot, Grenache (synonym Garnacha), the fertilized berries, on the other hand, is and Gewu¨ rztraminer, whereas cultivars such as called “millerandage” and results in clusters the Pinots, Chardonnay, or Sylvaner seem to be having the appearance of “hens and chickens” much less susceptible. The process of meiosis (Fouge`re-Rifot et al., 1995; Galet, 2000; May, appears to be particularly sensitive to stress 2004). The term “hens” refers to large, normal conditions that curtail the supply of carbohy- berries with at least one viable seed, and drates to the inflorescences. Optimum condi- “chickens” describes small berries with tiny, tions for flowering and fruit set are thought to degenerated seeds (i.e., seed traces) that often be similar to those governing inflorescence ini- lack endosperm (Ebadi et al., 1996; Staudt and tiation (see Chapter 6.1), namely high light Kassemeyer, 1984). Although the expansion of 78 2. PHENOLOGY AND GROWTH CYCLE

“chickens” is arrested within approximately with naturally tight clusters can be manipu- 3 weeks after anthesis due to seed abortion, lated using viticultural practices that tend to these berries apparently ripen normally. By decrease cluster compactness, although such contrast, so-called “shot berries” are essentially practices usually come at the cost of decreased arrested at the ovary stage; remain small, yield. Potential strategies include light or no green, and hard; and are high in tannins pruning resulting in large numbers of clusters (Fouge`re-Rifot et al., 1995). The term is unfortu- and, therefore, relatively poor fruit set (see nate: Because these ovaries fail to differentiate a Chapter 5.1); removing a portion of the leaves discernible mesocarp and exocarp, shot berries before or soon after bloom, which also are technically not berries at all. They usually diminishes fruit set (Candolfi-Vasconcelos and contain at least one sterile ovule (e.g., due to Koblet, 1990; Poni et al., 2006); or cutting incomplete or failed meiosis) in addition to through inflorescences around bloom time degenerated fertile but unfertilized ovules. Per- (i.e., removing the distal cluster portion), which haps shot berries are prevented from abscising leads to elongation of the remaining rachis. by the initial, although very limited, growth of In seeded grapes, fertilization is followed by the nucellus of the sterile ovule (Kassemeyer a second period of cell division (the first period and Staudt, 1982). Coulure results in very is before bloom and determines the cell number loose clusters, and millerandage leads to highly of the ovary), which is probably stimulated by variable seed numbers and berry sizes, a brief increase in cytokinin in addition including seedless berries, on the same cluster. to auxin produced in the developing seeds. Variations in the degree of fruit set together The berry cytokinin concentration, it seems, with the extent of inflorescence branching and declines after anthesis throughout berry devel- variations in the rate and/or extent of rachis opment (Chacko et al., 1976). During the first 3 cell expansion during cluster development weeks after anthesis, the polar nucleus of the before and after bloom lead to wide variation seeds’ endosperm divides several times before in cluster size and architecture, both between the endosperm becomes cellular so that endo- species and cultivars and within them (e.g., sperm nuclei become polyploid (Kassemeyer due to environmental influences). Such differ- and Staudt, 1983). By contrast, the zygote cells ences in morphological features have implica- giving rise to the embryo only begin dividing tions not only for yield formation (see Chapter approximately 2–4 weeks after anthesis. The 6.1) but also for the clusters’ susceptibility to rate and duration of cell division in the berry fungal attack. For instance, cultivars with tight pericarp are controlled by the seeds (i.e., the clusters (e.g., Chardonnay, Riesling, and Pinot) embryos) so that berries containing more seeds have small rachis cells and a short, compact will become larger than berries with fewer branching pattern. They are much more sus- seeds (Gillaspy et al., 1993). Whereas the cell ceptible to infection by Botrytis cinerea than number successively doubles approximately those with loose clusters (e.g., Cabernet Sau- 17 times before anthesis, only one or two more vignon, Syrah, and Thompson Seedless). This doublings happen after anthesis (Coombe, is probably because tight clusters have their 1976). The mesocarp cells cease dividing 3 or berries packed into a small volume and hence 4 weeks after anthesis (the innermost cells stop retain more rain (or irrigation) water and dry dividing within 2 weeks), whereas skin cells down more slowly than loose clusters. In addi- may continue to divide for up to 5 weeks after tion, berries touching and rubbing on each anthesis (Considine and Knox, 1981; Harris other may lose epicuticular wax and hinder et al., 1968; Nakagawa and Nanjo, 1965; Pratt, uniform fungicide coverage. Yet even cultivars 1971). Cell division is completed before the 2.3. REPRODUCTIVE CYCLE 79 berry enters a lag phase of slow or no fresh Chapter 6.2). In ripe grape berries, cell wall weight accumulation. Although it is frequently thickness ranges from >1 mm in the exocarp stated that berry growth is divided into a phase to <0.1 mm in the mesocarp and, if grapes of cell division and one of cell expansion, this is behave like tomatoes, the resistance of both not accurate. Growth is always by cell expan- the epidermis and the cuticle against defor- sion because plants (like any other organism) mation, their so-called modulus of elasticity or cannot add fully grown cells like bricks to a elastic modulus increases strongly during fruit wall. Although dividing cells usually remain ripening (Bargel et al., 2006). As first described small and have small vacuoles, cell division is by Winkler and Williams (1935), the growth or accompanied and followed by cell expansion increase in fresh weight of seeded berries traces (Gillaspy et al., 1993; Pratt, 1971). As the cells a double-sigmoid pattern; that is, the growth expand, the cell wall becomes thinner and the curve is bent in two directions, like the letter vacuole becomes predominant. Therefore, the S, and can be divided into three more or less vast majority of the volume gain of growing distinct phases or stages (Figure 2.11): berries is due to cell expansion (i.e., an increase in vacuole volume; see Chapter 3.1), especially Stage I: The first rapid increase in size of the in the mesocarp, which is stimulated by seed- seeds and pericarp. The first part of seed produced auxin. The lag phase is followed by development is termed morphogenesis and another period of cell expansion so that the vol- consists of embryo formation ume of mesocarp cells may increase more than (embryogenesis) and endosperm growth, 300-fold from anthesis to maturity (Coombe, but there is little growth of the embryo 1976) or 15-fold between fruit set and maturity. (Ebadi et al., 1996; Nitsch et al., 1960). The At the same time, the fruit surface area may seeds are green, and the berry is green and increase approximately 400-fold from anthesis hard and accumulates organic acids (mainly to veraison and almost doubles again from ver- tartrate and malate) but little sugar. The aison to maturity (Considine and Knox, 1981). growth hormone auxin, the cell division Cell division seems to be mostly under hormone cytokinin, and the cell expansion genetic control (i.e., fruit from different culti- hormone gibberellin (GA) produced by the vars should have different cell numbers), embryos and released into the pericarp reach whereas cell expansion is predominantly high concentrations during this period driven by environmental factors (i.e., berries (Iwahori et al., 1968; Sakakibara, 2006; of the same cultivar grown under different con- Scienza et al., 1978). Auxin and gibberellin ditions should have different cell sizes). This are also exported in the phloem to the means that fruit size can vary due to variations pedicel and peduncle, where gibberellin in both cell number and cell size. It also means induces cell elongation and auxin induces that cell division is comparatively immune to the production and differentiation of environmental influences so that viticultural vascular bundles to ensure that the transport attempts to manipulate berry size will have to capacity of the vascular system does not aim mostly at cell expansion, whereas breeding limit growth of the developing berry. In efforts should address cell division. The maxi- addition, import from the “mother” vine of mum extent of cell expansion, and hence berry the germination-inhibiting hormone ABA size, is limited by the elastic properties of the prevents seed abortion and promotes normal skin, which in turn may be related to the thick- embryo development (Nambara and ness and/or stiffness of the exocarp cell walls Marion-Poll, 2005). This phase lasts 6–9 or the cuticle (Matthews et al., 1987; see also weeks and ends when the skin cells stop 80 2. PHENOLOGY AND GROWTH CYCLE

FIGURE 2.11 Cabernet Sauvignon berry growth over 3 years (left; note 50% anthesis occurred on day 160 in year 1 and on day 150 in years 2 and 3; M. Keller, unpublished data), and beginning of ripening (veraison) in Concord clusters with berries displaying various stages of color change (right; photo by M. Keller).

dividing. By this time, the berry has attained gibberellin production in the seeds at least half its maximal size and fresh (Gutierrez et al., 2007; Nambara and Marion- weight. Poll, 2005). At the same time, there seems to Stage II: The lag phase during which the seed be a fleeting increase in ethylene (though far enters its maturation stage (just after the less pronounced than in so-called climacteric embryo’s genes take over control from the fruits; Chervin et al., 2004) that is mother vine), the embryo grows rapidly, and accompanied by heightened sensitivity to by 10–15 days before veraison the seeds ethylene (Chervin et al., 2008) and a dramatic reach their final size, maximum fresh and persistent rise in brassinosteroids (Pilati weight, and maximum tannin content et al., 2007; Symons et al., 2006), a class of (Adams, 2006; Niimi and Torikata, 1979; growth-promoting hormones probably Pratt, 1971; Ristic and Iland, 2005, Viala and produced in the epidermis. These two Vermorel, 1909). Moreover, the innermost hormones may trigger the changes in cell cells of the outer integument lignify, and wall chemistry that lead to berry softening the outermost cell walls thicken to form a and expansion. Berry turgor pressure also cuticle; the integuments now restrict further declines approximately 10-fold during this seed expansion. Although the pericarp period (Thomas et al., 2006). The length of the grows only insignificantly, the concentration lag phase (1–6 weeks) largely depends on of the growth hormone auxin (and ripening the cultivar and is important in determining inhibitor; Davies et al., 1997) peaks during the time of fruit maturity; late-ripening this phase and then declines sharply cultivars seem to have a long lag phase (Alleweldt and Hifny, 1972; Alleweldt et al., (Alleweldt and Hifny, 1972; Currle et al., 1975; Nitsch et al., 1960). The influx of ABA 1983). Seedless grapes tend to have a less increases toward the end of this phase, pronounced lag phase than seeded grapes suppressing embryo growth by blocking (Iwahori et al., 1968; Nitsch et al., 1960). 2.3. REPRODUCTIVE CYCLE 81

Stage III: The ripening period, which lasts expansion (Matthews et al., 1987). Red, 5–10 weeks. Its onset, which typically occurs purple, or blue pigments called over a period of 7–10 days within a grape anthocyanins accumulate in the exocarp cluster, is termed veraison and signals a (and in “teinturier” cultivars, such as fundamental shift from partly Alicante Bouschet, also in the pericarp), and photosynthetic to wholly heterotrophic sugars (glucose and fructose) accumulate in (Greek heteros ¼ other, different; trophe´ ¼ the pericarp, whereas organic acids (malate) nutrition) metabolism. Veraison is marked and chlorophyll are degraded. Chloroplasts by berry softening and an increase in sugar are converted into chromoplasts. Unlike in content, followed by a rapid change in skin climacteric fruits, the “fruit-ripening” color from green to red, purple or blue in hormone ethylene does not appear to play a dark-skinned cultivars (see Figure 2.11), and prominent role in grape ripening, and berry to more or less yellow in some white respiration apparently declines during this cultivars. Softening (i.e., a decrease in fruit period (Coombe and Hale, 1973; Harris et al., firmness or increase in deformability) 1971). Embryo growth stops early in this mainly arises from the disassembly of the phase (Nitsch et al., 1960; Pratt, 1971), the mesocarp cell walls (Huang and Huang, seeds turn from green to yellow and finally 2001). Although the cell walls remain brown due to oxidation of tannins in the functionally intact, they become more open, parenchyma cells of the outer integument, more hydrated, less flexible, and weaker so and they become hard and desiccated that they can no longer hold the cells (Adams, 2006; Ristic and Iland, 2005). Storage together very well (Brummell, 2006). Water reserves,suchasstarchandlipids,and import through the xylem declines gradually mineral nutrients are accumulated in the while the berry changes color, and water endosperm until the seeds become dormant import via the phloem increases rapidly (Rogiers et al., 2006). Thus, the seed fresh (water being the solvent for the incoming weight declines while the dry weight may sugar). The xylem now probably serves continue to increase until the berry reaches mostly to recycle excess phloem water back its maximum size and weight; seeds that out of the berry (Keller et al., 2006; Patrick, are hard and brown and have attained 1997). Berry turgor pressure is low (<0.5 bar) their maximal dry weight are called but remains positive and relatively constant “mature.” The concentration of ABA throughout ripening (Lang and Du¨ ring, increases rapidly and peaks in the seed 1990; Thomas et al., 2006). This last phase is and berry flesh during seed maturation, characterized by a further increase in berry decreases due to degradation during seed size, which is somewhat unusual because desiccation, and is relatively low in mature the fruit of most other species reach their seeds (Inaba et al., 1976; Lund et al., 2008; final size before ripening begins (Gillaspy Scienza et al., 1978). ABA, which now et al., 1993). The volume increase is initially accumulates mainly as a result of glucose- very rapid but slows progressively toward induced production by the embryo, helps fruit maturity; berry size may plateau or coordinate seed maturation and is necessary decrease due to evaporative water loss to induce seed dormancy, in which during later stages of ripening. The metabolism ceases until the seed is extensibility of the skin temporarily rehydrated for germination (Gutierrez et al., increases at veraison to accommodate berry 2007; Nambara and Marion-Poll, 2005; Wobus expansion, but the skin later restricts further and Weber, 1999). 82 2. PHENOLOGY AND GROWTH CYCLE

In contrast to seeded grapes, seedless grape degradation, facilitates breakage of the seed berries often do not show clearly discernible coat, and induces remobilization of nutrients phases of growth, and their volume increases from the endosperm (Nambara and Marion- much more “smoothly” (Nitsch et al., 1960; Poll, 2005; Olszewski et al., 2002). Winkler and Williams, 1935). The length of The evolution of a fleshy, sweet, and dark- each stage of fruit growth and the final berry skinned berry during the late Cretaceous size depend on the cultivar but are strongly period approximately 100 to 65 million years modified by rootstocks and environmental ago was probably an adaptation to the simul- conditions. Optimum conditions for rapid taneous evolution of birds and mammals berry development are similar to those gov- (Hardie, 2000). Birds are the predominant seed erning the other phases of reproductive dispersers of European wine grapes, whereas development: high light intensity, warm tem- bears are major dispersers of American juice perature, and adequate soil moisture and grapes, whose distinctive aroma birds find nutrient availability (see Chapter 6). unattractive. The only role grape berries play Harvest of grapes by birds or mammals, in the life of a vine is to spread its genes including humans, normally completes the (DNA). The ability to advertise seed maturity reproductive cycle. If grapes remain on the by visual and olfactory cues and reward seed vine for an extended period, the continued cell vectors with energy-rich sugar greatly wall disassembly leading to diminished cell increases the chances for gene dispersal. As wall strength and loss of intercellular adhesion Charles Darwin (2004) remarked, a fruit’s eventually results in tissue failure, cell separa- "beauty serves merely as a guide to birds and tion, and senescence (Considine and Knox, beasts in order that the fruit may be devoured 1979a), which sets the seeds “free”—often and the manured seeds disseminated." Berries aided by infection by opportunistic fungi such accumulate pigments and aroma volatiles to as B. cinerea and others (Brummell, 2006). advertise to potential seed vectors their con- Seeds are ready to germinate at or soon after tent of nutritionally valuable components, veraison (Pratt, 1971; Winkler and Williams, such as sugars, amino and fatty acids, vita- 1935); in fact, it is thought that fruit ripening mins, and antioxidants (Goff and Klee, 2006). is not initiated until seed maturation has been These compounds, in turn, constitute the completed (Cawthon and Morris, 1982; “ticket price” to cover the costs of transporta- Gillaspy et al., 1993). Whereas seeds may ger- tion by means of wings and legs of seed minate immediately if postveraison berries dispersers. Low concentrations of (volatile) drop in a moist environment, they lose this ethanol produced by yeasts, which have been ability within a day upon removal from a living on and in fruits since the Cretaceous, berry, when their water content drops from may also form part of the aroma bouquet of approximately 50% to 20% (Currle et al., ripe berries (Dudley, 2004). Although fruit 1983). Once dehydrated, the seeds require a color is certainly an important attractant dur- stratification period of 4–12 weeks at chilling ing the day, volatiles may be more important temperatures (0–5 C) and 40–50% relative at night, when colors are difficult to see. At humidity to overcome embryo dormancy. thesametime,tanninsandothersecondary Light (especially red light) induces water compoundsmaydetermicrobesthatalso imbibition and subsequent germination by would like to consume the fruit but do not dis- stimulating the embryo cells to produce gib- perse the seeds (Levey, 2004). Likewise, the berellin and expand rapidly, which overcomes high acidity and unpleasant aroma (caused the ABA-related dormancy by accelerating ABA by methoxypyrazines and other volatile 2.3. REPRODUCTIVE CYCLE 83 compounds) of unripe berries prevents the characteristic accumulation of sugars and berries from being eaten before the seeds are other compounds also means that fruit ripen- mature. This strategy enables grapevines to ing is different from the senescence process explore new habitats beyond the small area in leaves that involves disassembly of proteins around an existing immobile plant that simply (called proteolysis) and recycling of nutrients drops its fruit (seed) to the ground. The to other plant parts (Gillaspy et al., 1993). CHAPTER 3

Water Relations and Nutrient Uptake

OUTLINE

3.1. Osmosis, Water Potential, and Cell 3.3. Water and Nutrient Uptake and Expansion 85 Transport 92 3.2. Transpiration and Stomatal Action 89

3.1. OSMOSIS, WATER POTENTIAL, AND CELL EXPANSION channel proteins, called aquaporins, in the mem- branes (Katsuhara et al., 2008; Maurel et al., 2008; Tyerman et al., 1999). In contrast to the diffusion Between 70 and 95% of a grapevine’s fresh pathway, the rate (but not the direction) of water mass consists of water (H2O). Even the woody tis- transport across aquaporins can be fine-tuned by sues of trunk and roots have a water content of active regulation because these proteins are able approximately 60%. Most of this water serves as to open or close their pores in a process called gat- a solvent for ions and organic molecules in the ing. Additional regulation is provided by varying interior of the vine’s cells. Water can diffuse freely, the number of aquaporin proteins in the mem- albeit slowly, across the phospholipid bilayer of branes. Aquaporins are relatively temperature cell membranes, which are approximately 5–10 insensitive and can speed up water diffusion nm thick, and diffusion accelerates with rising across membranes more than 10-fold; in other temperature. More important, however, H2O words, they strongly raise the membranes’ molecules are small enough (0.3 nm diameter) to (osmotic) water permeability (also called hydrau- be able to just barely (i.e., in single file), but lic conductivity). At the same time, most of them extremely rapidly (up to 1 billion H2Omolecules are impermeable to mineral nutrient ions and per second), penetrate pores formed by water even to protons. In fact, cells use protons, in

The Science of Grapevines 85 Copyright # 2010 Markus Keller. Published by Elsevier Inc. All rights reserved. 86 3. WATER RELATIONS AND NUTRIENT UPTAKE

þ addition to calcium ions (Ca2 ) and removal of thermodynamic (or absolute) temperature in phosphate groups (so-called dephosphorylation) Kelvin (K ¼ C þ 273). from the protein, to close aquaporins (Johansson Sugars such as sucrose, organic acids such as et al., 2000; Luu and Maurel; 2005, Maurel et al., malate, and inorganic ions such as potassium þ 2008; Tournaire-Roux et al., 2003). In addition to (K ) and chloride (Cl ) are the major osmotic size exclusion (i.e., filtration), dissolved ions are solutes (osmolytes) of plant cells. Thus, solutes also repelled by charged residues at the entrance have an osmotic function in addition to their þ to the pores; thus, cations cannot pass through metabolic functions. For instance, cells use K a “gate” guarded by a positive charge, and anions to neutralize the negative charge of inorganic cannot pass through a gate guarded by a negative and organic anions (i.e., organic acids) and charge. In other words, membranes are selec- anionic groups of macromolecules because the tively permeable or semipermeable (Latin semi total charge of all anions and cations inside a ¼ half). Specialized gates consisting of transport cell must balance (Clarkson and Hanson, proteins and ion channels are required for 1980). When solutes attract water into a cell, molecules other than H2Otopassthrough the cell swells and causes the cell membrane membranes (see Chapter 3.3). to exert force on the cell wall. This, in turn, Because the concentration of dissolved ions leads to a balancing wall pressure, which raises (from dissociated salts) and organic molecules the energy of the water inside the cell until it inside a cell is almost always higher than that of equals that of water outside. At this point, the the exterior (i.e., cell walls and intercellular space), cell’s internal hydrostatic pressure (more there is relatively less water inside the cell than out- accurately, the pressure difference between side (i.e., the cellular water is “diluted” by the inside and outside of the cell), termed turgor solutes). This water concentration gradient causes pressure (P) but often abbreviated as turgor water to move passively through aquaporins and (Latin turgere ¼ to be swollen), is equal to the across the membrane into the cell, and this move- difference in osmotic pressure between the cell P ¼ p p ment is termed osmosis (Greek osmos ¼ impulse and its surroundings (i.e., inside outside), or thrust). In other words, the presence of solutes and net water influx stops (Figure 3.1). Thus, in a cell exerts a “pull” on the water molecules sur- osmotic pressure is defined as the hydrostatic rounding the cell, and this tension is termed pressure required to stop the net flow of water osmotic potential. The greater the concentration across a membrane separating solutions of of solutes, the more water will move into the cell differing concentration. In other words, osmo- to restore (hydraulic) equilibrium. This leads to a sis can only work as a driving force for water buildup of pressure due to the incompressibility flow (see Chapter 3.3) when two compartments of water and the rigidity of plant cell walls resisting are separated by a semipermeable membrane. an increase in volume. Osmotic pressure (p, Turgor pressure, in turn, is the osmotically expressed in MPa) of an aqueous solution is thus caused by dissolved solutes and increases linearly as the solute concentration (c, expressed in mol L 1; i.e., the number of dissolved molecules per unit volume) increases, and also as the temperature (T) increases, according to the following equation: FIGURE 3.1 During diffusion, solutes (red) and water p ¼ RTc (blue) move in opposite directions (left), whereas during osmosis water moves across a semipermeable membrane R ¼ 1 where is the universal gas constant ( 8.31 J K (brown) to balance the water concentration deficit created 1 1 1 mol ¼ 0.00831 MPa L K mol ), and T is the by the solutes (right). 3.1. OSMOSIS, WATER POTENTIAL, AND CELL EXPANSION 87 maintained hydrostatic pressure inside living as their natural trellis system (see Figure 1.2). plant cells. The turgor pressure of growing Pure water has the highest possible water cells typically reaches approximately 0.3–1 potential, which by convention equals zero MPa—that is, 3–10 times atmospheric pressure (C ¼ 0) at atmospheric pressure and 25C. P ( a 0.1 MPa) (Cosgrove, 1997). Opening sto- Therefore, the water potential of aqueous matal guard cells can generate over 4 MPa; solutions is always negative (C < 0). In other nevertheless, the deformation of these cells words, C is a measure of water concentration, and their cell walls is completely reversible and p is a measure of how much the dissolved (Franks and Farquhar, 2007; McQueen-Mason, molecules, termed solutes, decrease C. The C 2005; Roelfsema and Hedrich, 2005). of a cell describes the potential tension (nega- The solute and pressure forces in plants and tive pressure or suction) that the cell solution soils are conveniently described as free energy exerts on surrounding pure water. In the per unit volume (i.e., equivalent to force per unit absence of other forces, the movement of water area, or pressure) that is generated by the during osmosis is always from a region of lower random movement and collision of molecules. solute concentration (i.e., greater water poten- The free energy of water or, more accurately, of tial) to one of higher solute concentration (i.e., an aqueous solution, per unit molar volume lesser water potential). In other words, osmosis of liquid water (18 mL mol 1), is termed water is driven by a water potential gradient. potential (C, expressed in J m 3 or as its pressure Plant cells use osmotic solutes, especially equivalent Pa ¼ 10 5 bar) and is the sum of the sucrose and potassium, although in ripening component potentials arising from the effects of grape berries it is predominantly the hexoses C ¼ P turgor pressure (i.e., turgor potential, P ) glucose and fructose with smaller amounts of and solutes (i.e., solute potential or osmotic potassium, to lower their C in order to attract potential, Cp ¼p) in addition to interactions water into the cells. Therefore, solutes have a with matrices of solids (e.g., cell walls) and major role in building P, which is necessary C macromolecules (i.e., matrix potential, M) and for cell expansion and hence plant and organ effects of gravity (i.e., gravitational potential, growth. In fact, cell growth is mainly a function C G), as described by the following equation of turgor and the mechanical properties of the (Boyer, 1969): cell walls. Cell expansion is made possible by cell wall loosening and accumulation of solutes C ¼ P p þ C þ C ¼ C þ Cp þ C þ C M G P M G inside the cell vacuole, which draws water into Although the matrix potential is very impor- the vacuole to create turgor pressure. Thus, tant in soils (due to the adhesion of H2O mole- water movement into cells (i.e., across mem- C þ C cules to soil particles) and cell walls, it is very branes) is determined by P p (i.e., it is close to zero inside the cells of well-watered driven by both pressure and osmotic compo- plant tissues and is therefore negligible unless nents). In contrast, water movement outside of the tissue is severely dehydrated (e.g., loss of cells (i.e., through the apoplast; see Chapter 50% of tissue water; see Hsiao, 1973). The 3.3) is driven solely by pressure differences gravitational potential increases with height at (because water does not cross membranes in a rate of 0.01 MPa m 1. Although this may be the apoplast). The P of growing cells is typi- of little significance in the relatively short- cally approximately 0.3–1 MPa, and this pres- statured cultivated grapevines that rarely sure irreversibly extends (i.e., stretches) and exceed a height of approximately 2 m, the “pushes” the plastic cell walls outward C P downward pull of G is a factor in their taller (Cosgrove, 1997). Nonetheless, although causes wild relatives, as well as in the trees serving tension, it does not induce extension of the cell 88 3. WATER RELATIONS AND NUTRIENT UPTAKE walls, which is controlled by the cross-linking which are then converted by the enzyme of xylem-delivered calcium with pectic acids peroxidase into hydroxyl radicals (•OH), which produced by the expanding cells (Boyer, 2009; in turn may attack the bonds between cell wall Schopfer, 2006). Although P must be high polysaccharides (Mller et al., 2007). Slipping of enough to expand the cell, it must also be the parallel microfibrils and production and low enough to sustain water influx (i.e., maintain insertion of new wall material is necessary for a gradient of C). Continued water influx is the expanding cell to enclose its increasing necessary in expanding cells because the incom- volume; cell walls usually maintain their thick- pressibility of water means that P would ness during expansion (Boyer, 2009; Cosgrove, otherwise drop to zero as soon as the cell walls 2005). Because cells must increase in volume become stretched. Indeed, a decrease in P by before they can divide, cell expansion, in turn, is as little as 0.02 MPa can stop cell expansion but also a prerequisite for cell division. As much as not reverse it (Taiz, 1984). 90% of the entire volume gain during plant Cell expansion also requires loosening of the growth is due to the expansion of cell vacuoles cell walls of turgid cells before they can be and concomitant stretching and addition of cell extended (Boyer, 2009; Schopfer, 2006). Wall wall material. Hence, most of the increase in loosening, and thus wall stress relaxation, is volume during cell expansion is due to water thought to be achieved by the “relaxing” activ- uptake by the vacuole, whereas the amount of ity of proteins known as expansins that are cytoplasm increases only insignificantly. activated at low pH (Cosgrove, 1997, 2000, The term water potential is convenient 2005; Li et al., 2003). According to the “acid because it can be applied not only to (cell) growth theory,” the cell wall is acidified by solutions but also to soil solution and air—that þ protons (H ) pumped from the cell interior to is, to any medium with variable water content. the cell wall in exchange for potassium Water potential indicates the availability of þ ions (K ) that maintain the electrical charge water in any aqueous system. The water poten- balance inside the cell (Rayle and Cleland, tial of air can be estimated from the air’s rela- 1992; Stiles and Van Volkenburgh, 2004; Van tive humidity (RH; expressed in percentage). Volkenburgh, 1999). As a consequence, the cell Relative humidity is the amount of water vapor wall pH can temporarily decrease from the in air at a particular temperature in relation to normal 5.5–6 to as low as 4.5 (compared with the total amount the air could hold at that tem- a cytosol pH of 7.5; Kurkdjian and Guern, perature (i.e., the saturation vapor density or 1989), which equates to a more than 10-fold saturation vapor pressure). The water-holding þ increase in H in the cell wall. The ATP- capacity of air (and thus its saturation vapor þ powered H pumps in turn are activated pressure) increases exponentially with incre- by the growth hormone auxin and light (via asing air temperature. In the 10 to 35 C range, phytochromes and other photoreceptors; see an increase in air temperature of 12C doubles Chapter 5.2). Expansins may temporarily the water vapor concentration of saturated air; disrupt the chemical associations (hydrogen that is, air at 22C can hold twice as much bonds) between the parallel cellulose micro- water vapor as air at 10C, and air at 34C can fibrils and the cross-linking xyloglucans hold four times as much. In other words, if (hemicelluloses) in the cell walls, which makes saturated air (100% RH) is heated by 12C, its the walls flexible enough to allow expansion RH drops to 50% (i.e., the air develops a satu- C and insertion of new cell wall material. In addi- ration deficit). The relationship between air tion, auxin also seems to stimulate the production (in MPa) and RH (in %) can be approximated of reactive oxygen species (see Chapter 7.1), by the following equation: 3.2. TRANSPIRATION AND STOMATAL ACTION 89

TABLE 3.1 Typical Values of RH and C for Leaves leaf depends on two major factors: the differ- and Atmosphere at 25C ence in absolute water vapor concentration, or water potential, between the leaf air spaces RH (%) C (MPa) C C ( leaf) and the external air ( air) and the resis- r Air spaces in leaf 99 1.4 tance ( ) of this pathway to diffusion. As water evaporates from the mesophyll cell walls and is Boundary layer 95 7.0 discharged as water vapor into the atmosphere Atmosphere 100 0 (see Figure 1.14, Chapter 1.3), it meets a series 90 14.5 of resistances that work in the opposite 50 95.1 direction of its movement. A resistance slows 10 315.9 the H2O molecules down and thus limits their rate of diffusion. The major ones are the resis- tance at the stomatal pore and the resistance C : T ð = Þ due to the thin film of still and relatively moist air 0 46 ln RH 100 air at the surface of the leaf (the so-called where T is the thermodynamic (or absolute) boundary layer), but the stomatal resistance r temperature in K. ( s) is usually much greater than the boundary- C r As this equation shows, of saturated air is layer resistance ( b). The boundary layer (also always zero (ln1 ¼ 0), regardless of temperature, known as the unstirred layer) acts like an whereas C of drying air drops dramatically additional (cuticular) membrane in series, but (Table 3.1). Even relatively humid air exerts an wind increases turbulence in the boundary r enormous tension on the water inside a plant layer, making it thinner and decreasing b, which r or in the soil. This tension is responsible for increases transpiration dramatically even as s transpiration (see Chapter 3.2). In other words, declines (Freeman et al., 1982). On the other r transpiration is also driven by a water potential hand, b can be very high in dense canopies so gradient. Incidentally, the relationship between that transpiration of such canopies may be C r temperature and air is also responsible for the determined mainly by b and is not easily so-called rain shadow effect of the Cascades influenced by atmospheric conditions. Trans- mountain range in eastern Washington and piration rate per unit leaf area (E; expressed in Oregon and the Andes range in Mendoza mol m 2 s 1) increases as the leaf-to-air vapor (Argentina). As moist air from the Pacific Ocean concentration difference (i.e., the driving force) rises up the western slopes of the mountains, increases, and it decreases as the total resistance it cools and drops excessive moisture as rain- increases. This relationship can be described by fall or snow so that the saturation deficit (or the following equation, which is analogous to evaporative demand) of the now drier air Ohm’s law (I ¼ Vr 1,whereI is the current, increases further as the air warms down the and V is the electrical potential difference or eastern slopes of the mountains. voltage) used in electricity and thermodynamics: E ¼ DCðr þ r Þ1 s b 3.2. TRANSPIRATION AND DC ¼ C C STOMATAL ACTION where leaf air. Water vapor concentration is proportional to water vapor pressure, and the difference in The evaporation of water from plants is water vapor pressure (actually the vapor termed transpiration (Latin trans ¼ on the other pressure difference divided by the atmospheric side, spirare ¼ to breathe). Transpiration from a pressure) between the inside and the outside 90 3. WATER RELATIONS AND NUTRIENT UPTAKE of the leaf (i.e., the water potential gradient, DC) causes stomata to close partially to protect the is often called the vapor pressure deficit. As dis- xylem conduits from cavitation (see Chapter cussed in Chapter 3.1, an increase in air tempera- 3.3). This effect can override the influence of ture strongly increases the air’s water-holding high light intensity on stomatal opening so that capacity and thus results in a greater vapor even relatively mild soil water deficit in a vine- pressure deficit. The steeper concentration gradi- yard often leads to a midday depression in ent stimulates water evaporation from the leaf stomatal conductance (Du¨ring and Loveys, g mesophyll. Therefore, temperature is an impor- 1982). This temporary decline in s is usually tant determinant of the transpiration rate, and not observed in well-watered vines, except for transpiration increases linearly with increasing vines growing in hot climates (Downton et al., temperature. 1987; Williams et al., 1994). The aperture of the In a typical grapevine leaf, only 5–10% of the stomatal pores also decreases as air humidity total transpiration occurs across the cuticle, declines (i.e., as the vapor pressure deficit although the cuticular portion can reach up to between leaf and air increases), although this 30% in old leaves that have low overall transpi- could be a response to transpiration rather than ration rates compared with younger leaves to humidity per se. As would be expected from (Boyer et al., 1997). The remainder of the water the preceding “transpiration equation,” this vapor escapes through the stomatal pores, even stomatal closure can compensate for variations C though they cover less than 5% of the leaf sur- in humidity, holding transpiration and leaf face. Stomata are thus very important for regu- constant. Nevertheless, a decrease in relative lating diffusional water loss from a leaf. They humidity from 85 to 45% (at constant tempera- function like a pressure regulator, limiting ture) can decrease the transpiration rate by C pressure changes (i.e., leaf) by controlling flow more than 80% via partial stomatal closure. rate (transpiration), and respond very sensi- A rise in temperature initially increases stomatal tively to environmental variables (Sperry et al., aperture due to its stimulating effect on photo- 2002). The most conspicuous of these responses synthesis, but very high temperature leads to is the light-induced opening of stomata at sun- stomatal closure, possibly due to its stimulating rise and their closing at sunset in response to effect on respiration (see Chapter 5.2). An photosynthetically driven changes in the sub- increase in leaf nitrogen also leads to a wider stomatal CO2 concentration (Raschke, 1975; stomatal aperture because nitrogen also stimu- Roelfsema and Hedrich, 2005). This minimizes lates photosynthesis (see Chapter 5.3), but this water loss at night, when there is insufficient effect levels off at high nitrogen content. light energy to fuel photosynthesis (see Chapter Stomata are hydraulically driven gas valves 4.1) and hence no need for CO2 diffusion into because, as discussed in Chapter 1.2, they open the leaf (see Chapter 4.2). During the course of when the guard cells take up water and swell, the day, stomatal opening (i.e., stomatal con- causing their turgor pressure to rise (Franks g ¼ r 1 ductance, s s ) normally follows the daily and Farquhar, 2007). This clever mechanism is change in light intensity, which peaks around possible because the guard cells lack plasmo- midday (Du¨ ring and Loveys, 1982). Similarly, desmata connecting them to neighboring stomatal aperture is wider in bright light than epidermis and mesophyll cells; that is, they in dim light such as prevails under cloudy con- are symplastically isolated (Wille and Lucas, ditions or in the interior of dense canopies 1984). The osmotic uptake of water is driven (Shimazaki et al., 2007). Conversely, stomata close by a DC (see Chapter 3.1) caused by movement þ when a leaf runs out of water. Therefore, low of potassium ions (K ) from surrounding cells C þ leaf (indicating water stress; see Chapter 7.2) into the guard cells across K channels in 3.2. TRANSPIRATION AND STOMATAL ACTION 91

þ exchange for hydronium ions (H3O ) or pro- 2009). In other words, ABA modifies the per- þ tons (H ), which are driven out by a light- meability of the guard cell membrane by way þ þ activated pump called H -ATPase embedded of a Ca2 signal. in the plasma membrane (Roelfsema and The reaction of stomata to ABA is rapid Hedrich, 2005; Shimazaki et al., 2007). The (within approximately 10 min) and indepen- þ þ C reverse exchange (K out/H in) causes water dent of leaf (although ABA is also produced C to diffuse out of the guard cells so that the in the leaves in response to decreasing leaf) decrease in turgor causes the stomata to close. and vapor pressure deficit, and it does not þ The H may come from the organic acid involve starch degradation (Roelfsema and malate, which is produced from starch stored Hedrich, 2005). However, it is related to an in the guard cell chloroplasts and, courtesy of increase in the pH of the xylem sap (from the its double-negative charge, also serves as the “normal” pH 5–6 to 6.5–7), which enhances þ counterion to K . The starch, in turn, is pro- ABA delivery to the guard cells by reducing duced by guard cell photosynthesis or from uptake of ABA by intervening tissues (Bacon sucrose imported across the cell walls from et al., 1998; Hartung and Slovik, 1991; Wilkinson the underlying mesophyll cells (Lawson, and Davies, 2002). In other words, at higher 2008). In addition, sucrose derived from starch pH, less ABA becomes “trapped” inside the usu- þ breakdown also supplements K as an osmoti- ally more alkaline parenchyma and phloem cells cum, especially in the afternoon (Roelfsema lining the xylem vessels. It appears that a and Hedrich, 2005; Shimazaki et al., 2007; decrease in the flow rate of xylem sap leads to Talbott and Zeiger, 1996). Thus, guard cells an increase in the sap’s pH, perhaps because actively, and rapidly, regulate stomatal opening there is more time for the adjacent cells to and closing via a dynamic balance of starch/ remove protons from the sap (Jia and Davies, þ þ malate and H /K . The hydraulic effect of 2007). Even when only a portion of the roots C leaf on stomatal opening or closing can be experience dry soil, the ABA produced by that amplified by abscisic acid (ABA), which is portion is sufficient to trigger strong stomatal produced from carotenoids in dehydrated cells, closure despite the remainder of the roots C vascular tissues, and guard cells (Nambara and taking up enough water to maintain high leaf Marion-Poll, 2005; Wilkinson and Davies, 2002). (Comstock, 2002; Lovisolo et al., 2002a; In addition to the ABA produced in drying Stoll et al., 2000). When rainfall or irrigation leaves, the amount arriving from the roots can replenishes soil moisture, the roots stop increase considerably in response to reduced producing ABA, whose content in the xylem soil moisture (Bray, 1997; Davies and Zhang, therefore decreases rapidly. Nonetheless, the 1991; Davies et al., 2002). Thus, ABA acts as a transpiration-driven rise in water flow up the messenger from the roots, indicating water xylem after such water supply may “flush” stress when the soil dries out (see Chapter 7). residual ABA to the leaves, where it may keep C As soil declines, the roots produce increasing the stomata partially closed for 1 or 2 days, per- amounts of ABA, which is transported in the haps to enable any embolisms that may have xylem sap to the leaves’ guard cells (Loveys, formed in the xylem to be repaired (Lovisolo 1984). ABA triggers stomatal closure by induc- et al., 2008; see Chapter 3.3). Because the ABA ing an increase in reactive oxygen species that, that is present in the leaves is quickly degraded þ in turn, lead to a brief rise in calcium (Ca2 ) (mainly to phaseic acid; Nambara and in the cytosol, which blocks influx into and Marion-Poll, 2005), the stomata can then reopen. þ enhances efflux from the guard cells of K The turnover of water in a rapidly transpiring (Allen et al., 2001; McAinsh and Pittman, leaf occurs within 10–20 min; that is, the leaf 92 3. WATER RELATIONS AND NUTRIENT UPTAKE loses the equivalent of its entire water content than the leaf temperature. Nevertheless, every 10–20 min (Boyer, 1985; Canny, 1993). even in bright sunlight, leaves can radiate Although transpiration is sometimes described 50–80% of the energy they absorb. as a necessary evil (due to a plant’s dependence Convection: Heating or cooling of ambient air on gas exchange), it has some useful side effects. via sensible heat—that is, heat that can be For instance, the negative pressure (tension) in felt and measured with a thermometer. The the xylem created by transpiration aids in energy of molecules on the leaf surface is extracting water from the soil (see Chapter 3.3) exchanged with that of air molecules with that can be used for plant growth. This, in turn, which they are in direct contact. The attracts nutrients, which can then be taken up by warmed air becomes lighter and rises, which the roots. Although the increased flow of water leads to cooling. Convection is driven by during transpiration leads to a corresponding the temperature difference between the air increase in the rate at which dissolved nutrients and the leaf, and the boundary layer is the move upward in the xylem conduits, trans- main resistance to this process. Wind piration is not necessary for this nutrient move- decreases the thickness of the boundary ment (Tanner and Beevers, 2001). In fact, plants layer, which increases convective heat can absorb and transport nutrients just as well transfer and thus convective cooling. at night when transpiration is minimal as they Transpiration: Release of latent heat during do during the day. More important, evaporation the vaporization of water into the air, also of water is a powerful cooling process, which referred to as transpirational cooling. Water is why this principle is also used in air evaporation consumes energy called the conditioning. This heat loss prevents leaves latent heat of vaporization (2.45 kJ g 1 at exposed to sunlight from overheating. The 20 C) because water vapor contains more temperature inside actively transpiring leaves energy than liquid water. In other words, is ordinarily no more than 2 or 3C higher than evaporation removes heat that cannot be felt the ambient temperature. Nevertheless, leaf or measured with a thermometer. temperature can rapidly fluctuate by more than Transpiration is driven by the water 10C in direct sunlight (Sharkey et al., 2008): potential difference between the inside of a Water evaporation cannot completely prevent leaf and the outside. The water potential “heat spikes.” Grapevines have several cooling depends on temperature (which in turn tactics to lose (dissipate) excessive heat gained depends on solar radiation) and relative from absorbed solar radiation. The three humidity. main pathways are as follows (Mullins et al., 1992; Taiz and Zeiger, 2006): Radiation 3.3. WATER AND NUTRIENT : Direct transfer (re-radiation) of heat UPTAKE AND TRANSPORT as long-wavelength (infrared) radiation to surrounding objects or the sky. The percentage of incoming solar radiation Although the main force driving water reflected by a surface is termed albedo. movement through plants is water pressure, Leaves absorb ultraviolet, visible, and water uptake by the roots and transport to the infrared radiation and radiate infrared leaves is largely brought on by passive forces energy, which is a cooling process. This because the hydraulic system of plants has no happens mostly during the night, when the moving parts (Tyree and Zimmermann, 2002). temperature of the surrounding air is cooler The direction of movement is always from high 3.3. WATER AND NUTRIENT UPTAKE AND TRANSPORT 93 to low pressure or, if the movement is across grapevines oscillates with a minimum at night membranes, from a region with low solute con- and a maximum at approximately midday centration to one with high solute concentra- and why vines with a large canopy sustain tion. As discussed in Chapter 2.2, water higher flow rates than do small vines (Tarara absorption and movement in early spring is and Ferguson, 2006). The transpirational pull driven by positive root pressure induced by is possible because water forms a continuous remobilization of stored nutrients and starch system from evaporating surfaces in the leaves, and release of osmotically active ions and shoots, tendrils, and flower and fruit clusters to organic molecules into the xylem sap. Under absorbing surfaces in the roots and out into the these conditions, water moves from the soil soil. This system is termed the soil–plant–air into the root by an osmotically generated gradi- continuum. The chemical and physical proper- ent (difference per unit distance) of water ties of water (i.e., high surface tension, cohesion potential and is then pushed up the vine or the strong tendency of H2O molecules to through the xylem. Although an osmotic gradi- “stick” together by forming hydrogen bonds ent (Dp)—that is, a gradient in solute concentra- between the negatively charged oxygen and tion—has virtually no effect on the flow rate of the two positively charged hydrogen atoms, water inside the xylem because vessels have no and strong adhesion of H2O molecules to the membranes, it drives water inflow into the surfaces of xylem conduits) keep it in this con- xylem from the soil across the root cell mem- tinuum so that water exiting the leaves by tran- branes (Wegner and Zimmermann, 2009). This spiration is continually replaced by water being Dp usually collapses after leaf expansion pulled up the xylem from the roots (Steudle, (although it can remain important at night or 2001; Tyree and Zimmermann, 2002). The tran- during rainfall or overhead irrigation), and spiration–cohesion–tension theory of xylem water loss to the atmosphere by transpiration sap flow states that the evaporation of water from the unfolding leaves begins to hydrauli- from cell walls inside the leaf creates tension, cally produce a negative pressure (tension) in and adhesion of water to the walls of the xylem P the xylem ( x), which maintains the upward helps counteract gravity (i.e., the gain in gravi- flow of water in the vine. At night or under tational potential energy with increasing light conditions low enough to lead to stomatal height). Due to cohesion, the tension extends closure, such as during overcast periods, and down the xylem, into the roots, and out into P with plentiful soil water supply x can remain the soil. Therefore, plants directly utilize solar positive, albeit below atmospheric pressure energy (which causes water to evaporate from P ( a 0.1 MPa at sea level), but drops below the leaves) to drive water uptake and distribu- P < zero (i.e., x 0 MPa) as soon as the stomata tion. Again, the water moves along a gradient open even slightly to let water vapor escape of water potential (from high to low). This and declines as light intensity and transpiration gradient is intermediate within the root rise (Wegner and Zimmermann, 2009). xylem (0.3–2.5 MPa m 1 with lower values in Approximately 95–98% of all water absorbed well-watered and higher values in water- by the roots is lost to the atmosphere in the pro- stressed vines), gentle within a shoot (0.1–0.5 cess of transpiration; very little water is MPa m 1), and quite steep within the petiole required to build the plant body through cell (1–5 MPa m 1) of a transpiring leaf (Lovisolo expansion. Thus, transpiration is the main et al., 2008). driving force for water uptake and movement For xylem sap to sustain the tension required in the xylem up the vine to the leaves. This is in a grapevine to pull water from the soil, the why water flow through the trunk of hydraulic system has to be air-tight. The water’s 94 3. WATER RELATIONS AND NUTRIENT UPTAKE surface tension acts like a seal, keeping water removal), or if it receives some other physical inside the xylem and air out. However, the xylem shock (e.g., a bump by machinery), it snaps is a vulnerable pipe, and water is saturated with back like a piece of elastic in a rigid tube. This air at atmospheric pressure (Steudle, 2001; Tyree phenomenon is termed cavitation, in which the and Zimmermann, 2002). If the water column water column breaks and fills with H2O vapor inside the xylem is put under too much tension or air bubbles (Figure 3.2). The air can be sucked (e.g., excessive transpiration due to drying wind to adjacent vessels through the holes of the or heat combined with dry soil), if the tension is interconnecting perforation plates. Such gas suddenly relieved (e.g., by shoot, leaf, or cluster blockages are called embolisms and render the

End wall of vessel element with bordered pits

Gas-filled cavitated vessel Scalariform perforation plate

Pit

Liquid water

FIGURE 3.2 Cavitation of xylem vessel by water vapor bubble (left; reproduced from Taiz and Zeiger, 2006), bleeding of xylem sap during budbreak (top right); and guttation at night during the growing season (bottom right; photos by M. Keller). 3.3. WATER AND NUTRIENT UPTAKE AND TRANSPORT 95 vessels nonfunctional; in severe cases, this can Trifilo` et al., 2003). Moreover, the vessels and lead to leaf wilting and even canopy collapse tracheids in leaves and petioles are narrower (Schultz and Matthews, 1988a; Tyree and and much shorter than those in the rest of the Zimmermann, 2002). For the gases to dissolve plant, and the pits in the adjoining end walls P and conduits to refill with liquid water, x must of these tracheary elements normally do not rise to within approximately 0.1 MPa of atmo- allow air to pass into the next vessel—that is, P spheric pressure or above (i.e., x 0 MPa) pits function as hydraulic safety valves (Choat (De Boer and Volkov, 2003; Sperry et al., 2002, et al., 2008). This prevents spreading of embo- 2003). More accurately, gas emboli dissolve at lisms in the xylem network following leaf P > tr1 t x 2 (where is the surface tension of damage and sacrifice, a protective mechanism water, and r is the radius of curvature of the dubbed the “lizard’s tail” strategy (Tyree and air–water interface). If cavitation occurs in the Zimmermann, 2002). Finally, the high number trunk or shoots, repair normally requires of parallel tracheary elements makes it unlikely positive root pressure or starch remobilization that the entire transport pathway is interrupted in the surrounding xylem parenchyma cells at any one time. The fact that roots, trunks, and loading of sugar into the vessels to decrease and shoots are not simply hollow pipes ensures the osmotic potential (Cp) of the xylem sap. that some residual water flow persists under These metabolic processes require an input of all but the most severe drought conditions energy to proceed. In addition, the parenchyma (see Chapter 7.2), and this flow can rapidly cells may also release water into the vessels by rehydrate the leaves upon restoration of soil opening the aquaporins in their cell membranes moisture (Lovisolo et al., 2008). (Lovisolo and Schubert, 2006). Vitis species are The root xylem of grapevines is much more thought to be able to generate root pressure vulnerable to cavitation than their shoot xylem >0.1 MPa, which is sufficient to push water to (Lovisolo and Schubert, 2006; Lovisolo et al., more than 10 m above ground level (Tibbetts 2008). This can be a problem with shallow root and Ewers, 2000). Nonetheless, embolism repair systems because roots in the surface soil are the C in grapevines may require both a rise in leaf and first to experience dry soil, and their failure can a cessation of xylem flow so that it typically trigger leaf wilting and canopy collapse. If dee- occurs only at night, provided soil moisture is per roots are present, cavitation in the surface high enough, or during rainfall (Holbrook et al., roots will simply shift water uptake down to 2001). The trunk and shoots, at least after wetter soil layers so that shoot damage can be they have produced some lignified secondary avoided (Sperry et al., 2002). Moreover, the xylem, are much less prone to cavitation than expansive mycelium network created by sym- the petioles and minor veins in the leaves biotic mycorrhizal fungi also facilitates water (Choat et al., 2005; Lovisolo et al., 2008; Nardini uptake into the roots (Stahl, 1900) and helps et al C ., 2001). However, leaf recovers very avoid cavitation by osmotic water “lifting,” rapidly upon water supply, which implies that which is possible due to the high carbohydrate only a fraction of the vessels are cavitated at concentration in the fungi supplied from any one time. the plants. Plants, it seems, can also reciprocate Although the minor veins can suffer from the “favor” at night by transferring water from embolisms even during an average sunny day, the roots to the symbionts (Querejeta et al., the high concentration of inorganic ions and 2003). In a “compensation game” fungal coloni- organic molecules in the leaves can generate zation of the roots tends to increase as soil the positive pressure needed to refill the veins moisture, and hence root growth, decreases relatively easily (De Boer and Volkov, 2003; and vice versa (Schreiner et al., 2007). The fungi 96 3. WATER RELATIONS AND NUTRIENT UPTAKE

DC ¼ C C r can even transfer water, as well as sugar and where soil leaf, h is the hydraulic l nutrients, between neighboring plants (includ- resistance, and h is the hydraulic conductance. ing grapevine–weed transfer) either directly Inside the xylem conduits (vessels and trac- (through the mycelium connecting the roots of heids), the osmotic component of C is insigni- two plants) or indirectly (through uptake of ficant because there are no membranes in the water that was released by the roots of another pathway; hence, water flow follows a pressure et al DC DP plant) (Egerton-Warburton ., 2007). difference over distance (i.e., x). Some- Grapevines growing under conditions of times a negative sign is added on the right side moderate water deficit develop narrower xylem of the equation to indicate that water flows in vessels than vines growing with abundant the opposite direction to that of DC—that is, water supply (Lovisolo and Schubert, 1998; from high to low C (Tyree and Zimmermann, Mapfumo et al., 1994b). Narrower vessels might 2002). Although this equation is surely an over- be a consequence of curtailed supply of assi- simplification, it is quite convenient to explain milates to the cambium resulting from lower most phenomena involved in water flow r photosynthesis due to higher s. Although the through plants. The hydraulic conductance is r smaller vessel diameter increases h, which a measure of transport capacity or water may lead to reduced water loss and increased permeability. At the whole-plant level, it deter- C cavitation resistance, this effect cannot be very mines leaf at any specific transpiration rate. large because the few large vessels typically The term “conductance” should not be confused L K present in grapevines account for the majority with “conductivity” ( h or ), which refers to r DP of the s (Tibbetts and Ewers, 2000). The vulnera- the water flow normalized to over a specific x l ¼ L x1 bility to cavitation also varies among species length ( ) of the flow path (thus, h h ). L and cultivars (Schultz, 2003); less vulnerable In addition, “specific conductivity” ( s) applies cultivars (e.g., Syrah) probably have denser and to the cross-sectional area (A) of a unit flow L ¼ L A1 stronger wood and less permeable pits (i.e., path ( s h ) and is roughly proportional more resistant xylem) than more vulnerable to the number of xylem conduits passing cultivars (e.g., Grenache, which nevertheless through that cross section and the fourth thrives in hot, arid, and windy conditions). power of their radius (r4; the rate of water The flow of water from the soil toward the flow per unit cross-sectional area is termed roots is maintained by suction at the root flux, J ¼ FA1) (Tyree and Zimmermann, L L surface, which is caused by root pressure (i.e., 2002). Therefore, h and s are independent of l osmotically) or transpiration (i.e., hydrostati- the total length of the pathway, whereas h r cally). As transpiration removes water, the soil decreases (and h increases) with increasing adjacent to the roots (rhizosphere) becomes path length (e.g., increasing plant height). The r drier than the more distant (bulk) soil. Thus, term h reflects the resistance to water flow water flow to the roots, and from the roots to due to friction between water and conduit the leaves, is caused by a water potential walls and between the H2O molecules. It is a gradient. In analogy to Ohm’s law (and to the measure of how much a component of the flow transpiration equation discussed previously), path hinders the free flow of water and thus the flow rate (F; volume per unit time) of water slows water flow. There are both short-distance from the soil to the leaves can be described by radial and long-distance axial or longitudinal r the following equation: components of h; the axial resistance to water flow is orders of magnitude lower than the F ¼ DC r 1 ¼ DC l ðbecause l ¼ r 1Þ r h h h h radial resistance. The radial h is the resistance 3.3. WATER AND NUTRIENT UPTAKE AND TRANSPORT 97 water encounters when it flows across the root given vine but varies for several reasons. In r into the xylem and out of the xylem into the long term, h usually increases with increas- r leaf and fruit cells, much of it imposed by cell ing plant size and age (as does s), but it membranes and regulated by aquaporins decreases with increasing shoot number on oth- (Maurel et al., 2008; Vandeleur et al., 2009). erwise similar vines. In the short term, a rise in r r Most of the axial h is imposed by the vine’s temperature decreases h (by approximately “plumbing layout,” termed hydraulic architec- 2–2.5%/C due the decrease in water viscosity), ture, which is mainly determined by the num- which enables more rapid water delivery for ber, shape, size, and arrangement of xylem transpirational cooling (see Chapter 3.2) during conduits and their interconnections (e.g., pits), heat periods. Moreover, transport of nutrient þ as well as the total length of the flow pathway ions, especially of cations such as K , may r and the number and shape of bends in the decrease h by increasing pit permeability, pathway (i.e., plant size and shape) (Tyree possibly due to ion-induced shrinking of the and Zimmermann, 2002). Due to their small gel-like pectin matrix (“hydrogel”) in the so- size and fine pore meshwork, however, the pits called pit membranes, which are specialized connecting individual xylem conduits usually modifications of the primary cell wall, rather r account for more than 50% of a plant’s total h than proper cell membranes, with pores et al r < m et al (Choat ., 2008). The increase in h with 0.2 m (van Ieperen ., 2000; Zwieniecki et al increasing plant height partly accounts for the ., 2001). Nitrate (NO3 ), but not other tendency of photosynthesis to decrease as anions such as phosphate (H2PO4 ) or sulfate 2 r plants grow taller because their leaves must (SO4 ), also decreases h, perhaps by altering close their stomata earlier (i.e., at less severe the status of aquaporins either directly or via C water deficit) to maintain leaf above a species- a rise in pH brought about by NO3 assimila- and cultivar-specific minimum (Hubbard et al., tion (Gloser et al., 2007; Gorska et al., 2008; see 2001; Ryan and Yoder, 1997). In other words, also Chapter 5.3). This means that increasing the photosynthesis in tall vines, such as those concentration of some nutrients in the xylem climbing in trees, may be at least partly sap increases the sap flow rate (and hence hydraulically limited. Note, however, that water uptake by the roots) and, conversely, that growth is more sensitive to water stress than nutrient deficiency strongly reduces sap flow is photosynthesis, so it may be more correct to even under well-watered conditions. It also say that growth, rather than photosynthesis, is means that vines may be able to actively, et al r hydraulically challenged (Ryan ., 2006; see rapidly, and reversibly modify h by altering also Chapter 5.2). Moreover, the larger vines’ the concentration of ions in the xylem sap, for more extensive root system and their usually instance, by remobilizing/resorbing nutrients greater trunk diameter tend to counter this in the surrounding parenchyma cells. hydraulic limitation trend. Grapevines sustain very rapid water flow up As in electricity, resistances encountered in their trunks that matches the canopy trans- series (e.g., in the roots, trunk, cordon, shoots, piration rate plus the water “bound” by the r ¼ r þ r þ and leaves) are additive (i.e., h h1 h2 growing tissues. The flow rate in field-grown ... þ r hn), whereas for those acting in parallel vines has been estimated to fluctuate daily from (e.g., several shoots on the same cordon) it is low values at night (<0.1 L h 1) to high values the individual conductances that are additive during days with clear sky (1–3 L h 1), but r 1 ¼ r 1 þ r 1 þ ... þ r 1 (i.e., h h1 h2 hn ). Never- it decreases to 10–20% of clear-sky values r theless, whole-plant h is not constant for a during rainy or overcast days (Tarara and 98 3. WATER RELATIONS AND NUTRIENT UPTAKE

r r Ferguson, 2006). Other estimates, however, are which in turn is correlated with s. Thus, h considerably lower at 2 or 3 L day 1 averaged defines how wide the stomatal pores can be over the growing season (Currle et al., 1983). open without desiccating the leaves (Brodribb r Aquaporin activity in the root cell membranes, and Holbrook, 2003; Jones, 1998). A larger h r C and hence radial h, approximately matches will result in a greater decrease in leaf when the diurnal oscillations in flow rate to enable transpiration increases. Water will flow from C appropriate water uptake by and flow across the soil to the vine as long as xylem is lower et al C the roots (Luu and Maurel, 2005; Maurel ., than soil, and it will flow from the xylem to et al C C 2008; Vandeleur ., 2009). The rate of sap the leaf as long as leaf is lower than xylem. C flow also varies with canopy size and water Even in well-watered vines, leaf can fluctuate availability: For instance, maximum values widely during the day (by as much as 1.5 may be approximately 1 or 2 L h 1 in deficit- MPa, depending on cultivar; see Chapter 7.2) irrigated Cabernet Sauvignon (declining to following both opening and closing of stomata <0.5 L h 1 as the soil dries down), but can at dawn and dusk and depending on the evap- reach 3 or 4 L h 1 in well-watered Concord orative demand (i.e., vapor pressure deficit) of et al C (Dragoni ., 2006; Tarara and Ferguson, the air. These changes in leaf (i.e., changes in 2006). A pressure gradient >10 kPa m 1 is C of the cell walls) must be balanced by required to maintain such flow rates in the changes in turgor pressure for the leaf cells to trunk. Rapid transpiration favors hydrostatic maintain volume and solute concentration, (and hence apoplastic) water flow through the which is achieved due to vacuoles and rigid r root and thus reduces h (Steudle, 2000, 2001; but elastic cell walls. Because transpiration- C Steudle and Peterson, 1998). Conversely, when driven water flow is minimal at night, leaf there is little or no transpiration, such as at reaches a maximum (Schultz and Matthews, r night or with dry soil, h increases substantially 1988a). In other words, soil water status deter- C because water flow is driven by osmotic gradi- mines the baseline leaf in the near absence of ents (i.e., across membranes). Of course, cavita- transpiration (Tardieu and Simonneau, 1998). r tion in the xylem also greatly increases h—to As a soil dries, the extraction of water by the infinity in the affected vessel. If cavitation is roots and transport to the shoots become to be avoided, the flow of water from the soil increasingly more difficult for grapevines. To to the leaves must balance the water lost maintain the water potential gradient that through the stomata. By combining the transpi- drives water flow, the C in the shoot must C ration equation with the flow equation, this can decrease. Therefore, the predawn leaf can be C be written as follows: used as a robust indicator of the soil to which ðC C Þðr þ r Þ1 ¼ðC C Þr1 the roots are exposed. Transpiration during the leaf air s b soil leaf h C day decreases leaf below the predawn value C As long as transpiration rate and soil-to-leaf so that leaf at a particular time of day is the C flow rate remain constant, leaf will remain result of both soil water status and transpira- C constant. For a given microclimate ( air), which tion driven by evaporative demand (Smart, determines the vapor pressure deficit, and a 1974; Tardieu and Simonneau, 1998). The water C r r given soil water status ( soil), b and s deter- potential equation introduced in Chapter 3.1 mine the transpiration rate, whereas whole- also applies to the soil solution, although, in r C C vine h determines leaf at that transpiration contrast to plant cells, the p of the soil water rate (Tyree and Zimmermann, 2002). In other is generally negligible (approximately 0.01 C C words, the difference between soil and leaf MPa), except in saline soils (see Chapter 7.3). r C is determined by the transpiration rate and h, Consequently, soil is determined mainly by 3.3. WATER AND NUTRIENT UPTAKE AND TRANSPORT 99

C M, which is close to zero in wet soils but can soil–root–shoot pathway. Although nutrients decrease to 3 MPa in dry soils due to the are often concentrated in the biologically active surface tension resulting from expanding air surface soil, water and nutrient availability var- spaces. In drying soil, a point will eventually ies greatly in both space and time. To compli- be reached at which the resistance to water cate matters further, different nutrients are flow is so great that the vine can no longer often not readily available in the same location. DC maintain a sufficient to sustain transpiration For example, NO3 diffuses through the soil þ and becomes drought stressed (see Chapter 7.2). approximately 10 times faster than K and 500 Drought stress can also be induced by low soil times faster than H2PO4 , so percolating water temperature, which increases the resistance to moves and drains (i.e., leaches) nitrate down water uptake and flow inside the roots (i.e., it to the subsoil much more rapidly than other r increases the root’s radial h), ostensibly due to nutrients. As a consequence, superficial roots closure of aquaporins (Maurel et al., 2008). This often take up soil-immobile nutrients (e.g., can be a problem when cold irrigation water is potassium and phosphorus) from the topsoil, applied to rapidly transpiring vines, especially whereas deeper roots tap water and soil-mobile at high atmospheric vapor pressure deficit nutrients (e.g., nitrate) that leach deeper into (see Chapter 3.2). The resulting imbalance the soil profile. These deep roots can also trans- between water uptake by the roots and water port water to the surface roots in a process loss from the leaves can induce cavitation called hydraulic lift or hydraulic redistribution, in the xylem, which may lead to leaf wilting which occurs mostly at night when transpira- and injury (Scheenen et al., 2007). When vines tion is minimal (Bauerle et al., 2008a). Deep are grown in pots, especially in dark pots roots need not even be grapevine roots; some heated by the sun, whose soil temperature can cover crops might also be able to lift water to be several degrees above air temperature, app- the topsoil, where it may be available to the lying cold irrigation water can inhibit growth vines, although this does not apply to covers (Passioura, 2006). dominated by grass species (Celette et al., Water influx is proportional to the surface 2008; Patrick King and Berry, 2005). Hydraulic area of the root system (Steudle, 2001). redistribution keeps the surface roots alive Grapevines often have dense root systems in and, by locally increasing soil moisture due to C < C the topsoil and can extract water effectively water loss from the roots when soil root, from the surface soil layers. Extracting water even enables them to sustain nutrient uptake from lower soil layers is more difficult so that in drying soil. In addition, roots can passively the surface soil dries more quickly than the move water laterally (horizontally) and down- subsoil. Under nonirrigated conditions, roots ward (e.g., when rainfall or irrigation make continue to grow into deeper, wetter soil layers, the surface soil wetter than the subsoil) accord- C et al whereas the roots of irrigated plants proliferate ing to local gradients of soil (Bauerle ., mostly in the topsoil; in both cases, the C of the 2008a; Smart et al., 2005; Stoll et al., 2000). Water advancing root tips remains high as long as redistribution by roots tends to even out local the roots can find water (Hsiao and Xu, 2000). differences in soil moisture (e.g., due to varia- Root water uptake shifts to deeper soil layers tions in soil texture or organic matter), delays as the soil dries and back to the upper layers soil drying, and may assist in maintaining after rainfall or irrigation. symbiotic mycorrhiza. However, water that is Because plant-available nutrient ions are lost from vine roots by diffusion can also be dissolved in the soil solution, nutrient uptake reabsorbed by competitors (e.g., some weeds also depends on water flow through the or cover crops). 100 3. WATER RELATIONS AND NUTRIENT UPTAKE

Epidermis

Casparian band

Endodermis Root hair

Vessel

FIGURE 3.3 Water and nutrient flow from the soil through a root into the xylem via the symplastic (top blue line) and apoplastic (bottom blue line) pathways. Illustration by A. Mills and M. Keller.

As shown in Figure 3.3, water that enters the Steudle, 2001). In other words, cell walls resem- root initially moves through the epidermis and ble dense aqueous gels. Nonetheless, because cortex tissues along both symplastic (or sym- cell walls comprise only approximately 5% of plasmic) and apoplastic (or apoplasmic) routes a tissue’s total volume, they have very limited (Steudle, 2000, 2001; Steudle and Peterson, water storage capacity. In addition, the cell 1998). The symplast is everything inside the walls’ dense network of microfibrils also acts plant that is bound inside a membrane (i.e., like a sieve (pore sizes of 4–14 nm) because it the interconnected cytoplasm of the cells); thus, permits passage only to small molecules such the symplastic pathway is an intracellular path- as nutrient ions, amino acids, and sucrose. way. Within the symplast, water and solutes Water on an apoplastic route flows around the move from cell to cell via small connecting cell membranes; hence, the apoplastic pathway pores called plasmodesmata (Lough and Lucas, is an extracellular pathway. Because there are 2006). Short-distance cell-to-cell movement of no membranes along the apoplast, hydraulic, solutes within the symplast thus occurs by dif- nonselective water flow dominates along this fusion but is also influenced by osmotic gradi- pathway (Steudle, 2000, 2001). Rather than ents across membranes, and long-distance moving exclusively in one or the other com- movement between symplastic domains occurs partment, a third possibility for water flow is by mass flow through the interconnecting straight across cell walls, membranes, and phloem (see Chapter 5.1). The apoplast, on the through cells. In other words, water moves other hand, is everything that is outside the alternately in the apoplast and the symplast. membrane and includes cell walls, intercellular This route is termed the transcellular pathway spaces, and xylem conduits. Cell walls consist and is probably the preferred pathway for of a porous framework of the hydrophilic (i.e., water on its way to the xylem. However, the wettable; from Greek hydros ¼ water, philos ¼ endodermis cells that separate the cortex from loving) polymers cellulose and hemicellulose the stele have thickened, hydrophobic radial (matrix glycans) embedded in a polymer and transverse cell walls called Casparian matrix of pectin, lignin, and proteins, which is strips or Casparian bands, which act like a imbued with water like a sponge so that gasket due to their impregnation with lignin approximately 65–75% of the cell wall is water and suberin (Clarkson, 1993; Steudle, 2000). (Carpita and Gibeaut, 1993; Cosgrove, 1997; This waterproofing blocks diffusion and 3.3. WATER AND NUTRIENT UPTAKE AND TRANSPORT 101 hydraulically separates the cortical and stelar inward rectifying or outward rectifying. apoplasts, forcing water and dissolved nutrient Inward-rectifying channels allow ions to move ions to pass directly through the cell mem- into cells, whereas outward-rectifying channels branes into the cell interior—that is, into the facilitate ion movement out of cells (Tyerman symplast. Thus, on their way to the xylem, et al., 1999). Such one-way movement water and solutes must pass at least two cell allows channels to function like valves, which membranes (i.e., in and out of at least one cell), prevents unnecessary nutrient loss. Because of regardless of whether the initial route is sym- their ability to control the passage of ions, plastic or apoplastic (Tester and Leigh, 2001). membranes also act as electrical isolators, Unlike cell walls, cell membranes are semi- which leads to electrical potential gradients permeable (see Chapter 3.1); that is, they are, across membranes (negative charges typically in principle, impermeable to solutes, including dominate inside the cell) in addition to concen- even small ions such as protons. In reality, they tration gradients. The sum of the gradients of act like selective sieves, permitting entry to concentration and of electric charge of an ion some ions and not to others. The degree of is called the electrochemical potential gradient. selectivity or semipermeability is termed the Some channels, termed ion channels, are reflection coefficient(s), which can vary from specific for cations in general, and some ion 0 to 1. Membranes with s ¼ 0 behave nonselec- channels are specific for particular cations, tively and are equally permeable to water and whereas solute carriers are responsible for the solutes, whereas at s ¼ 1 membranes retain transport across membranes of neutral solutes. solutes completely and have high water but Some neutral solutes (e.g., urea and boric and no solute permeability (Steudle and Peterson, silicic acid) and gases (e.g., CO2 and NH3) 1998). A high s in the endodermis ensures, may also move through aquaporins (Maurel for instance, that nutrients taken up by the et al., 2008). A channel is called specific if it is roots do not leak back out into the soil solution, selective, or discriminates, for a particular mol- which also permits the buildup of root pressure ecule or group of molecules such that other under conditions of slow transpiration. Pores molecules cannot pass through the channel, created by special channel and transport pro- regardless of whether their movement is pas- teins embedded in the membranes regulate sive or active. Ions moving passively simply the passage of water and nutrient ions, and diffuse across channels down the electrochemi- each cell type or tissue seems to be equipped cal potential gradient. As with water flow, the with a unique set of these proteins. Many chan- direction of movement is from high to low nel proteins, especially in the parenchyma tis- potential, typically from high to low concentra- sue around vascular bundles, are specific for tion. Ions moving actively must be transported water (aquaporins; see Chapter 3.1) and control (pumped) across membranes by transport pro- water flow by opening or closing (Maurel et al., teins called transporters or carriers against their 2008; Steudle, 2000). Thus, they control the rate electrochemical potential, ordinarily from low but not the direction of the flow, which simply to high concentration (Grossman and Takaha- and passively follows the DC (Tyerman et al., shi, 2001). Active movement typically draws þ 1999). For instance, aquaporins close at night on a proton (H ) gradient across the membrane but open during the day; this opening and requires an input of energy in the form of r decreases the radial h, which enables the roots ATP, which is hydrolyzed to ADP and phos- to meet the water demand for transpiration. phate (Gilroy and Jones, 2000; Lalonde et al., In contrast with the bidirectional aquapor- 2004). However, rather than burning ATP ins, many ion channels are said to be either directly for nutrient transport, the ATP is used 102 3. WATER RELATIONS AND NUTRIENT UPTAKE

þ to power pumps, termed H -ATPases, which reached after nitrogen fertilizer applications þ export H from the cell interior to the apoplast and are relatively common for potassium; þ þ at a rate of approximately 1 H per “consumed” moreover, the passive uptake of NH4 and þ ATP (Britto and Kronzucker, 2006; Sondergaard Na can sometimes result in ion toxicity (see et al., 2004). The resulting decrease in apoplast Chapter 7.3). Some transporters (e.g., those for þ pH (to pH 4 or 5) generates an electrochemical NO3 and K ) can function in both active and gradient of -50 to 200 mV that, in turn, fuels passive mode, depending on the external con- nutrient uptake via the membrane’s channels centration, and anion channels can also trans- þ and carriers. Thus, H -ATPases act as ener- port organic acids (Britto and Kronzucker, gizers that convert the chemical energy released 2006). Although controlled ion uptake is also by ATP hydrolysis into chemiosmotic energy; important to maintain a neutral electrical the resulting ADP is then recharged by respira- charge balance in the plant, uptake rates of tion. Whereas in the so-called antiport one cations and anions are rarely precisely equal; þ proton is pumped out of the cell for each charge roots restore neutrality by releasing H , HCO3 , of an incoming cation, two protons are needed or OH into the soil immediately surrounding per charge of an incoming anion that then them, termed the rhizosphere (Clarkson and enters together with the backflowing protons in Hanson, 1980). In addition, the high uptake the so-called proton cotransport or symport capacity of the passive channels is usually (Amtmann and Blatt, 2009). accompanied by nutrient efflux from the roots. Active transport enables grapevines to Whereas anions can leak back passively in concentrate nutrient ions inside the roots well exchange for protons, cations must be pumped above their concentration in the surrounding back out actively (Britto and Kronzucker, 2006). soil solution (Keller et al., 1995, 2001b), Therefore, because either uptake or efflux is although some concentration is also possible active, even passive nutrient uptake could be simply due to the electrical potential gradients called active in terms of energy expenditure (Amtmann and Blatt, 2009). Most ions occur for the related active efflux. For most nutrient in the soil water at much lower concentration ions save phosphate, this efflux increases than would be required for plant cellular with rising external concentration and becomes functions, and without a concentration mecha- almost equivalent to ion uptake at high nism nutrient uptake would not be possible. soil nutrient concentration, resulting in 2 Whereas anions (e.g., NO3 ,SO4 ,orCl )must considerable—and apparently futile—nutrient always be taken up actively against their elec- cycling across the membranes (Britto and trochemical potential gradient, some cations Kronzucker, 2006). þ þ þ (e.g., K ,NH4 ,orNa ) are taken up passively Once inside the stele, most water and dis- across relatively generic (i.e., nonselective) ion solved nutrients ultimately return to the apoplast channels when their availability in the soil solu- when the water is released into the xylem tion is high and actively and very selectively conduits across the pit membranes separating across carriers when availability is low (Tester the vessels and tracheids from the surrounding and Leigh, 2001; Ve´ry and Sentenac, 2003). For parenchyma cells (see Chapter 1.3), which most macronutrients, the carriers become themselves have acquired the solutes via their saturated at approximately 1 mM in the soil plasmodesmata from other parenchyma cells or solution, and the channels are switched on from the cell wall apoplast (Sattelmacher, 2001; when the nutrient concentration rises above Sondergaard et al., 2004). Because the cell walls this threshold (Britto and Kronzucker, 2006). of the xylem conduits are waterproof around the Such high nutrient concentrations may be pits, release into the xylem is another bottleneck 3.3. WATER AND NUTRIENT UPTAKE AND TRANSPORT 103

r of high radial h in the vine’s hydraulic system the nonxylem apoplast, but this diffusion is at after the passage through the endodermis. In least one order of magnitude slower than the addition, the presence of aquaporins and selec- movement in the xylem (Kramer et al., 2007). tive channel or transport proteins in the paren- This is because cations interact with, and chyma cell membranes provides a chance to hence are slowed by, the negatively charged car- actively control access to the xylem (De Boer boxyl groups of the cell wall pectins that func- and Volkov, 2003; Tester and Leigh, 2001). Such tion like a cation exchanger and also because controlled release and reversible exchange the pores in the apoplast hinder the passage of between parenchyma cells and xylem conduits solutes and increase their path length (Sattelma- also enables the vine to modify the xylem sap cher, 2001). composition and hence to fine-tune nutrient The long-distance transport of water in the supply with the changing demand by the shoots. xylem, the so-called transpiration stream, Once inside the xylem conduits, the tension occurs by bulk flow, in which large numbers P (negative x) induced by transpiration and the of H2O molecules move together, and is r very low axial h of the tracheary elements driven by a hydrostatic pressure gradient 1 DP facilitate rapid transport of water (up to 2 m h ( x) caused by transpiration, which “sucks” during a sunny day) and its dissolved nutri- water up the vine against gravity and friction. ents to the shoots, where they are delivered The flow rate increases exponentially (actually initially to the apoplastic compartment of to the power of 4) with increasing diameter of transpiring organs. the xylem conduits, and so does the velocity of Even with the concentration mechanism dis- the flow (actually to the power of 2), due to the r cussed previously, xylem sap is very dilute decreasing h with increasing diameter. Thus, (i.e., it has a low p) compared with phloem sap the large vessels formed in spring can trans- (see Table 5.1) and the contents of shoot and port water much more rapidly than the leaf cells. Nevertheless, due to the transpiration narrower ones formed during summer. More- stream, it transports large amounts of nutrient over, older vines with more annual rings and ions by mass flow, although the nutrient con- a larger trunk diameter, and hence more large centration can fluctuate severalfold between vessels, are able to transport much more water day and night due to the diurnal change in to support a larger leaf area than can young transpiration rates. Lower concentrations are vines, and their generally deeper and more associated with higher flow rates, which in turn extensive root system is able to access soil are associated with greater water uptake by the moisture at deeper layers, which is especially roots; despite the lower concentration, how- important later in the season as the soil dries ever, the total nutrient flow to the shoots is down from the top. The roots, trunks, and cor- greater because transpiration-driven water dons also act as important water storage com- influx is strongly coupled with nutrient uptake partments; that is, they behave as hydraulic (Wegner and Zimmermann, 2009). In addition capacitors. These reservoirs, which are also to nutrient ions, the xylem can also transport called hydraulic capacitance, are depleted in- sugars, especially during springtime remobili- the morning, when the stomata open and zation of storage reserves as discussed previ- transpiration rises, and replenished in the ously, as well as other organic molecules such afternoon (Schultz and Matthews, 1988a; as amino acids as nitrogen carriers (see Chapter Steppe and Lemeur, 2004). Depletion and 5.3), organic acids as carriers of metal ions (see replenishment of the internal water reserves Chapter 7.3), and hormones such as abscisic leads to daily cycles of shrinkage and expan- acid and cytokinins. Solutes can also diffuse in sion of the trunk diameter. Older vines can 104 3. WATER RELATIONS AND NUTRIENT UPTAKE store more water due to their higher number nonxylem) resistance predominates. This of xylem (and phloem) parenchyma cells; this means that the distance between the veins and buffering capacity could make them less vul- the evaporative surfaces is directly correlated r nerable to xylem cavitation so that they can with, and controls, leaf h, which in turn limits cope with drought stress better than young photosynthesis (Brodribb et al., 2007). Uptake vines. Moreover, the xylem parenchyma cells of water and solutes into the leaf mesophyll mayalsobeabletoabsorbnutrientionssuch cells from the xylem vessels occurs initially as nitrate and potassium during periods of through pits (from the apoplast back into the abundant supply and release them back into symplast of neighboring parenchyma cells) the xylem in times of starvation. In other and then through plasmodesmata or, across words, these cells may serve as short-term the cell membranes, via aquaporins (for water) storage reserves of both water and nutrients. and energy-dependent carrier proteins (for The xylem constitutes more than 99% of the solutes) that are stimulated by the growth total length of the transpiration stream; this hormone auxin. Therefore, the mesophyll is proportion increases with increasing plant size somewhat hydraulically isolated from the (Sperry et al., 2003). Nevertheless, a few dozen remainder of the transpiration stream. micrometers in the roots and in the leaves exert During the day, grapevines lose most of the r the vast majority of whole-vine h, even though absorbed water in transpiration, but a small the entire transpiration stream can be several fraction (1%) is used for cell expansion, cell r meters long, and h increases with increasing metabolism, and phloem transport. At night, transport distance (i.e., with increasing plant when the stomata are essentially closed and height) (Sperry et al., 2002). Thus, one of two transpiration is drastically reduced, those small major resistances to water flow through a amounts may become the dominant component grapevine is not in the trunk or shoots but in of water flow. In fact, water is circulated the roots, where the radial (i.e., nonxylem) between the phloem and xylem, and this circu- rather than axial (i.e., xylem lumen) resistances lation maintains water flow in the xylem in the dominate (Steudle and Peterson, 1998); within absence of any significant transpiration, even the xylem, the pit membranes between adjoin- at night (Ko¨ckenberger et al., 1997; Windt ing vessels and tracheids can account for half et al., 2006). The water taken up to balance this r et al or more of the total h (Choat ., 2008). The “growth water” and phloem counterflow other main resistance is in the leaves, at the ter- amounts to a transpiration-independent water minal section of the transpiration stream, flow that is sufficient to transport nutrients where the water flows through orders of veins absorbed by the roots; hence, nutrient uptake in series and in parallel, leaves the xylem net- and long-distance transport in the xylem are work at the veins across and around the bundle not dependent on transpiration (Tanner and sheath parenchyma encircling the veins, and Beevers, 2001). Of course, the increase in moves into and around the mesophyll cells water flow due to transpiration results in a before it evaporates into the air spaces, chang- corresponding increase in the speed at which ing from the liquid to the vapor phase, and dif- dissolved molecules move up the xylem (Peuke fuses out of the stomata (Comstock, 2002; Sack et al., 2001; Wegner and Zimmermann, 2009). and Holbrook, 2006; Sack et al., 2003; see also Rapid transpiration thus increases nutrient r Chapter 1.3). In a transpiring grapevine, h of uptake by the roots, especially when nutrient the leaves accounts for approximately one- availability in the soil is high (Alleweldt et al., fourth of the whole-plant resistance to water 1984a; Hsiao, 1973; Keller et al., 1995). Over flow, and within the leaf the post-vein (i.e., the course of a day, however, root water uptake 3.3. WATER AND NUTRIENT UPTAKE AND TRANSPORT 105 must exceed transpiration sufficiently to also DC is required to sustain growth (Schultz and supply water for growth because plant growth Matthews, 1993). If water ceases to move out is mainly caused by cell expansion, which of the xylem, shoot and leaf growth stops results almost entirely from an increase in the immediately because cells must take up water cell’s water content (Boyer, 1985; Hsiao and to expand, whereas root growth is less affected Xu, 2000; Schopfer, 2006). Water import into (Boyer and Silk, 2004; Hsiao and Xu, 2000; Wu C cells is driven by cell wall loosening and and Cosgrove, 2000). Lower xylem is also accumulation of solutes inside the cell and thus responsible for the smaller berry size of water- depends on a water potential gradient between stressed vines. Moreover, it explains why pre- DC ¼ C the cell and the supplying xylem ( xylem veraison water deficit has a larger effect on C cell; see Chapter 3.1). Therefore, expanding berry size than post-veraison water deficit: C tissues normally have lower cell than mature Water influx into the berry changes at veraison (nongrowing) tissues. Because growth com- from predominantly via the xylem to pre- petes with transpiration for xylem water, cells dominantly via the phloem (see Chapters 1.3 C must also maintain lower cell during the day and 6.2). Photosynthesis of mature leaves is less than at night if they are to sustain expansion affected by decreasing C than is expansion of during the day (Boyer and Silk, 2004). Indeed, young leaves (Hsiao and Xu, 2000), and solute grape berries, which must remove water from transport in the phloem can continue at the transpiration stream surging up the shoot decreasing C. As a consequence, the import of and past the fruit clusters, grow mostly at solutes may exceed their use so that solutes night, especially before veraison and when the often accumulate in sink organs, such as roots water demand by the transpiring canopy is or berries, of vines that experience mild water high (Greenspan et al., 1994, 1996; Matthews deficit. Therefore, berries grown on mildly and Shackel, 2005). water-stressed vines are not only smaller but Growth rates of all tissues change rapidly also have higher sugar content. Yet the C with fluctuating xylem (i.e., with extracellular decrease in photosynthesis under more severe water status), and growth is extremely sensitive water stress leads to a reduction in berry sugar to water deficit (Boyer, 1985; Boyer and Silk, (see Chapter 7.2). As in the berries, some of the 2004; Hsiao and Xu, 2000). Shoot growth of water for root growth comes from the phloem C grapevines declines with decreasing xylem in addition to direct influx from the soil (Boyer C and stops completely when xylem around mid- and Silk, 2004). Moreover, because xylem cells day reaches approximately 1.0 to 1.1 MPa. are differentiated behind the growing root tip, C Because water stress decreases xylem, a major water uptake for transpiration also occurs cause for growth inhibition under water deficit behind the expanding cells. Due to this hydrau- may simply be the smaller DC, which reduces lic isolation, the root tip usually experiences water uptake by the expanding cells (Nonami slightly higher soil moisture than the mature et al., 1997). Moreover, due to the small, imma- root. All of this makes root growth somewhat C ture xylem conduits near the shoot tip and in less vulnerable to fluctuations in xylem and r young petioles and expanding leaves, the h in ensures that it is favored over shoot growth the “growth zone” is much greater than in the when the soil dries (Boyer and Silk, 2004; Hsiao mature portion of the shoot so that a steeper and Xu, 2000). CHAPTER 4

Photosynthesis and Respiration

OUTLINE

4.1. Light Absorption and Energy 4.4. Respiration 118 Capture 107 4.5. From Cells to Plants 121 4.2. Carbon Uptake and Assimilation 111 4.3. Photorespiration 117

4.1. LIGHT ABSORPTION AND enables us to describe light energy as a stream of ENERGY CAPTURE energy-carrying particles called quanta (singular: quantum) or photons. The window of the spectrum called visible Photosynthesis is the most important way of light, because our eyes are sensitive to it, also obtaining energy for all plants including grape- happens to be the range of wavelengths vines. As the name suggests, photosynthesis (approximately 400–700 nm) that is important (Greek photos ¼ light, synthesis ¼ building a for photosynthesis. This region is only a small whole) is a process by which sunlight is con- portion of the entire spectrum of electromag- verted to chemical energy that is used to pro- netic radiation, and other organisms can see duce (synthesize) organic compounds within light of other wavelengths. Insects and birds, the plant from inorganic compounds acquired for instance, often distinguish between objects from outside the plant. Sunlight is a form of such as flowers or eggs according to their color electromagnetic radiation that has properties of patterns in the ultraviolet (UV) range, and both waves and particles. The wave nature of plants can detect both UV and infrared radia- light can be visualized with a prism separating tion in addition to visible light (see Chapter a ray of visible or white light into a continuous 5.2). All forms of electromagnetic radiation array of different colors according to their travel at the speed of light (c 300,000 km s 1), wavelengths (Figure 4.1). Light’s particle nature but the energy content (E) of photons is higher

The Science of Grapevines 107 Copyright # 2010 Markus Keller. Published by Elsevier Inc. All rights reserved. 108 4. PHOTOSYNTHESIS AND RESPIRATION

the conversion of CO2 (which is discussed in Chapter 4.2) occurs in the fluid chloroplast matrix referred to as stroma. This chapter pro- vides a brief summary of the most important aspects of the photosynthetic processes. Because of their importance for life on Earth, the majority of these processes are virtually identical in all plants and are covered in great FIGURE 4.1 A portion of the electromagnetic spectrum. detail in general texts on plant physiology, Reproduced from Taiz and Zeiger (2006). from which most of the following information is derived (e.g., Atwell et al., 1999; Salisbury the shorter their wavelength (l), or the higher and Ross, 1992; Taiz and Zeiger, 2006). their frequency (u), within the spectrum. This Before the light energy can be used, the light can be written as follows: must first be absorbed and translated into a flow of electrons that are derived from the E ¼ hu ¼ hcl 1 splitting of water. The green chlorophyll is where h is the Planck constant (6.63 10 34 Js). the main light-absorbing pigment that makes The energy contained in the photons is light energy capture possible. It is the world’s exploited by plants during photosynthesis, most abundant pigment. Chlorophyll is green which proceeds in two stages. The first is the because it absorbs predominantly blue (430 nm) photochemical capture of solar energy, trans- and red (680 nm) light and reflects most of ported in the form of photons, and its tempo- the green light with wavelengths intermediate rary storage in high-energy chemical bonds of between blue and red (Figure 4.2). In plants, 0 adenosine 5 -triphosphate (ATP; the universal chlorophyll comes in two slightly different biological energy currency) and nicotinamide versions called chlorophyll a and chlorophyll adenine dinucleotide phosphate (NADPH). b, both of which, along with various other The second stage uses this energy to enzymati- pigments called accessory pigments (e.g., caro- cally convert carbon dioxide (CO2) and water tenoids such as carotenes and xanthophylls), (H2O) to sugar (carbohydrate), which in turn are located in the thylakoid membranes. When is used to produce all other organic compounds a chlorophyll molecule absorbs a photon, it is throughout the plant. Of course, in the absence converted from its low-energy state, called of light there is no energy source for photosyn- ground state, to an excited state by abruptly thesis, and there is no carbohydrate produc- shifting one of its electrons from a lower tion. The two stages of photosynthesis occur energy atomic orbital, or shell, close to the in different regions of specialized organelles atomic nucleus to a more distant, higher termed chloroplasts that contain the photosyn- energy orbital. This instantaneous electron thetic machinery. Although the cells of all “jump” from one energy level to another is green plant organs and tissues have chloro- termed a quantum leap. The excited chloro- plasts, the leaves are their main residence, and phyll is extremely unstable and returns to its they are well designed to intercept the maxi- ground state within nanoseconds (10 9 s) by mum amount of light (see Chapter 1.3). The releasing its available energy as either heat or conversion of light energy into chemical energy fluorescent radiation (in which the photon is happens in the membranes of small, disc- transferred to xanthophylls and reemitted), or shaped, stacked sacs called thylakoids (Greek by transferring either the energy or an electron thylakos ¼ sac) inside the chloroplast, whereas to another pigment molecule. 4.1. LIGHT ABSORPTION AND ENERGY CAPTURE 109

FIGURE 4.2 Structure of the chlorophyll a molecule (left) and absorption spectra for different light-absorbing pigments (right).

Approximately 250 chlorophyll molecules are times faster (i.e., in picoseconds, 10 12 s, grouped together in the so-called photosystem II making it one of the fastest known chemical reac- (PSII), draped on the framework of a multiprotein tions) than the other processes. The chlorophyll complex in the thylakoid membrane. The major- molecule replaces the lost electron (e ) by captur- ity of these chlorophylls, called antenna pig- ing an electron from water, which is thereby oxi- ments, are responsible for absorbing photons dized (i.e., split), releasing oxygen as a waste þ (i.e., for “harvesting” light) and transferring their product and a proton (H ). This water-splitting ! þ þ þ energy from one pigment to another until it reaction (2H2O 4H 4e O2)iscatalyzed arrives at a single specialized chlorophyll mole- by a protein complex that contains manganese, cule called the reaction center. The antenna pig- calcium, and chloride ions at a ratio of 4:1:1 ments transfer only their excitation energy so (Nelson and Yocum, 2006). Thus, water is the that the reaction center is the only chlorophyll source of all electrons (and protons) involved þ molecule that actually releases an electron in a in photosynthesis (both e and H are later mechanism called photochemistry. It wins out incorporated into a sugar molecule by the in the race of possible energy-releasing mechan- enzyme rubisco; see Chapter 4.2); for each elec- isms, and thus makes photosynthesis possible, tron that is released, one water molecule is con- because it occurs at a rate approximately 1000 sumed and its oxygen released to the air (the 110 4. PHOTOSYNTHESIS AND RESPIRATION oxygen in photosynthetically produced sugar recycles all protons back to the chloroplast therefore comes from CO2,notH2O). stroma, is called photophosphorylation. The electron released from the reaction center In essence, energy transfer and photochemis- chlorophyll is transferred to an acceptor molecule try convert light energy into chemical energy in in a process termed photochemical quenching the form of NADPH and ATP, which provide because it “quenches” the excitation energy of the energy for carbon assimilation, as well as the chlorophyll (Baker, 2008). The acceptor in for the assimilation of nitrogen (see Chapter turn passes the electron on to a secondary accep- 5.3), phosphorus, and sulfur and other meta- tor and so on down a cascade of steps called the bolic processes in the chloroplast (Baker, electron transport chain until it arrives at the reac- 2008). The proportion of absorbed photons that tion center of a second photosystem, confusingly are used for photosynthesis is termed quantum named photosystem I (PSI), operating in series yield or quantum efficiency, and it decreases as with PSII. PSI is an extremely efficient nano- incident light intensity increases (Baker, 2008). photoelectric machine that is also composed of a Although inevitable, the release of oxygen so multiprotein complex and contains approxi- close to the PSII reaction center is problematic mately 175 chlorophyll molecules, which also because excited chlorophyll can cause this oxy- absorb light and transfer its energy (Nelson and gen to be transformed into the very reactive 1 Yocum, 2006). The electron is transferred from singlet oxygen ( O2), which can damage mem- the excited PSI reaction center to yet more branes and cause mutations or cell death by acceptors until it is accepted by an iron sulfur- oxidizing lipids, proteins, and DNA (Apel and containing protein called ferredoxin, which passes Hirt, 2004; Halliwell, 2006; Mller et al., 2007). þ it to NADP that is thereby reduced to NADPH. In other words, chlorophyll is a “Jekyll and The protons produced during the oxidation Hyde” molecule, enabling the crucial light of water are released into the thylakoid’s inte- absorption for photosynthesis but acting as a rior, the so-called thylakoid lumen (Baker, phototoxin when it becomes overly excited 2008). In addition, the electron transport chain (Ho¨rtensteiner, 2009). Excessive oxygen is toxic between PSII and PSI also “pumps” protons due to the formation of free radicals termed from the stroma into the thylakoids using some active (or reactive) oxygen species (see Chapter of the energy released by the electrons “flow- 7.1). Fortunately, under normal conditions the ing” down the chain. This results in a relative carotenoids (especially the yellow xanthophyll shortage of protons in the stroma (pH 8), creat- pigments violaxanthin, antheraxanthin, and ing an electrochemical potential (i.e., a pH dif- zeaxanthin) associated with the reaction center ference equating to a 3000-fold difference in and antenna complex quickly “scavenge” sin- protons) across the thylakoid (pH 4.5) mem- glet oxygen in addition to dissipating excess brane. This so-called proton motive force consti- light energy absorbed by the antenna pigments tutes a source of energy that powers another and converting it into heat. This dissipation is protein complex in the thylakoid membrane, termed non-photochemical, energy-dependent called ATP synthase or ATPase, to produce quenching or thermal dissipation, and it ATP from ADP and inorganic phosphate (Pi): employs the so-called xanthophyll cycle, which þ ! ADP Pi ATP. Working like a turbine in a converts violaxanthin via antheraxanthin to hydroelectric power plant, each rotation of the zeaxanthin when there is too much light but enzyme ATP synthase releases three molecules runs in the reverse direction when light inten- of ATP while transferring 14 protons back out sity declines (Baker, 2008; Demmig-Adams to the stroma (i.e., the “waterfall” or flow of pro- and Adams, 1996; Noctor and Foyer, 1998). tons drives the “turbine”). This process, which This is why the amount of antheraxanthin and 4.2. CARBON UPTAKE AND ASSIMILATION 111 zeaxanthin in grapevine leaves often rises in takes place inside the leaf cells, CO2 must move the morning and declines in the evening, from the atmosphere to the leaf interior. This whereas violaxanthin follows the opposite happens by way of diffusion through the sto- trend (Du¨ ring, 1999). In addition, the carote- mata (see Chapter 1.3), then through the inter- noids also assist with light harvesting by cellular air spaces, and finally into cells and absorbing photons at wavelengths not covered chloroplasts. As discussed in Chapter 3.3, the by chlorophyll (Bartley and Scolnik, 1995). rate of diffusion is dependent on a driving When the photosynthetic pigments absorb force (a potential gradient or concentration gra- more light energy than can be converted to chem- dient) and the resistance (the inverse of con- ical energy and used in CO2 assimilation, the ductance) to diffusion. The diffusion path for “energy overload” will damage the photosyn- CO2 is almost entirely through the stomata, thetic apparatus (especially PSII) by “knocking whereas the path for water vapor involves both þ out” manganese ions (Mn2 )fromtheprotein the stomata and the cuticle (Boyer et al., 1997). complex and inactivating the reaction center. CO2 diffusion through the stomata follows the Such damage is an unavoidable ancillary cost of same path as H2O during transpiration (E)in the business of doing photosynthesis and the reverse direction (see Chapter 3.2), but the increases in proportion to the light intensity path for CO2 is longer because it must cross (Takahashi and Murata, 2008). It reduces the effi- additional cell walls and membranes (i.e., the ciency of photosynthesis and is called photoinhi- plasma membrane and the chloroplast mem- bition, which also is a form of non-photochemical branes) before it can be assimilated in the chlor- quenching (Baker, 2008; Gamon and Pearcy, oplasts. Thus, although CO2 may be able to use 1990). The damaged proteins are normally certain aquaporins (which are normally degraded (i.e., they undergo proteolysis) and reserved for water transport and hence have recycled to build new PSII proteins; thus, photo- been renamed cooperins if they are permeable inhibition results from the balance between dam- to CO2) to cross membranes, it meets more age and repair. When the photosynthetic fixation points of resistance than does water (Evans of CO2 is limited, the demand for ATP and et al., 2009; Katsuhara et al., 2008; Maurel et al., NADPH declines. Oxygen can seize the resulting 2008). Moreover, the concentration gradient of surplus electrons from PSI, especially in strong CO2 from the outside air to the leaf’s interior light, and the ensuing superoxide is converted is very small: The current atmospheric CO2 to hydrogen peroxide (H2O2). Such reactive oxy- concentration (Ca) is approximately 0.038% gen species strongly inhibit the repair process (¼ 380 mmol mol 1 ¼ 380 mbar ¼ 380 ppm), by interfering with the assembly of proteins whereas the concentration inside the leaf (Ci) (Takahashi and Murata, 2008), which can cause cannot be less than 0%. Grapevines literally problems under conditions of environmental have to extract their main source of food and stress that curtails CO2 fixation (see Chapter 7.1). energy out of thin air. By contrast, air at 20 C and 50% relative humidity contains approxi- 4.2. CARBON UPTAKE AND mately 1.25% H2O, whereas the air inside the ASSIMILATION leaf is at almost 100% relative humidity and contains approximately 2.5% H2O. This means that the gradient for H2O diffusion out of the Just as it does for other plants, uptake of CO2 leaf is more than 40 times steeper than that by leaves presents a special dilemma for for CO2 diffusion into the leaf. To make grapevines. Because CO2 is available in the matters worse, H2O molecules are smaller than atmosphere around plants and photosynthesis CO2 molecules, so H2O diffuses approximately 112 4. PHOTOSYNTHESIS AND RESPIRATION

FIGURE 4.3 Relationship between photosynthetic CO2 uptake and transpirational H2O loss in mature leaves of two grapevine cultivars (M. Keller, unpublished data).

1.6 times more easily through air than does CO2. In the dark or under drought conditions, the As a consequence, the “exchange rate” of mature stomata are almost closed (gs 0), and photo- leaves is only approximately 1 CO2 molecule synthesis ceases. However, as discussed previ- fixed for every 200–600 H2O molecules lost; the ously, opening of the stomata not only allows current value depends on cultivar and soil CO2 to diffuse into the leaf but also allows conditions (Figure 4.3). This exchange rate is also water vapor to escape from the leaf. Thus, the termed water use efficiency (WUE ¼ AE 1). dilemma for the vine is that it cannot simulta- Leaves develop fewer stomata when they neously maximize CO2 uptake and minimize unfold under elevated atmospheric CO2 con- H2O loss. Once CO2 has entered the chloro- centrations (Woodward, 1987); the unfavorable plast, the chemical energy generated by the exchange rate between CO2 and H2O might be chloroplast pigments is used to convert (assim- the cause of this adaptive response. ilate) CO2 and water into carbohydrate (e.g., The net uptake of CO2 by the leaf equals the the hexose sugar glucose C6H12O6). In the over- net rate of photosynthesis per unit leaf area (A; all process of photosynthesis, the leaf absorbs m 2 1 usually expressed in mol CO2 m s ) and CO2 molecules and releases O2 molecules as a can be approximated by the following formula: waste product of the process (see Chapter 4.1), as shown in the following equation: A g1DC 6CO þ 12 H O ! C H O þ 6O þ 6H O where DC ¼ (C C ), and g is the leaf 2 2 6 12 6 2 2 a i l D ¼þ 1ð ¼ Þ conductance. G 2840 kJ mol 1 mol glucose 180 g The term gl reflects the combined gas con- The incorporation of CO2 into carbohydrate is ductances of the boundary layer (gb) and the achieved by the action of 13 enzyme proteins in stomata (gs) and increases with increasing sto- the Calvin cycle (named after one of its matal aperture (see Chapter 3.2). As with water discoverers, the American biochemist Melvin flow, conductance is the reciprocal of resistance Calvin, but also called photosynthetic carbon (g ¼ r 1). The stomata are open in the light, and reduction cycle or reductive pentose phosphate CO2 diffuses into the leaf and into the meso- pathway) located in the chloroplast stroma. The phyll cells to be assimilated in photosynthesis. Calvin cycle proceeds in three successive stages 4.2. CARBON UPTAKE AND ASSIMILATION 113

Ribulose-1,5-bisphosphate 2002). Rubisco commandeers an extremely CO2 ADP prominent position in grapevine physiology because almost all of the carbon that is assimi- lated is initially “captured” by this protein. As its ATP 3-phoshoglycerate full name suggests, the enzyme “works” in both directions so that CO competes with oxygen for ATP 2 1 the same acceptor. Although under normal 3 conditions the “forward” reaction (carboxylation) 2 is much faster than the “reverse” reaction (oxyge- ADP nation), the fact that O2 is present in the atmo- NADPH sphere at much higher concentration (21%) than CO2 (0.036%) means that the competition results in a loss of CO2 from the cells (Spreitzer NADP+ and Salvucci, 2002; see also Chapter 4.4). Glyceraldehyde-3-phosphate The second stage, called reduction, forms a triose phosphate carbohydrate (glyceralde- Sucrose + starch hyde-3-phosphate) using ATP and NADPH FIGURE 4.4 The three stages of the Calvin cycle that from photochemistry. The two reactions converts CO2 into carbohydrates (illustration by M. Keller). involved in this reduction oxidize ATP to ADP (i.e., one phosphate group is taken from þ ATP) and NADPH to NADP , which then have to be returned to the thylakoid membranes to (Figure 4.4). The first stage is termed carboxy- be “recharged.” During the final stage of the lation and combines CO2 and water with an ini- cycle, termed regeneration, the initial CO2 tial carbon acceptor (ribulose-1,5-bisphosphate) acceptor molecule is restored in a further series to generate two intermediate molecules (3- of enzymatic reactions involving 10 of the 13 phosphoglycerate), thereby “converting” the Calvin cycle enzymes and various intermediate inorganic CO2 into an organic molecule. As its sugar phosphates. The last of these steps also name suggests, this initial organic compound, uses ATP. Thus, the cycle consumes two like all other intermediates of the Calvin cycle, is NADPH molecules and three ATP molecules a sugar phosphate; thus, despite the previous for every CO2 molecule fixed into carbohy- simplified and convenient equation, glucose is drate. The regeneration stage allows continued not a direct product of the Calvin cycle. Because uptake of CO2, but it also means that only one 3-phosphoglycerate is the first stable product of out of every six triose phosphates produced CO2 fixation and contains three carbon atoms, can be either used for starch production within grapevines are grouped with most other crop the chloroplast or exported to the cell’s cytosol plants as so-called C3 plants. The carboxylation for the synthesis of sucrose (Paul and Foyer, step is catalyzed by the enzyme ribulose bispho- 2001). Three turns of the Calvin cycle are sphate carboxylase/oxygenase (rubisco). Rubisco required to form one 3-phosphoglycerate mole- occurs at very high concentration (often in cule, and six turns are required to produce one substantial excess): It represents approximately glucose molecule. However, the cycle operates one-third of a leaf’s total protein complement, very rapidly; free sugars such as sucrose appropriating up to half of leaf nitrogen, which appear within less than 30 s of “feeding” CO2 arguably makes it the most abundant proteins to a leaf. Note that animal cells carry out all nature ever invented (Spreitzer and Salvucci, reactions of the Calvin cycle with the exception 114 4. PHOTOSYNTHESIS AND RESPIRATION of the first and the last step (i.e., the two neces- Sucrose is produced in the cytosol rather than sary enzymes are missing), which is why the chloroplast. The glucose phosphate mole- animals cannot convert CO2 into sugar. cule is energized (as UDP-glucose) before it is The organic product of photosynthesis, photo- linked with the fructose phosphate molecule. synthate, is often given as (CH2O)n, which is the In this case, the required energy is not basic component of carbohydrates. The simple provided by ATP but instead comes from a sugars (usually glucose or fructose) are termed related compound, uridine triphosphate hexoses or monosaccharides and are produced (UTP). The Pi released during sucrose synthesis by putting six of the basic units together (i.e., is transported back into the chloroplast for n ¼ 6). They can be formed either directly inside continued photophosphorylation (Paul and the chloroplast or in the cytosol from triose Foyer, 2001). A special protein embedded in phosphates exported from the chloroplast. Sev- the chloroplast membrane, the phosphate eral enzymes are involved in hexose synthesis, translocator or triose phosphate transporter some of which remove the phosphate groups (Figure 4.5), is responsible for exchanging triose (Pi), which can then be used to recharge ADP phosphate (out) for inorganic phosphate (in). into ATP. Sucrose is exported from the cell to the phloem Combining glucose and fructose gives the and distributed throughout the plant (see disaccharide sucrose (n ¼ 12), which is the Chapter 5.1) for use in the production of other most abundant sugar found in nature. Sucrose, organic components, maintenance processes, a glycoside, is the major end product of photo- and growth of the various sink organs. synthesis and the predominant organic trans- In contrast to sucrose synthesis, the produc- port compound in the phloem of grapevines. tion of starch inside the chloroplast requires

FIGURE 4.5 The dynamics of sugar production, export, and starch accumulation inside a leaf cell. TPT, triose phosphate transporter. Reproduced from Ensminger et al. (2006). Physiologia Plantarum, 126, 37. Wiley-Blackwell Publishing. 4.2. CARBON UPTAKE AND ASSIMILATION 115

ATP to form ADP-glucose. The starch “assembly (Hendrickson et al., 2004b; see also Chapter line” links thousands (5000–500,000) of glucose 7.3). Starch also accumulates in leaves when molecules together, resulting in the long, osmot- the phloem of a shoot is obstructed or injured. ically inert chains of glucose that are called This can happen due to physical injury from starch. Depending on the nature of the bonding strong wind or trellis wires, crown gall or virus between the glucose molecules, the chains can infection (e.g., leafroll virus), or tendrils coiling be linear (a1!4-glucans), in which case they around a shoot. Such shoots can be easily are termed amylose, or branched (a1!4- and recognized in a vineyard because the leaves a1!6-glucans), in which case they are called turn red due to anthocyanin production from amylopectin. Starch is organized into semicrys- surplus sugar (see Figure 2.6). Accumulation talline grains or granules of alternating amor- of light-absorbing anthocyanin pigments may phous and crystalline regions that can grow by occur in order to protect the leaves’ photosyn- adding layers (resulting in growth rings) from thetic machinery from damage due to excessive less than 1 mm to more than 100 mm in diameter light absorption because sugar accumulation (Emes and Neuhaus, 1997; Martin and Smith, leads to feedback inhibition of photosynthesis 1995; Tetlow et al., 2004). to bring the supply of sugar back in balance The Calvin cycle feeds carbon into starch with the demand for sugar. production when the export of sucrose from The rate of sucrose export depends on the the cell cannot keep pace with photosynthesis rate of photosynthesis; the greater the rate of (Paul and Foyer, 2001). This is necessary photosynthesis, the greater the rate of concur- because osmotic pressure is proportional to rent export (Wardlaw, 1990). As discussed pre- the number of dissolved particles (see Chapter viously, however, grapevines, like other plants, 3.1): n glucose molecules cause n times the use starch production as an overflow mecha- osmotic pressure than a polymer composed of nism when sucrose formation exceeds the n identical glucose units. Accumulation of the leaves’ export capacity or the demand for osmotically active sucrose or glucose (i.e., sucrose by the various plant organs. The starch many small solute particles) would therefore can be degraded (remobilized) by removing cause water to flood the cell and it would burst. glucose units simultaneously from the many To avoid this potentially lethal problem, chain ends in a starch granule and converting sucrose that accumulates in the leaf cells slows them, at least partly via intermediary maltose its own formation in a process called feedback that serves as the transport form out of the inhibition. The Pi released during sucrose pro- chloroplast, back to sucrose (Smith et al., duction assumes the function of “regulator”; a 2005). Little or no starch turnover normally high Pi concentration indicates a high rate of occurs in leaves during the day, but degrada- sucrose export and promotes triose phosphate tion is switched on at night when there is no export from the chloroplast (Woodrow and photosynthesis to provide carbon for continued Berry, 1988). Conversely, a low Pi concentration sucrose export, as well as energy and reducing indicates that sucrose export is slow (e.g., due agents in the leaf (Geiger and Servaites, 1991; to low sucrose demand by sink organs) and Geiger et al., 2000; Tetlow et al., 2004). In the promotes starch accumulation for temporary absence of environmental limitations, the starch storage in the chloroplast. Therefore, phosphate pool is depleted by sunrise and replenished by deficiency resulting from low Pi uptake by the sunset, so that sucrose export remains rela- roots also leads to starch accumulation and pre- tively constant over the course of a day–night vents starch mobilization for export, and it may cycle. An intricate, albeit poorly understood, lead to feedback inhibition of photosynthesis control mechanism ensures that this starch 116 4. PHOTOSYNTHESIS AND RESPIRATION accumulation and turnover is adjusted such cell walls, the wall matrix forms the plastic ele- that it compensates for changes in day length ment that holds the microfibrils together and during the growing season to maintain a steady enables cell walls to move and expand (see supply of sucrose over a 24-h period (Geiger Chapter 3.1). The viscous matrix is also com- et al., 2000; Stitt et al., 2007). Because more posed of polysaccharides (e.g., the hydrophilic starch builds up during long than short days, pectin and the cellulose-binding matrix gly- more is broken down during the ensuing short cans, also called hemicellulose, which is mainly nights. This means that the total amount of composed of xyloglucan) in addition to struc- sucrose export from a leaf increases as day tural and enzyme proteins (Cosgrove, 2005; length increases so that more sugar is poten- Reiter, 2002). During periods of rapid growth, tially available for growth, yield formation, cellulose production can be a major drain on a and fruit ripening at higher latitudes. Daytime cell’s resources because both new (dividing) starch degradation, on the other hand, appar- and expanding cells require the insertion of ently occurs only when dense clouds or canopy new (primary) cell wall material. Indeed, most shade prevent net carbon assimilation (Smith of the carbon fixed by photosynthesis may be et al., 2005). Due to its ability to channel assimi- incorporated into cell walls (Reiter, 2002). In lated carbon into the fairly large short-term secondary cell walls, which fully grown cells storage pools of starch and sucrose, the vine insert between their primary cell wall and the can insulate the rate of sucrose supply to other cell membrane, the carbohydrate polymers are organs from the changes in the rate of photo- complemented by the three-dimensional, synthesis that occur during the course of day water-repellent polymer lignin. Lignin consists and night (Woodrow and Berry, 1988). of phenolic building blocks that originate from Glucose molecules (from split sucrose) can the aromatic amino acid phenylalanine and also be linked together as linear b1!4- are linked together in the process of lignifica- glucans forming chains (polymers) of 2000– tion that often follows cell death and employs 25,000 glucose units called cellulose. Cellulose enzymes such as peroxidase along with hydro- is arranged in approximately 35 parallel chains gen peroxide as an oxidizing agent (Boerjan that form a network of long and very strong et al., 2003). By displacing water, lignin fills crystalline microfibrils held together by hydro- the spaces between the polysaccharides, adding gen bonds. These inert, inextensible fibrils, extra mechanical support and waterproofing, whose tensile strength is greater than that of especially to the xylem cells of the wood. Its steel, form the basic framework (i.e., the stiff importance as a stiffener and strengthener of structural elements) of all plant cell walls, the cell wall structure makes lignin the world’s which make up a large portion of a vine’s dry second most plentiful polymer, comprising weight. In fact, cellulose is the most abundant approximately 30% of all organic carbon organic polymer on Earth, making up approxi- (Boerjan et al., 2003). Once lignified, cells mately half of the total organic matter. For become unable to expand further and are glued instance, dry wood consists of 40–50% cellulose to their cell neighbors by the pectin-rich middle (in addition to 25% hemicellulose and 25–35% lamella. lignin; Plomion et al., 2001), and cotton fibers A chloroplast in a young, expanding leaf is used in the textile industry are made up of basically a biosynthetic organelle manufacturing approximately 95% cellulose; cellulose is also compounds for cell growth and development. In the stuff that makes the paper of this book. a mature leaf, on the other hand, a chloroplast Whereas the cellulose microfibrils are responsi- fixes CO2 for the production and export to other ble for the stiffness and mechanical strength of parts of the plant. Photosynthesis therefore 4.3. PHOTORESPIRATION 117 increases with leaf age and in grapevines reaches ATP for detoxification (i.e., reassimilation). þ a maximum approximately 5 or 6 weeks after The flux of NH4 due to photorespiration is at leaf unfolding, gradually declining thereafter, least 10-fold higher than that due to root nitrate as senescence (Latin senescere ¼ to grow old) uptake and assimilation (see Chapter 5.3). sets in (Bertamini and Nedunchezhian, 2003; The glycolate pathway recovers approximately Williams and Smith, 1985). Senescence is the 75% of the lost carbon and returns it to the end phase of differentiation and eventually ends Calvin cycle. in death and abscission of the leaf. Photorespiration reduces the efficiency of photosynthesis, and approximately one out 4.3. PHOTORESPIRATION of every five CO2 molecules entering the chlo- roplast is released back to the atmosphere (Du¨ ring, 1988). The biological function of pho- As previously discussed, rubisco, the first torespiration is not understood. One possible enzyme in the Calvin cycle, can catalyze the function could be the use of electrons when addition of both CO2 and O2 to ribulose-1,5- energy supply exceeds demand, such as in very bisphosphate. The latter is called oxygenation bright light, or during drought conditions and is the main reaction occurring during a pro- when the photosynthetic pigments capture cess known as photorespiration (Ogren, 1984; more photons than the Calvin cycle can use to Spreitzer and Salvucci, 2002). Because the cur- fix CO2 (Apel and Hirt, 2004; Foyer et al., rent atmosphere contains vastly more O2 (21%) 2009; Lawlor and Cornic, 2002; Niyogi, 2000; than CO2 (0.036%) and due to the release of O2 Ogren, 1984). The “venting” of surplus elec- in the water-splitting reaction (see Chapter 4.1), trons substantially reduces a potential “energy the O2:CO2 ratio in the chloroplast fluid is overload” that would otherwise damage the approximately 24:1 at 25C. Moreover, because photosynthetic machinery and lead to photoin- the solubility of CO2 decreases with increasing hibition (see Chapter 4.1). temperature, the O2:CO2 ratio also increases so Nonetheless, it is likely that this seemingly that higher temperatures favor photorespiration. wasteful pathway may be an evolutionary Photorespiration is a problem for grapevines “hangover.” Photosynthesis, and hence because the process works in the opposite rubisco, evolved very early in the existence of direction of photosynthesis and therefore com- life on Earth (more than 3 billion years ago) petes with it for ATP and NADPH and results when the atmosphere was rich in CO2 but in a loss of CO2 that could otherwise be used almost devoid of O2 (Foyer et al., 2009; Xiong for sugar production. Part of this CO2 can be and Bauer, 2002). As a remarkable aside, it recovered in a series of reactions called the was the “invention” of photosynthesis with photorespiratory carbon oxidation cycle or gly- oxygen as its waste product (in addition to colate pathway after the initial product of the injection of oxygen into the air from abiotic oxygenation reaction. This process is quite sources) that enabled the evolution of all complex and involves interconversion and oxygen-breathing life-forms, including humans transport of several amino acids and coopera- (Xiong and Bauer, 2002). This innovation was a tion between three distinct cell organelles—the momentous breakthrough event in the history chloroplast, peroxisome, and mitochondrion of life because it enabled the photosynthetically (Foyer et al., 2009; Ogren, 1984). It also costs endowed organisms to exploit limitless energy in the form of ATP and results in heat supplies of water and solar energy. Of course, loss. In addition, the release of toxic ammonium not all life depends on oxygen for respiration, þ (NH4 ) from amino acids requires yet more as viticulturists and winemakers should know. 118 4. PHOTOSYNTHESIS AND RESPIRATION

Brewer’s or baker’s yeast (Saccharomyces cerevi- lower stomatal density of leaves grown at higher siae) is one of many anaerobic microorganisms [CO2] (Woodward, 1987). Grapevines are no that cannot or prefer not to use oxygen. exception to this general trend (Du¨ring, 2003; Although yeast is a facultative anaerobe and Schultz, 2000; Tognetti et al., 2005). All other plant thus is not poisoned by oxygen, unlike obligate processes affected by elevated [CO2] are thought anaerobes, it relies on energy-inefficient fer- to be an outcome of these two basic responses mentation rather than respiration of sugar for (Long et al., 2004). In the long term, however, pho- energy generation (Rolland et al., 2006). This is tosynthesis can only increase if the vine can uti- why Louis Pasteur described fermentation as lize the extra sugar produced, for example, by “life without oxygen.” Ethanol production increasing shoot vigor or yield, both of which occurs only under anaerobic conditions; in the have been predicted to increase under higher presence of oxygen, ethanol is oxidized to [CO2](Bindiet al., 1996). acetic acid (i.e., vinegar) by other (aerobic) Some plant species long ago “invented” yeasts or Acetobacter bacteria. solutions to the problem posed by high [O2]: The rising atmospheric CO2 concentration The most prevalent of these are the so-called ([CO2]) that is a consequence of rapid burning of C4 plants, which were named for the four- fossil fuels, forest clearing, and soil cultivation carbon oxaloacetate, rather than the usual three- and is responsible for global climate change carbon 3-phosphoglycerate, as the product of [Huang et al., 2000; Intergovernmental Panel on CO2 fixation (Foyer et al., 2009; Ogren, 1984). Climate Change (IPCC), 2007] will, to some Among other adaptations, the C4 plants use a extent, turn the clock back, as it were, to benefit biochemical “pump” to concentrate CO2 near the carboxylation function of rubisco and favor the rubisco enzymes, which almost eliminates photosynthesis over photorespiration (Foyer photorespiration and speeds up the rates of et al., 2009; Paul and Foyer, 2001). Whereas during photosynthesis and growth, especially in high at least 400,000 years before industrialization— light and at high temperature. Because of their that is, twice as long as Homo sapiens has roamed competitive advantage in warm climates, these Earth—[CO2] never exceeded 290 ppm (Petit plants include not only a few important com- et al., 1999), it has increased from approximately mercial crops (e.g., maize, sorghum, and sugar- 270 ppm prior to the beginning of industrializa- cane) but also many highly successful weed tion to approximately 380 ppm today and is pre- species (e.g., crabgrass, switchgrass, pigweed, dicted to reach up to 600 ppm by the end of the and nutgrass). Photosynthesis in C4 species 21st century (IPCC, 2007). The increase in ambi- benefits less from the current rise in atmospheric ent [CO2] results in a steeper CO2 concentration [CO2] so that they can be expected to become gradient, which favors carboxylation by rubisco somewhat less competitive in the near future— and photosynthesis, while decreasing gs. Indeed, although it is far from clear whether this will across all C3 species investigated thus far, an make weed control any easier. increase in [CO2] from approximately 370 ppm to approximately 570 ppm was associated with 4.4. RESPIRATION a one-third increase in both light-saturated and daily photosynthesis that was accompanied by a greater than 80% increase in leaf starch content, Respiration is the series of three processes whereas rubisco content and stomatal conduc- (glycolysis, citric acid cycle, and electron trans- tance declined by approximately 20% (Ainsworth port chain) responsible for releasing the energy and Rogers, 2007; Long et al., 2004). At least some stored in carbohydrate and incorporating it into of the decrease in gs might be a consequence of the a form (ATP) that can be readily used to power 4.4. RESPIRATION 119 þ þ ! þ most energy-requiring metabolic processes in C6H12O6 6O2 6H2O 6CO2 12 H2O the plant. In addition, it produces a number of DG ¼2840 kJ mol 1 intermediate organic components (“carbon ske- Because sucrose, rather than glucose, is the letons”) that can be used for the manufacture of transport sugar in grapevines, and because res- other compounds. In the process, glucose is piration also occurs in nonphotosynthetic tis- completely oxidized to CO2 and its electrons sues that rely on sucrose import, sucrose is are transferred to oxygen, which is reduced to the sugar used as a starting point (substrate) water. In other words, respiration is the oxida- for respiration. However, sucrose must be bro- tion of carbon reduced by photosynthesis. ken down into the two hexoses glucose and Therefore, the chemical equation that sum- fructose, both of which can then enter glycoly- marizes this process, which occurs in all living sis, which occurs in the cells’ cytosol. Stored tissues, is basically the reverse of that used to starch can also be used for respiration after it describe photosynthesis: has been hydrolyzed to glucose (Figure 4.6).

Sucrose Starch

Glucose

Cell walls Hexose-phosphates Cellulose Pentose phosphate Nucleic acids, ATP, NADPH, cytokinin

Glycerol Triose-phosphates Fatty acids

ysis Serine Cysteine Proteins

Phosphoenolpyruvate Shikimate Phenylalanine Phenolics Glycoly Tryptophan Auxin

Pyryuuvate ate Alaanin e Proteinsote s Fatty acids Lipids Membranes Acetyl-CoA Isoprenoids (carotenoids, terpenoids, gibberellin, abscisic acid) Phenolics Citrate Aconitate Oxaloacetate Aspartate

Isocitrate Pyrimidines Other Other Proteins amino acids amino acids Malate TCA cycle

2-Oxoglutarate Glutamate Glutamine Nucleic Proteins acids Fumarate Succinyl-CoA Chlorophyll, phytochrome

Succinate

FIGURE 4.6 Glycolysis and TCA cycle are of central importance for the manufacture of many organic compounds throughout the plant. Modified after Salisbury and Ross (1992). 120 4. PHOTOSYNTHESIS AND RESPIRATION

Although the oxidation of one sucrose mole- the glycolysis intermediate phosphoenolpyr- cule with its 12 C atoms yields a net energy uvate by the enzyme phosphoenolpyruvate equivalent of 60–64 ATP molecules, the maxi- carboxylase (see Figure 4.6). In a series of three mum respiration efficiency is probably slightly enzymatic reactions, the TCA cycle reduces the less than 5 molecules of ATP per molecule of resulting molecule completely to CO2 and H2O, CO2 released (Amthor, 2000). Glycolysis (Greek generating a substantial amount of chemical glykos ¼ sugar, lysis ¼ splitting) degrades (oxi- energy or reducing equivalents (10 NADH per dizes) glucose in a series of enzymatic reactions glucose) in the process. The remaining three to the three-carbon compound pyruvic acid (or steps of the cycle restore oxaloacetate, which pyruvate). This process initially requires input is then ready to accept the next transformed of energy in the form of ATP but later regener- pyruvate (i.e., acetyl-CoA), allowing the ates twice as many ATP molecules in addition continued operation of the cycle. to NADH. Pyruvate still contains more than The electron transport chain transfers elec- 75% of the energy contained in glucose and trons from NADH to oxygen in a stepwise can be used as the starting material for the manner involving several carrier proteins citric acid cycle. bound to the inner membrane of the mitochon- The next steps of respiration are carried out drion. This mitochondrial electron transfer by the citric acid cycle (also called Krebs cycle chain is structurally very similar to the photo- after its discoverer, the German physician Hans synthetic electron transfer chain in the chloro- Adolf Krebs, tricarboxylic acid cycle, or TCA plast and also involves a transfer of protons cycle) and an electron transport chain known from the interior of the mitochondria to the as oxidative phosphorylation. It occurs in cytosol (see Chapter 4.1). The free energy specialized organelles termed mitochondria released during this process (actually the (singular: mitochondrion), which can be potential gradient resulting from the move- regarded as the cell’s power plants. The num- ment of protons across the mitochondrial ber of mitochondria per cell varies and is membrane) is used to power an enzyme directly related to the metabolic activity of the termed ATP synthase that recharges ADP to tissue to which the cell belongs (i.e., energy ATP using Pi (Fernie et al., 2004). Working like generation is driven by demand), but there a turbine in a hydroelectric power plant, are hundreds of mitochondria scattered whose rotation is driven by a proton flow through most cells. This is possible because (each rotation of the enzyme releases three mitochondria have their own genome and rep- molecules of ATP), ATP synthase is life’s uni- licate independently of their “mother cell” by versal power generator that is responsible for simple division. In other words, they reproduce almost all of the ATP produced by living cells. asexually like bacteria, and they can do so The production of 15 ATP equivalents per many times while the cell (and its nucleus) pyruvate molecule by oxygen-consuming does not divide. Not surprisingly, the enzymes mitochondria (burning sugar as fuel) is by far carrying out glycolysis are attached to the the most efficient means of biological energy outside of the mitochondria (Fernie et al., generation. However, because respiration also 2004). Pyruvate is transported into the mito- produces a small amount of highly reactive chondria, where it is oxidized to acetic acid and damaging oxygen intermediates (so-called and linked to a sulfur-containing coenzyme reactive oxygen species; see Chapter 7.1), it called acetyl coenzyme A (acetyl-CoA). Ace- may slowly but surely lead to oxygen poison- tyl-CoA is then combined with oxaloacetate ing that results in aging and, eventually, already present in the cycle or supplied from senescence (Logan, 2006). 4.5. FROM CELLS TO PLANTS 121

In addition to the formation of ATP, respi- leaves in the phloem to other growing organs ration generates various intermediate carbon also requires energy (“transport cost”). A sub- compounds and thus is central to the pro- stantial portion of growth respiration involves duction of a wide variety of components of the production of carbon skeletons for nitrogen the plant’s metabolism (Fernie et al., 2004; see assimilation (see Chapter 5.3). The cost of also Figure 4.6). These include cellulose, production for plant tissues varies with the nucleotides (and thus nucleic acids, cytoki- nature of the chemical compounds that are nins, etc.), amino acids (and thus proteins, fla- being produced (Table 4.1). For instance, vonoids, anthocyanins, lignin, etc.), fatty acids producing proteins or phenolic compounds is (and thus lipids, etc.), isoprenoids (and thus much more expensive than producing carbohy- terpenoids, carotenoids, cytokinins, abscisic drates; thus, plant parts with a high protein acid, gibberrellins, etc.), and porphyrins (and content, such as leaves, are relatively expensive thus chlorophylls, phytochromes, etc.). For to build (Vivin et al., 2003). The rate of growth example, all 20 standard amino acids formed respiration increases as the rate of growth in plants for the manufacture of proteins are increases. The other component, maintenance assembled from the carbon skeletons provided respiration, is needed to keep existing, mature by the organic acid products of glycolysis, the cells in a viable, functional state and is coupled TCA cycle, and the Calvin cycle. Therefore, to energy generation, carbon and nutrient a large portion of the sugar that is metabo- transport, protein and lipid turnover (i.e., lized by glycolysis and the TCA cycle is not production–breakdown–recycling–resynthesis), oxidized to CO2 but instead is diverted to and adjustment or acclimation to changing biosynthetic purposes. Moreover, both ATP environmental conditions. Protein turnover and ADP are also used as the original precur- and adjustment/acclimation are linked so that sor molecules for the assembly of all compounds plants do not have to maintain a complete set that comprise the group of cell division of enzymes and transport proteins at all times, hormones called cytokinins (Sakakibara, 2006). just to be ready for changing conditions. It is much more economical for them to produce 4.5. FROM CELLS TO PLANTS specialized proteins, or groups of proteins, “on demand” and recycle them when not

Although photosynthesis is the ultimate TABLE 4.1 Average Construction Costs of Major source of carbohydrates and supplies some Chemical Plant Components ATP and NAD(P)H in photosynthetic tissues, a respiration is the powerhouse of plants, which Component Cost provides the driving force for biosynthesis (production of new materials), cell mainte- Carbohydrates 1.17 nance, and active transport (including nutrient Organic acids 0.91 uptake). It is generally broken down into two Lipids 3.03 components: growth respiration and mainte- Amino acids, proteins 2.48 nance respiration (Amthor, 2000; Penning de Phenolics 2.60 Vries, 1975; Penning de Vries et al., 1974). In Inorganic compounds (minerals) 0 growth respiration, reduced carbon is pro- cessed to fuel the accumulation of new biomass aExpressed as grams of glucose used to produce 1 g of each (“construction cost”): Growth is not possible compound class. without respiration. Sugar export from the From Vivin et al. (2003). 122 4. PHOTOSYNTHESIS AND RESPIRATION needed so that their amino acid building blocks respiration constitutes a compromise between can be reassigned to other temporary jobs. Scien- energy generation and carbon gain. A high res- tists do not have a good understanding of energy piration rate means that more ATP is produced utilization by maintenance respiration, but esti- (and thus more energy is available for metabo- mates suggest that it may represent more than lism), but this comes at the expense of carbon 50% of the total respiratory CO2 release. lost to accumulation of biomass. Conversely, a Respiration can consume a significant por- low respiration rate improves the carbon bal- tion (30–80%) of the photosynthetically fixed ance but reduces the energy available to utilize carbon each day over and above the losses this additional carbon. The increase in photo- due to photorespiration, and it releases large synthesis associated with the elevated atmo- amounts of CO2 back to the atmosphere spheric [CO2] that is driving global climate (Amthor, 2000; Atkin and Tjoelker, 2003; Atkin change (see Chapter 4.3) may also stimulate et al., 2005). Grapevine leaves respire at a rate of respiration by increasing leaf sugar and starch approximately 5–10% of the rate of photosyn- concentrations (Amthor, 2000; Leakey et al., thesis. However, all plant tissues, whether pho- 2009). Although this will diminish the net plant tosynthetic or not, respire, and they do so 24 h carbon balance, it may nevertheless lead to a day. Respiration rates vary with the age of greater carbon export for growth and yield for- the plant and differ from one organ to another mation, which is associated with higher growth depending on the availability of carbon sub- and maintenance respiration. The rise in tem- strates, stage of development, and temperature. perature that accompanies the higher atmo- For instance, a temporary 10 C rise in tem- spheric [CO2] may also stimulate respiration perature usually leads to at least a doubling of and growth, although probably not as much ¼ the respiration rate (i.e., Q10 2). Young grape- as short-term, temporary increases in tempera- vines lose approximately one-third of their ture, due to the effects of acclimation to long- daily photosynthate to respiration, and this term temperature changes (Amthor, 2000). loss increases in older plants as the amount Although nutrient ions per se do not cost of nonphotosynthetic tissue (e.g., in cordon, any glucose for their construction (they are trunk, and roots) increases. inorganic chemicals), grapevines spend much As a general rule, the greater the metabolic of the energy generated during respiration on activity of a given tissue or organ and/or the nutrient acquisition—that is, on the uptake of higher its growth rate, the higher its respiration nutrient ions from the soil solution and rate. Meristems have the highest respiration their assimilation into organic compounds. rates so that young, rapidly growing plant Nitrate and sulfate acquisition incurs particu- parts, such as developing buds, shoot and root larly high respiratory costs and causes an tips, unfolding leaves, or flowers, can respire increase in respiration rates to supply carbon up to 10 times more rapidly than older parts skeletons for the production of amino acids that rely mainly on maintenance respiration. (see Chapter 5.3). Vigorous species or cultivars Between 70 and 90% of imported carbon is are characterized by spending less respiratory incorporated in new material in growing energy for nutrient acquisition and more for organs; the remainder is released as CO2 growth. In addition, their maintenance costs (Amthor, 2000). This means that during and also seem to be slightly lower, although this after budbreak, vines will literally lose weight difference is small compared with the differ- (dry matter) until the new leaves become pho- ence in energy allocated to nutrient acquisi- tosynthetically competent to replace the lost tion. However, differences in maintenance carbon (Buttrose, 1966b). It appears that respiration become more important when 4.5. FROM CELLS TO PLANTS 123 vines are grown under unfavorable condi- in the growth of roots, which have higher res- tions. For example, low soil nitrogen availabil- piration rates than shoots. Thus, rather than ity decreases the absolute rates of whole-plant having higher photosynthetic rates, vigorous respiration but increases the proportion of car- plant species often respire a lower proportion bon fixed during the day that is lost during of their acquired carbon than slow-growing respiration. This happens because vines with species; that is, the respiration:photosynthesis a low nitrogen status have reduced photosyn- ratio is smaller in vigorous species (Loveys thesis and invest more of their photosynthate et al., 2002). CHAPTER 5

Partitioning of Assimilates

OUTLINE

5.1. Photosynthate Translocation and 5.2.4. Humidity 155 Distribution 125 5.2.5. The “Ideal” Canopy 156 5.1.1. Allocation and Partitioning 134 5.3. Nitrogen Assimilation and Interaction 5.2. Canopy–Environment Interactions 140 with Carbon Metabolism 158 5.2.1. Light 143 5.3.1. Nitrate Uptake and Reduction 159 5.2.2. Temperature 150 5.3.2. Ammonium Assimilation 160 5.2.3. Wind 154 5.3.3. From Cells to Plants 162

5.1. PHOTOSYNTHATE TRANSLOCATION AND demand). A source is any plant organ that DISTRIBUTION exports material. The typical source organ is the mature leaf that produces more photosyn- thate than it needs for its own growth and Grapevines, like all other plants, employ a metabolism, but all green (i.e., chlorophyll- division of labor between different tissues and containing) tissues (including those in the shoot organs that is coupled to their structural frame- and cluster) can contribute to a vine’s total work and relies on an efficient transport system assimilate production. Other sources of grape- connecting the various parts. The organic com- vines include the woody structures (canes, cor- pounds produced during photosynthesis and don, trunk, and roots), which function as nutrient assimilation (photosynthates and storage organs. However, all sources begin assimilates) and certain mineral ions must be their life as sinks. A sink is a nonphotosynthetic transported from their place of manufacture plant organ or an organ that produces insuffi- or storage (“source”: supply) to places of inter- cient photosynthate to supply its own needs. mediate or final use or storage (“sinks”: Sinks can be growing vegetative organs

The Science of Grapevines 125 Copyright # 2010 Markus Keller. Published by Elsevier Inc. All rights reserved. 126 5. PARTITIONING OF ASSIMILATES

(expanding leaves and root tips), storage from the growing shoot tip, although export organs (shoots or canes, trunks, and roots), or of leaves below a cluster becomes bidirectional reproductive organs (flowers and developing again after fruit set (but not beyond the cluster). fruits and seeds). Following the sink–source transition, a leaf will All leaves are initially sinks because they no longer import substantial amounts of sugar, need to build up their photosynthetic machin- even under conditions of heavy shading (i.e., ery before they can start producing and export- the leaf acts at most as a very weak sink). ing their own sugar. The construction phase Approximately 40 days after unfolding, the leaf entails an investment of carbon (and other reaches maturity. Thereafter, it slowly exports nutrients) in both building material and energy less photosynthetically fixed carbon as it ages, (respiration). The time period required for a although some leaves can remain fully photo- leaf to make the transition from sink to source synthetically active for more than 100 days is important because it determines how quickly (Hunter et al., 1994; Intrieri et al., 1992; Schultz the leaf starts paying dividends on the carbon et al., 1996). Old leaves, although still photosyn- invested to construct it. It seems as though thetically active, do not seem to export assimi- leaves unfolding in spring pass through this lates but, rather, retain them for their own period more rapidly than leaves unfolding in metabolic demands (Koblet, 1969). Instead, the fall; thus, late-season leaf growth may be too leaves become a major source of mineral nutri- slow to make a significant contribution to fruit ents (particularly nitrogen, phosphorus, and ripening or carbohydrate storage. This is one potassium) toward the end of their life as a reason why viticultural practices aim at sup- result of the resorption of proteins and other pressing shoot growth late in the growing sea- constituents during senescence (Thomas and son. Such growth may compete for resources Stoddart, 1980). with the ripening fruit as well as with the The woody organs of the vine are generally replenishment of the reserve pool and cold regarded as sinks because they do not photo- acclimation of the vine. The sink-to-source tran- synthesize. They rely on imported photosyn- sition happens gradually; a grapevine leaf gen- thate and nutrients for growth and erally starts exporting assimilates when it has metabolism. However, sugar and other nutri- reached approximately one-third its final size ents imported and stored as starch and other but continues to import carbon up to half its material (e.g., amino acids and proteins) in final size (Koblet, 1969), which requires bidirec- these tissues during the growing season are tional assimilate transport in the petiole. The remobilized in spring to support budbreak oldest, most basal leaf on a shoot typically and the initial shoot and root growth before begins exporting assimilates when the shoot the new leaves start to export assimilates has approximately five or six leaves (Hale and (Wardlaw, 1990; Williams, 1996). This starch Weaver, 1962; Koblet, 1969; Koblet and Perret, remobilization (i.e., hydrolyzation to glucose 1972; Yang and Hori, 1979, 1980). Export is and glucose phosphate that are then converted initially directed toward the shoot tip, but as back to sucrose for export) is stimulated by the soon as the next leaf above makes the transition plant hormone gibberellin. Although the oldest from sink to source, the older leaf also begins to leaf on a new shoot begins exporting some of export a portion of its assimilates toward the its assimilates to the woody parts of the vine shoot base and into the permanent structure as early as 2 or 3 weeks after budbreak, remobi- of the vine. Only a few days later, export of lization and export from the wood to the devel- the older leaf becomes exclusively basipetal. oping shoots continues through bloom (i.e., This pattern is repeated as new leaves unfold transport is bidirectional). In addition, stored 5.1. PHOTOSYNTHATE TRANSLOCATION AND DISTRIBUTION 127 reserves also serve as a buffer by providing a TABLE 5.1 Composition of Phloem and Xylem Sap temporary supply of carbon that can be mobi- lized during stress periods, such as overcast Solute Xylem Sap Phloem Sap conditions or loss of leaf area, that interfere Sugars (mostly sucrose, 5gL 1 100–300 g L 1 with photosynthesis (Bloom et al., 1985). some glucose and 15 mM 300–900 mM The transport must be sufficiently flexible to fructose) supply the growing and metabolizing tissues of Amino acids (mostly 2gL 1 5–40 g L 1 roots, shoots, leaves, flowers, and fruits. It glutamine, some 15 mM 30–270 mM therefore must be relatively rapid to keep up glutamate, with the demand for “fuel” and “building asparagine, and blocks” by these growing tissues, which can aspartate) 1 be far away, and must be able to reverse the Proteins (mostly Absent 0.2–2 g L direction of flow depending on the relative glycolytic and structural proteins) needs of different organs at different times. Organic acids (mostly 0.1–0.5 g L 1 1–3 g L 1 This translocation of solutes occurs in the malate) 0.7–4 mM 7–20 mM phloem tissue (more precisely in the phloem’s Inorganic ions 0.2–4 g L 1 1–5 g L 1 sieve elements; see Chapter 1.3), and because þ solute concentration is usually higher in the Potassium (K ) 1–10 mM 40–100 mM þ source phloem than in the sink phloem, it fol- Sodium (Na ) 0.1–2 mM 0.1–5 mM þ lows a gradient of concentration (i.e., chemical Magnesium (Mg2 ) 1–2 mM 1–6 mM þ potential). Calcium (Ca2 ) 1–5 mM 0.1–2 mM By far the major compound (besides water) Phosphate (H2PO4 / 0.1–3 mM 7–15 mM 2- translocated in the phloem of grapevines is HPO4 ) sucrose (Koblet, 1969; Swanson and Nitrate (NO3 ) 0.01–20 mM Traces El-Shishiny, 1958), which accounts for 50–70% Total solute 10–100 mM 250–1200 mM of the sap osmotic pressure (Table 5.1). The concentration mass flow resulting from the steep osmotic Osmotic pressure (p) 0.02–0.2 MPa 0.6–3 MPa gradient created by sucrose also drags along pH 5.0–6.5 7.5 numerous other solutes, of which potassium followed by amino acids, glucose, fructose, Modified from Buchanan et al. (2000), Peuke et al. (2001), and malate make up much of the remainder and Keller et al. (2001b). of the osmotic components (Patrick, 1997; Peuke et al., 2001). The concentration of amino acids and organic acids (e.g., malate and ascor- bate) varies widely but is normally much lower expanding leaves under conditions of phospho- than the sucrose concentration. Nonetheless, as rus deficiency. The phloem also contains pieces the leaves change from carbon sources to min- of RNA and several peptides and proteins that eral nutrient sources during senescence, are involved, among other things, in sugar phloem sap composition shifts from predomi- metabolism and transport (sucrose carriers and þ þ nantly sucrose to mainly amino acids (Thomas ATPases), nutrient transport (K and Ca2 chan- and Stoddart, 1980). Some nutrient ions can nels), and water transport (aquaporins) across also be transported in the phloem. For instance, membranes. Because the sieve elements lack most of a grape berry’s potassium is imported the necessary machinery to manufacture pro- via the phloem, and phosphate can be retrieved teins, RNA pieces, which even include base from old leaves and recycled to young, sequences that act as a “zip code” that specifies 128 5. PARTITIONING OF ASSIMILATES the recipient tissue and ensures proper delivery, The composition of the phloem sap not only might be used as long-distance signals (even depends on species, cultivar, and rootstock but across graft unions) that promote the production also varies with changing physiological condi- of proteins in the target sink organs (Kehr and tions, location within the plant, and time of Buhtz, 2008; Lough and Lucas, 2006; Turgeon the growing season. Although the composition and Wolf, 2009). Phloem-specific proteins, man- also varies with time of day, the amount of ufactured in the companion cells, are also transported solutes remains more or less con- responsible for the almost instantaneous sealing stant over a 24-h period (Peuke et al., 2001). of the phloem’s sieve plate pores upon injury Solutes can also be exchanged between the (e.g., by pruning and leaf or cluster removal), phloem and xylem (Lalonde et al., 2004). whereas long-term plugging is achieved by Xylem-to-phloem transfer occurs particularly deposition of callose (see Chapter 1.3), whose in leaf traces (i.e., in the vascular bundles production is induced by a sudden increase in branching away from the shoot into a leaf) (cytosolic) calcium in the sieve elements (Furch and in the minor veins of a leaf, but it can also et al., 2007; Ku¨ hn et al., 1999; Oparka and Santa happen in the shoot, especially at nodes. It Cruz, 2000; Van Bel, 2003). Furthermore, almost enhances the delivery of nutrients to rapidly all plant hormones (auxin, gibberellins, cytoki- growing, but slowly transpiring, sinks (e.g., nins, and abscisic acid) are also translocated in meristems, fruits, and seeds). Phloem-to-xylem the phloem in addition to the xylem. By acting transfer is important in roots for redirecting as chemical signals, these hormones exert some solutes delivered by the phloem (e.g., þ remote controls on physiological processes in amino acids and K ) back to the shoots. organs and tissues distant from their site of pro- Phloem sap in grapevines typically moves duction. Even electrical signals may be transmit- at flow velocities of 0.2–1.5 m h 1 (Koblet, ted through the phloem (and the xylem, where 1969), which is much faster than would be they arise locally in response to hydraulic pres- expected from simple diffusion. Phloem flow sure waves or transported compounds due to can be bidirectional within a single internode physical injury), which gives the phloem proper- but is unidirectional within a given sieve ele- ties of a neural network (Fromm and Lautner, ment. In contrast to the xylem, the rate and 2007). Such signals are generated in response to direction of solute movement in the phloem sudden environmental stimuli (e.g., tempera- are under metabolic control and change over ture, light, touch, and wounding) and exert an time in response to source and sink develop- additional regulation of physiological functions. ment. Thus, the direction of phloem flow can The long-distance information transmission of be reversed over time and often is against the electrical signals is much more rapid (>10 m h 1) direction of the transpiration stream in the than that of chemical signals (1mh 1). Unfor- xylem. The pressure flow theory of phloem sap tunately, some undesired hitchhikers, such as movement developed by Mu¨nch (1930) states viroids, viruses, and some bacteria, also use the that sap moves from places of high (turgor) P ¼ C phloem-generated mass flow for transport and pressure ( P) to places of low pressure are able to slip through the intercellular plas- within the phloem (Figure 5.1). The volume flow J modesmata, enabling them to spread easily rate ( v) varies with the resistance of the flow r throughout the vine (Lough and Lucas, 2006; path ( h) and can be described by the following Oparka and Santa Cruz, 2000; Van Bel, 2003). equation (Patrick, 1997): Finally, the ability of the phloem to transport J ¼ DPr 1 fungicides, insecticides, and herbicides within v h DP ¼ P P r the plant is exploited in viticultural practice to where source sink, and h is the combat pathogens and weeds. hydraulic resistance of the phloem path. 5.1. PHOTOSYNTHATE TRANSLOCATION AND DISTRIBUTION 129

Xylem Phloem Source

ψ = −0.8 ψ = −1.0 P = −0.7 P = 0.6 π = 0.1 π = 1.6 Sucrose Water

Leaf cell

Companion cell

Sink

ψ = −0.6 ψ = −0.4 P = −0.5 P = 0.3 π = 0.1 π = 0.7

Vessel Sieve tube Root cell

FIGURE 5.1 Schematic representation of the movement of phloem sap from regions of high pressure in a source to regions of low pressure in a sink. All values are in MPa (illustration modified after Nobel, 1991, Evert, 2006, and Taiz and Zeiger, 2006). P It is important to realize that source refers to In other words, the transport rate in the P the pressure inside the source phloem and sink phloem is dependent on solute (assimilate) con- refers to the pressure inside the sink phloem; that centrations and a DP. The radius of a sieve is, the hydrostatic pressure difference or pressure tube, and hence the cross-sectional area (A), is gradient (DP) occurs within the sieve tubes rather important insofar as it determines the hydrau- l ¼ L A ¼ r 1 L than between a source organ and a sink organ. In lic conductance ( h h h , where h is other words, the sieve tubes are pressurized, and the hydraulic conductivity; Patrick et al., 2001). the buildup and dissipation of pressures in the Unlike the xylem (see Chapter 3), the phloem phloem are isolated from the turgor pressures in cannot take advantage of transpiration as the the source and sink cells to permit continued driving force for mass flow but instead relies phloem flow and translocation of solutes. It fol- on a pressure-driven mass flow in the opposite P et al et al lows that the flow rate increases as source direction (Lalonde ., 2004; van Bel ., P increases and/or as sink decreases. This means 2002). Ingeniously, sucrose is not only the that sinks compete for assimilates by lowering major cargo (i.e., the main transport form of the pressure in their sieve elements (Wardlaw, carbon) of the phloem system but also its fuel J 1990). The bulk flow rate of solutes ( s)through (i.e., the osmotic driving force for mass flow) the phloem can be written as follows (Lalonde (Hellmann et al., 2000; Mu¨ nch, 1930; Van Bel, et al., 2003; Patrick, 1997): 2003). The source cells “load” sucrose into the sieve elements, which results in an osmotic gra- Js ¼ Jv c dient (DCp) that generates a water potential where c is the solute concentration. gradient (DC). This draws water from the xylem 130 5. PARTITIONING OF ASSIMILATES into the sieve elements, increasing their hydro- that is, across cell walls—because the transport static (turgor) pressure (P). In other words, sugar in grapevines is sucrose (rather than the phloem transport depends on the osmotic gen- larger raffinose and/or stachyose) and, assum- eration of hydrostatic pressure in the source ing that the sucrose concentration inside the phloem. The sap moves toward the sink because phloem is much higher than in the surrounding sucrose is being removed (unloaded) there, cells, because diffusion against a concentration which leads to a corresponding loss of water gradient is not possible. If this is true, then (which passively follows the sugar by osmosis) sucrose must be pumped out to the cell walls from the sieve element so that P is lower at the (which are permeable to small organic mole- sink end of the sieve tubes. Water movement cules such as sugars and amino acids) and into and out of the phloem is driven by DC, across the cell membranes of the companion whereas water movement inside the phloem cells and sieve elements by proton cotransport DP ¼ P (from source to sink) is driven by ( source (or symport), a process that requires an input P et al sink), which in turn is generated by loading of energy in the form of ATP (Lalonde ., (at the source) and unloading (at the sink) of 2003; Patrick et al., 2001). Hexose sugars that sugar and other solutes. Whereas most of the may have leaked to the apoplast might be water entering the phloem in the minor veins transported back into the cells using hexose of source leaves is absorbed from the xylem, transporters (Hayes et al., 2007). The average the water leaving the phloem in the sink tissues energy demand for phloem loading has been is used in part for growth of the sink cells, and estimated at roughly one-third of the entire the remainder enters the xylem to be recycled respiratory energy demand of mature leaves back to the source (Bradford, 1994; Mu¨nch, (Amthor, 2000). Other scientists believe that in 1930; Patrick, 1997; Van Bel, 2003; see also contrast to most other crop species, this sucrose Figure 5.1). In transpiring sinks, such as expand- transport occurs entirely within the symplast ing leaves and young grape berries, some water rather than across the apoplast. According also evaporates to the atmosphere, although to this model, sucrose in the leaves moves berry transpiration rates decline during the by diffusion or mass flow (generated by a DP course of berry development (Blanke and between mesophyll and phloem cells) along a Leyhe, 1987; Du¨ring and Oggionni, 1986; continuous, membrane-bound pathway (through Rogiers et al., 2004a). plasmodesmata) from the mesophyll to the The phloem system is composed of three phloem, which is therefore called “open phloem” sequential sectors: collection phloem, transport (Turgeon, 2000; Turgeon and Wolf, 2009). phloem (also called path phloem), and release Provided the concentration of sucrose inside the phloem (Lalonde et al., 2003; Patrick et al., minor-vein phloem cells is lower than that in the 2001; Van Bel, 2003). Each of these sectors exe- surrounding mesophyll cells (unlike in many cutes a specific task. Sucrose produced in the other species), there is no need for active, mesophyll cells of source leaves moves (i.e., energy-dependent transport. This implies, how- diffuses) symplastically via plasmodesmata to ever, that the flow direction of the phloem is the bundle sheath cells of the leaf’s minor reversible, depending on the direction of the pres- veins. From there, it is loaded into the compan- sure gradient. Surprisingly, despite the proposed ion cells and sieve elements of the collection symplastic continuity, grapevines are somehow phloem (Lalonde et al., 2003, 2004). The way able to mostly exclude hexoses from the phloem, by which grapevines load sucrose into the although there are plenty of these sugars in the phloem is controversial. Some researchers mesophyll cells. Only traces of glucose and fruc- argue that loading occurs via the apoplast— tose have been reported to be transported in 5.1. PHOTOSYNTHATE TRANSLOCATION AND DISTRIBUTION 131 grapevine phloem (Koblet, 1969). One costly and in the sink, the export rate of sugar drawback of the open phloem design is that viral depends on both the sucrose production in the pathogens can also pass through plasmodesmata source leaf (which in turn depends on net car- and thus become systemic once a vector (usually bon assimilation; see Chapter 4.2) and the an insect or nematode) has deposited them near capacity of sink tissues to remove (and use) the phloem (see Chapter 7.5). It is also possible sucrose from the phloem. In contrast to the that both modes of loading operate in tandem in xylem (see Chapter 3.3), the transport phloem the leaves (Lalonde et al., 2003; Turgeon, 2000; is rather “leaky” due to the presence of plasmo- Turgeon and Wolf, 2009). desmata. Therefore, a considerable portion of Amino acids can be produced in the meso- the solutes are passively and slowly released phyll cells or delivered from the roots in the to the phloem parenchyma along the way to xylem along with potassium and other nutrient supply axial sinks (e.g., for maintenance respi- ions (see Chapter 5.3). These compounds can be ration, radial growth, and reserve storage in loaded into the phloem symplastically (leaf- woody organs), from where they can also be produced amino acids) or apoplastically actively and rapidly retrieved into the phloem, (xylem-delivered amino acids and potassium) for example, via starch hydrolysis and ATP- (Lalonde et al., 2003). The apoplastic step fuelled proton/sucrose symport (Lalonde requires channels (for potassium) and transpor- et al., 2003; Martens et al., 2006; Patrick, 1997; ters (for amino acids). The energy-dependent Van Bel, 2003). Such “pathway storage” can transporters are driven by ATPases that move buffer diurnal and environmentally induced a proton across the membrane along with, or fluctuations in assimilate supply from the in exchange for, an amino acid, depending on leaves (Wardlaw, 1990) so that the sugar con- whether a transporter functions in symport centration in the phloem entering distant sinks or antiport mode (Lalonde et al., 2004). Potas- varies little compared with that in the minor sium also seems to stimulate sucrose loading veins of the source leaves. Symplastic net into the phloem. Other compounds, such as release along the transport phloem is favored organic acids and hormones, probably enter by high source:sink ratios (i.e., under sink limi- the phloem mostly by diffusion, although tation, such as with light crop loads), whereas auxin may also be actively loaded, and are under low source:sink ratios (i.e., under source swept along the pathway by mass flow. The limitation) the plasmodesmata close and minor veins are also a rerouting system for solutes can only leave the phloem apoplasti- water; water entering the leaves in the xylem cally and hence slowly (Patrick, 1997; Patrick is carried away again in the phloem if it is et al., 2001). The transport phloem is in local not used for transpiration or cell expansion water potential equilibrium with the apoplast: C C (see Chapter 3.2). phloem and xylem are tightly coupled because Once the solutes have entered the phloem, water moves across the separating membranes they are exported away from the source in the (Thompson and Holbrook, 2003; see also Chap- transport phloem of the main veins, petioles, ter 3.1). Water enters the transport phloem shoots, trunk, and roots. This long-distance osmotically from the surrounding apoplast, transport occurs by mass flow rather than by including the xylem, along the pathway and diffusion because the pressure inside the progressively dilutes the sieve element con- phloem is lower in the distant sink organs than tents. The sucrose concentration in the phloem in the leaves, with water being provided by the consequently declines with increasing trans- xylem. Because phloem flow is driven by the port distance and has been estimated to be less DP between the sieve elements in the source than 50 mM in the pedicel of ripening grape 132 5. PARTITIONING OF ASSIMILATES berries (Zhang et al., 2006). This dilution gradu- base and export from the leaf tips, although C ally raises phloem and increases the transport import and export probably occur in different rate (Patrick et al., 2001). If the increase in flow vascular bundles (Wardlaw, 1990). The unload- rate cannot compensate for the decrease in sol- ing pathway is turned off after a leaf has com- ute concentration, however, then more distant pleted its sink–source transition so that even sinks will receive less sugar and will be at a heavily shaded mature leaves in the interior of competitive disadvantage compared with more dense canopies do not import assimilates, proximal sinks. unless there is no competition from sink organs Upon arrival in the sink tissue (e.g., shoot (Lalonde et al., 2003; Quinlan and Weaver, 1969, tips, root tips, fruit, and seeds), the solutes are 1970; Turgeon, 1984). unloaded from the phloem (release phloem) in Grape berries are special because although reverse order of the loading sequence and unloading initially follows mostly the standard metabolized or stored in the sink cells (Lalonde symplastic route, the apoplastic (extracellular) et al., 2004; Patrick, 1997; Patrick et al., 2001). In path of unloading (in which sucrose is released contrast with the transport phloem, this release to the apoplast) comes to predominate at or just is final—that is, retrieval stops in the release before veraison (Zhang et al., 2006). The peri- phloem. The sucrose concentration in the carp cells seem to become symplastically phloem is almost always much higher than in isolated from the phloem inside the fruit by the surrounding sink parenchyma cells (but partial blockage (probably by callose) of the lower than in the source), so unloading again plasmodesmata that connect the sieve ele- passively follows a pressure gradient. In most ment/companion cell complex with the sur- sinks (e.g., root and shoot tips and expanding rounding parenchyma cells inside the vascular leaves), the unloaded sucrose by and large fol- bundles and with the berry’s flesh cells. Even lows a symplastic pathway through plasmo- before veraison, however, there are no plasmo- desmata to the site of usage, although there desmatal connections between tissues of differ- may also be some additional apoplastic unload- ent genomes, such as a seed’s filial tissues ing (Lalonde et al., 2003; Patrick, 1997; Van Bel, (embryo and endosperm) and its maternal tis- 2003). The symplastic pathway is one of passive sues (which include the seed coat). Therefore, diffusion and bulk flow and is characterized by although unloading into the seed coat may be its large transport capacity and low hydraulic symplastic, unloading into the embryo and resistance. The ability to open or close the plas- endosperm is always apoplastic (Bradford, modesmata provides sink cells with a mecha- 1994; Patrick, 1997; Walker et al., 1999). Apolas- nism to control solute import (facilitated tic unloading also occurs at the interface diffusion) by changing the hydraulic resistance between leaf or berry cells and biotrophic of the pathway. In young, expanding leaves, pathogens, such as the powdery mildew fun- assimilate import seems to be restricted to the gus Erysiphe necator (see Chapter 7.5). In addi- larger veins (the minor veins are probably tion to the facilitated diffusion mentioned responsible for assimilate collection for export) previously, apoplastic unloading and post- and is maximal at approximately 15–20% of phloem transport require energy-dependent the leaf’s final size. The transition from sink to active transport to “pump” (again using source starts in the leaf margins so that during ATPases) the solutes across the phloem and the transition phase (from 30 to 50% of full leaf pericarp cell membranes (Giribaldi et al., 2007; size, corresponding roughly to leaf positions Sarry et al., 2004; Zhang et al., 2008). These four to six from the shoot tip; Koblet, 1969) ATPases include sucrose and hexose transporters there can be simultaneous import into the leaf (Ageorges et al., 2000; Davies et al., 1999; Fillion 5.1. PHOTOSYNTHATE TRANSLOCATION AND DISTRIBUTION 133 et al., 1999; Hayes et al., 2007; Vignault et al., 2005) sucrose released from the phloem to the apo- and, along with the invertases discussed later, are plast is broken down to hexoses there before activated by sugars and abscisic acid, whose con- being pumped into the cytoplasm, where some centration inside the berry increases at veraison of them may be reassembled to sucrose for (Pan et al., 2005). import into the vacuole, only to be split into As illustrated in Figure 5.2, the importing glucose and fructose again (Sarry et al., 2004; berry cells can retrieve sucrose from the apo- Terrier et al., 2005). During ripening, the “sur- plast unaltered, again with the (active) aid of plus” hexoses—that is, sugars that are not used sucrose transporter proteins in the cell mem- in further metabolism—are accumulated for branes, or sucrose can be split into hexoses storage inside the vacuoles. If grapes are simi- (glucose and fructose) by the enzyme invertase lar to tomato, then cultivars that accumulate located in the cell walls (apoplast). The hexoses different amounts of hexoses and therefore are then imported into the cell and then into the reach different degrees Brix at maturity should vacuole by membrane-based hexose trans- mainly differ in their rate of sugar transport porter proteins or directly into the vacuole across the mesocarp cell membranes (Patrick (i.e., bypassing the cytosol), via intracellular et al., 2001). Moreover, due to the dependence vesicles—a mechanism termed endocytosis. of phloem flow on DP, berries that are able to Sucrose can also be split inside the cell by a rapidly use or accumulate sugars are more cytoplasmic invertase or enter the vacuole competitive than their slower siblings and will unaltered. Inside the vacuole, it can again tend to develop and ripen more rapidly. remain unaltered or be split by a vacuolar Despite the continued import of sucrose into invertase (Famiani et al., 2000; Robinson and most sink tissues, its concentration in these tis- Davies, 2000). It appears that most of the sues remains low because it is “consumed” by respiration (for growth and metabolism), used as building blocks in the construction of new cell walls and other cell components, or con- verted to storage material such as starch (in shoots, trunks, and roots). However, this is not the case in postveraison grape berries, which accumulate large amounts of hexose sugars to concentrations that exceed that in the phloem of the pedicel by severalfold. The switch from symplastic to apoplastic phloem unloading before veraison isolates the sink symplast (berry pericarp cells) from the vascu- lar symplast (phloem) and enables the berries to accumulate osmotic solutes to high concen- trations without inhibiting phloem flow. FIGURE 5.2 Possible pathways for sugar from the Indeed, the temporary deposition of solutes in phloem to the vacuole of grape berry pericarp cells. HT, the apoplast and the activity of cell wall inver- hexose transporter; Inv-CW, cell wall invertase; Inv-N, cyto- tase lower the apoplast’s osmotic potential plasmic invertase; Inv-V, vacuolar invertase; ST, sucrose (Cp), leading to water influx from the phloem. transporter. Reprinted from Trends in Plant Science 9, T. Roitsch and M. C. Gonza´lez, Function and Regulation This decreases phloem pressure (i.e., turgor C of Plant Invertases: Sweet Sensations, Pages 606–613, potential, P), which in turn increases the Copyright 2004, with permission from Elsevier. berry’s sink strength and thus the flow rate of 134 5. PARTITIONING OF ASSIMILATES phloem sap to the berry. Because sugar and pathways within the leaf is termed allocation. other solutes are actively pumped into the Of course, allocation is also important in the berry vacuoles, they remain there for good or sink organs that import carbon. Allocation can at least until they are consumed by other be grouped into three main categories: organisms. Under conditions that limit sink Utilization: Fixed carbon is consumed in activity, however, less sucrose is unloaded respiration for energy (ATP) “generation” or from the release phloem, which consequently used as building blocks for the production of slows the flow rate in the transport phloem. other components (mainly amino acids) This leads to sucrose “congestion” in the collec- needed by the cell for metabolism and tion phloem, and sucrose becomes “stuck” in growth. the leaf apoplast (with apoplastic loading) or Storage: Fixed carbon is converted to starch the mesophyll cells (with symplastic loading), during the day and stored within the which ultimately leads to feedback inhibition chloroplast for remobilization at night and of photosynthesis (see Chapter 4.2). Such con- when environmental constraints limit ditions may include water deficit, nutrient defi- photosynthesis. ciency, or low temperatures (see Chapter 7). Transport: Fixed carbon is converted to sucrose Nutrient ions and other small solutes probably for temporary storage in the vacuole (mainly move from the phloem to most sink tissues pas- as a buffer against short-term fluctuations in sively by diffusion through the plasmodesmata sucrose formation) or export to sink organs. or by bulk flow. The pressure flow mechanism requires water to flow out of the phloem along As discussed in Chapter 4.2, carbon alloca- with the unloaded solutes. Therefore, in addition tion is regulated by the availability of triose to delivering nutrients, the bulk flow of phloem phosphate sugars and inorganic phosphate. sap provides a mechanism to meet the water The coordination of the allocation of fixed car- demands of expanding sink cells (Lalonde et al., bon to form either sucrose or starch is of partic- 2003; Patrick, 1997; Patrick et al., 2001) because ular importance because only sucrose is cells, and therefore tissues and organs, grow as available for immediate export. This coordina- water moves across the cell membrane down a tion is at least partly driven by the demand water potential gradient (see Chapter 3.1). This for sucrose by all the sinks on a vine. High sink is especially true of expanding grape berries (see demand removes sucrose from the source, Chapter 6.2), but even in growing roots a consid- which favors sucrose production over starch erable portion of water is derived from phloem production. Therefore, once the source’s own import. This is necessary because transpiration needs have been met (allocation to utilization), rates in sink tissues are typically too slow to drive the proportion of currently produced assimilate sufficient water import via the xylem to sustain allocated to storage in the leaf is determined by growth of these organs. the sink demand for sucrose (allocation to transport). On the other hand, an abundance 5.1.1. Allocation and Partitioning of fixed carbon in the source can also stimulate sink growth, apparently by inducing the pro- The amount of photosynthate available for duction of additional sucrose transporter export from a source leaf depends on the leaf’s proteins. Moreover, both the proportion and carbon balance, which is determined by its the total amount of carbon stored as starch in photosynthetic rate and metabolic activity. a leaf also depend on day length—that is, The way in which the leaf regulates the distri- season and latitude (Stitt et al., 2007). Although bution of fixed carbon to the various metabolic a greater portion of the fixed carbon is allocated 5.1. PHOTOSYNTHATE TRANSLOCATION AND DISTRIBUTION 135 to starch during short days, the total amount is As new leaves are added on the shoot, the smaller than that produced during long days. older leaves find themselves at increasing dis- This is followed by slower rates of starch deg- tance from the shoot tip and begin exporting radation during the longer nights so that the toward the shoot base (Figure 5.3). Assimilate overall sucrose export over a 24-h period is less transport is a dynamic process that can be in short days. This adjustment of starch turn- adapted to the developmental status of the over to the amount of carbon fixed during the vine and to changing requirements imposed day leads to slower growth rates during short by the environment. In other words, although days, which balances the utilization of carbon sources usually supply materials to nearby sinks, with its supply (Stitt et al., 2007). they do not do so to all sinks on a plant equally The distribution of exported assimilates to (Wardlaw, 1990). Certain sources preferentially the various sink organs is called partitioning, supply specific sinks, but the supply pathways and it occurs via source–sink turgor gradients are flexible. Moreover, as the various organs of within the phloem. Leaves that have recently a vine develop, they compete with each other become sources initially export their assimilates for space, light, and nutrients. Therefore, there to the growing shoot tip and unfolding leaves. is a hierarchy of relative priorities among the

FIGURE 5.3 Source–sink relations along a grapevine shoot change over the course of a growing season. Arrows indicate direction of movement. Reproduced from Koblet (1969). 136 5. PARTITIONING OF ASSIMILATES sinks, and this hierarchy is dynamic and sensi- meters away (Koblet and Perret, 1972; tive to environmental variables. Flowers are gen- Meynhardt and Malan, 1963), the closer a erally poor competitors (especially under source is to a specific sink, the more likely it source-limiting conditions), whereas after fruit will supply materials to that sink (Wardlaw, set the berries and seeds dominate the shoots, 1990). The mature leaves closest to the shoot which in turn often outcompete the roots tip generally export their assimilates to the (Buttrose, 1966a; Hale and Weaver, 1962; growing tip, whereas the more basal leaves Wardlaw, 1990). How much of the available preferentially supply the grape clusters and assimilates a particular sink organ receives (beginning around bloom) the permanent (imports) is a matter of supply and demand. parts of the vine. The intermediate leaves The import rate depends on the organ’s sink export in both directions (Hale and Weaver, strength (i.e., assimilate demand) relative to the 1962; Koblet, 1969). strength of all other sinks on the same vine and Connection: A source leaf favors a sink with also on the total amount of assimilate available which it is directly connected via vascular from the various sources (i.e., assimilate supply). bundles. Any leaf on a shoot is usually Sink strength is defined as the product of sink connected with the leaves and clusters above size (total weight of a sink) and sink activity and below it (i.e., on the same side of the (rate of assimilate import per unit sink weight). shoot). Therefore, a flower or fruit cluster is However, small sink strength is not always supplied mostly by the leaves located on the associated with low sink priority; seeds are too same side of the shoot as itself (Koblet, 1969; small to be strong sinks but are generally the Motomura, 1990, 1993; Yang and Hori, 1980). top-priority sinks (i.e., they are ranked higher This “unilateralism” even applies to the than the surrounding berry flesh), whereas supply of clusters with assimilates derived reserve storage pools (e.g., in the wood of canes, from lateral shoots (Koblet, 1975; Koblet and trunks, and roots) usually have the lowest sink Perret, 1971). priority (Minchin and Lacointe, 2005; but see later Interference: The “normal” pathways of discussion). The relative priority of a particular translocation can be altered by wounding or sink depends on its ability to lower the concen- pruning, which interrupts the direct tration of sucrose in the phloem—that is, on the connection between a source and a sink. rate of phloem unloading—and thereby maintain Following such interruption, vascular a favorable pressure gradient to the source interconnections (anastomoses) can provide (Patrick, 1997; Wardlaw, 1990). The sinks usually alternative connections (e.g., cross-transfers). convert most of the imported sucrose to the This has implications for canopy hexose sugars glucose and fructose, using inver- management. Hedging (shoot tipping or tase and/or sucrose synthase, before they can topping) stimulates cross-transfer of use the sugar for their own metabolic processes, assimilates to clusters on both sides of the including the reassembly of starch (Hawker shoot and induces even young leaves to et al., 1991). Modifying either the size or the switch from upward (acropetal) export to activity of a sink leads to changes in assimilate downward (basipetal) export that can extend transport patterns in a vine. The following to neighboring nontopped shoots (Koblet patterns are important in partitioning: and Perret, 1972; Quinlan and Weaver, 1970). This is probably why shoot tipping during Proximity: Although leaves on one shoot can bloom often improves fruit set (Coombe, export assimilates to fruit clusters on other 1959, 1962; Vasconcelos and Castagnoli, shoots, even if they are located several 2000), although the subsequent outgrowth of 5.1. PHOTOSYNTHATE TRANSLOCATION AND DISTRIBUTION 137

lateral buds may later reverse assimilate Mechanical elimination by shoot hedging of flow again. Defoliation of a shoot triggers the auxin signal required for root growth compensatory assimilate import into that may further enhance delivery to the clusters shoot’s clusters from neighboring shoots because lack of auxin flow may temporarily (Quinlan and Weaver, 1970). Leaf removal or inhibit root growth, so long as outgrowth of shade in the cluster zone force more distal lateral buds does not generate new shoot leaves to export their assimilates to the apices. clusters (but not to the shaded leaves), Competition: Competition determines the whereas loss (or shading) of younger leaves priority of each sink relative to all other induces older leaves to reverse the export sinks on the plant in terms of assimilate direction toward the shoot tip at the expense distribution. The greater the ability of a sink of the perennial parts of the vine. Fruit to store or metabolize imported assimilates removal on a shoot induces that shoot to (i.e., the higher its sink capacity), the better is export assimilates to neighboring shoots its ability to compete for exported with clusters (Quinlan and Weaver, 1970). assimilates. A rapidly growing sink is Girdling (phloem removal) of a shoot leads competitive because the consumption of to the leaves exporting assimilates to clusters assimilates lowers sink pressure. A on both the same and opposite sides of that competitive sink is said to have high sink shoot (Motomura, 1993). strength. Moreover, the greater the number Communication: Grapevines carefully balance of sinks competing for assimilates, the less their investment in growth and reproduction available for each individual sink. This has (reproductive productivity). Vegetative implications for winter pruning and canopy growth also must be balanced between shoot management. Leaving more buds per vine growth (photosynthetic productivity) and increases the number of shoots and clusters root growth (water and nutrient uptake). As but decreases the vigor of each shoot and the Goethe stated, “in order to spend on one proportion of flowers that set fruit. side, nature is forced to economize on the Moreover, increasing the number of berries other side” (cited in Darwin, 2004). This per plant or per unit leaf area can limit berry requires interaction between centers of expansion (Keller et al., 2008) or slow the rate supply and demand, which happens by way of fruit ripening. Conversely, applying of pressure gradients (low sink pressure gibberellin to table grape clusters increases stimulates import via the phloem), nutrients, not only berry size but also the amount of and hormones. Cytokinins produced by imported assimilates (Weaver et al., 1969). roots or seeds and transported to the leaves Development: The relative importance of in the xylem may integrate assimilate supply different sinks changes during plant with sink demand (Lalonde et al., 2003; Paul development. Shoot tips have high priority and Foyer, 2001). The same may be true for after budbreak, when the vine needs to auxin produced in shoot tips and seeds and establish its new canopy (Hale and Weaver, exported in the phloem and parenchyma 1962). By contrast, clusters are unimportant cells. A developing grape cluster can attract sinks until bloom. This is why shoot topping assimilates from nearby shoots, especially or trunk girdling during bloom may when assimilate production exceeds those improve fruit set, whereas loss of leaf area shoots’ own demand (Currle et al., 1983; reduces set (Coombe, 1959, 1962). After fruit Intrigliolo et al., 2009; Koblet and Perret, set, however, the clusters become powerful 1972; Quinlan and Weaver, 1970). sinks and dominate the sink hierarchy 138 5. PARTITIONING OF ASSIMILATES

during grape development, especially for from the “permanent” parts of the vine, such adjacent and nearby leaves (Hale and as canes, spurs, cordons, trunks, and roots, Weaver, 1962; Williams, 1996). After which are therefore depleted beginning before veraison, the clusters are initially very strong budbreak (Eifert et al., 1961; Loescher et al., sinks, but the vine then gradually shifts its 1990; Williams, 1996; Zapata et al., 2004). Even priorities to the wood and roots to replenish unfolding leaves, before becoming sources, storage reserves and acquire cold hardiness must compete with other sinks for assimilates, (Candolfi-Vasconcelos et al., 1994). yet they are stronger sinks than the cambium In summary, for an organ or tissue to be a of the cane, trunk, and roots (Wardlaw, 1990). strong sink, it pays to be large, close to a source, As more leaves are added on the shoot, they and have good connections. However, many increasingly support the shoot’s further other factors also modulate sink strength and growth, which soon becomes independent of assimilate partitioning. Environmental variables the parent plant. The growing shoots also begin play an important role in modifying the pattern to contribute to radial growth via the cambial of partitioning. For instance, soil water or nutri- activity in the permanent parts of the vine. ent deficits decrease overall vine growth but Beginning at approximately the time of bloom, increase the proportion of assimilates parti- assimilate export from the shoots normally tioned to the roots (see Chapters 7.2 and 7.3). begins to replenish the parent plant’s storage Source and sink organs are parts of a single reserves and also sustains secondary growth inseparable system, and an effect on one part of the cordons, trunk, and roots. is bound to have a consequential and concur- The ability to refill the storage pools also rent influence on the other. For instance, depends on the amount of fruit the vine has to removing a sink improves the availability of support because the berries generally dominate assimilates to adjacent sinks (Quinlan and the hierarchy of sink priorities between fruit set Weaver, 1970). In viticulture, we are mainly and seed maturity (Hale and Weaver, 1962; interested in maximizing or optimizing the Williams, 1996). Although the replenishment of vine’s investment in reproduction (i.e., fruit reserves is often described as an overflow mecha- production). In mature vines, the proportion nism that directs “surplus” assimilates to storage of total biomass (dry matter) partitioned to the only after all other assimilate-requiring pro- fruit can range from 10% (e.g., in nonirrigated, cesses, such as growth and fruit production, have relatively water-stressed, cluster-thinned Pinot been satisfied (Lemaire and Millard, 1999), this noir) to 70% (e.g., in heavily irrigated, mini- may depend on the vine’s developmental stage. mally pruned Concord). The dilemma for The woody parts of the vine may become high- grapevines as perennial plants is to maximize priority sinks late in the season (Loescher et al., annual seed dispersal (short-term reproductive 1990), when the plant “loses interest” in the fruit output) while at the same time ensuring long- and focuses on the replenishment of storage term survival of the plant (for long-term repro- reserves and on cold acclimation (see Chapter ductive output). The vine must coordinate the 7.4). This appears to happen after about midri- supply of photosynthate to seed production pening (Candolfi-Vasconcelos et al., 1994; and, hence, fruit development such that it does Wample and Bary, 1992) and can clash with a not occur at the expense of other essential pro- grower’s or winemaker’s desire to improve fruit cesses and structures. Early shoot growth dur- quality by delaying harvest (i.e., prolonged “hang ing and after budbreak is completely time”), especially when adverse environmental dependent on remobilized storage reserves conditions limit the availability of resources. 5.1. PHOTOSYNTHATE TRANSLOCATION AND DISTRIBUTION 139

The net assimilation rate of a grapevine can- Nitrogen that could be remobilized from the opy determines the export rate from the canopy, leaves and transported to the perennial parts of and the export rate increases with increasing the vine for storage is lost when leaves are source–sink turgor gradient. Therefore, the best removed before normal abscission. Alterna- way to increase assimilate translocation is to tively, inadequate carbohydrate reserves in the increase the rate of photosynthesis in source roots, by curtailing new root growth, could also leaves because an increase in the amount of lead to nitrogen deficiency (Loescher et al., photosynthate available for export increases 1990). However, even before such nitrogen defi- the turgor at the source, which results in a ciency may limit plant growth, it is possible that greater turgor gradient between source and an inadequate reserve status may interfere with sink. Leaves that unfold under intense competi- the plant’s ability to generate adequate root pres- tion from other sinks often achieve higher pho- sure prior to budbreak (Ame´glio et al., 2001; see tosynthetic rates than leaves that do not also Chapter 2.2), which would not permit the experience competition during development. vine to purge air bubbles from the xylem, poten- However, even in fully grown leaves, photo- tially leading to poor budbreak or early canopy synthesis can still be influenced by natural or collapse. manipulated changes in sink demand (Down- In contrast, excessive fruit removal (i.e.. sink ton et al., 1987; Flore and Lakso, 1989; Paul removal, for example, cluster thinning), espe- and Foyer, 2001). Thus, the presence of fruit cially if done early, may result in an assimilate on a vine tends to increase the leaves’ photo- surplus due to a shortage of demand and can synthetic rate, especially in vines with small decrease photosynthesis because of feedback leaf area that leads to source limitation (Edson inhibition from accumulating sugar (Currle et al., 1993; Eibach and Alleweldt, 1984; et al., 1983; Downton et al., 1987; Iacono et al., Hofa¨cker, 1978). The fruit also induces a shift 1995; Naor et al., 1997; Paul and Foyer, 2001; in assimilate supply to reproductive growth at Roitsch, 1999). Alternatively, the surplus sugar the expense of vegetative growth, especially may be used to stimulate vegetative growth root growth, keeping whole-vine biomass (vigor), especially of lateral shoots (Pallas constant (Edson et al., 1993; Petrie et al., 2000b; et al., 2008; Petrie et al., 2000b), which may Williams, 1996). This shift is particularly pro- result in dense, shaded canopies. However, nounced at low soil moisture (Eibach and vines without fruit often begin to “shut down” Alleweldt, 1985). Destruction or removal of leaves earlier in autumn, which is visible as leaf chlo- (i.e., source removal, for example, to improve rophyll content declines in response to fruit exposure to sunlight) also leads to a decreasing photosynthesis, and the leaves are compensatory increase in photosynthesis of shed before those on similar plants with fruit the remaining leaves and a delay of their senes- (Figure 5.4). Such premature leaf senescence cence (Buttrose, 1966a; Candolfi-Vasconcelos maybetriggeredbytheaccumulationof and Koblet, 1991; Currle et al., 1983; Hofa¨cker, surplus sugar in the leaves (Lim et al., 2007; 1978; Hunter and Visser, 1988; Iacono et al., Rolland et al., 2006; Wingler et al., 2009). 1995). However, this compensation is incom- Whereas the presence of fruit influences the plete, and severe defoliation may retard fruit physiology of the rest of the vine, fruit growth development and ripening (Petrie et al., 2000a). often compensates for changes in sink number, If defoliation occurs late in the season, it can andthisiscalledtheyieldcomponentcom- result in poor carbohydrate reserve status and pensation principle (see Chapter 6.1). Thus, in nitrogen deficiency the following growing early loss of fruit, such as due to poor fruit season (Loescher et al., 1990; Sartorius, 1973). set or early thinning, is partly compensated 140 5. PARTITIONING OF ASSIMILATES

export increases the production of vascular tis- sues in the pedicel so that more water and nutrients can be imported by the fruit (Else et al., 2004). Indeed, fruit size and pedicel cross-sectional area are strongly correlated, and the (dry) weight of a cluster can be estimated from the diameter of its peduncle (Castelan-Estrada et al., 2002). Therefore, berries that begin development with a head start (differences in the rate of cell division before bloom can result in differences in ovary FIGURE 5.4 Senescing Cabernet Sauvignon leaves in size among berries) usually remain more com- early November (Northern Hemisphere) from plants that petitive throughout their growth and ripening had all fruit removed at veraison in mid-August (left) or with the fruit still on the plant (right, photo by M. Keller). (Coombe, 1976). Grapevines often form large numbers of mer- istems (shoot and root tips and berries) capable by increased growth of the remaining fruit, of growth under favorable conditions. Thus, which tends to lead to an increase in berry size they respond strongly to variations in the avail- (Keller et al., 2008). If the loss in sink number ability of resources: They are said to be very occurs before bloom (e.g., due to cold injury), “plastic” (Lawlor, 2002). Environmental condi- then the proportion of flowers that set fruit tions, therefore, also play an important role in often increases in addition to berry growth, the regulation of partitioning. Low irradiance which can result in compact clusters with stimulates carbon supply to the shoots, whereas large berries. Conversely, vines usually self- low nutrient or water availability favors supply adjust to a large number of sinks (e.g., due to to the roots (Keller and Koblet, 1995a). In the high bud numbers following mechanical or case of water, this does not mean that root minimal pruning) by lowering the percentage growth actually increases in response to water of fruit set and decreasing berry growth so deficit. On the contrary, root growth decreases, that there may be many small, loose clusters but less so than shoot growth. These relation- with small berries. ships are explored in subsequent chapters. A puzzling phenomenon in grapes is that different berries on the same cluster seem to 5.2. CANOPY–ENVIRONMENT develop independently at different rates INTERACTIONS (Coombe, 1992; Coombe and Bishop, 1980) and to accumulate very different amounts of sugar, even though they may be supplied by Grapevine productivity (growth, yield, and the same phloem strand through the peduncle. fruit composition) ultimately depends on the This shows that individual berries, rather than photosynthetic capacity of the vine’s canopy, the cluster as a whole, are able to control deliv- integrated over the growing season. A canopy in ery of phloem solutes. Berries containing more the viticultural sense is defined as the above- seeds than others seem to be better able to ground parts of a vine (i.e., shoots, leaves, fruit, attract assimilates, possibly because each seed trunk, and cordon). Compared with leaves, shoot produces and exports auxin (see Chapter 2.3). and berry photosynthesis is minor and never Because auxin stimulates cambial activity and exceeds respiration. Therefore, canopy photo- xylem and phloem development, more auxin synthesis is the sum of the photosynthetic activity 5.2. CANOPY–ENVIRONMENT INTERACTIONS 141 of all the leaves minus the respiratory activity of an overview of the importance of aboveground the leaves and all nonphotosynthetic tissues. climatic variables. Whereas the term “weather” Although canopy photosynthesis is to a large refers to the daily, seasonal, and annual extent determined by the availability of fluctuations of temperature, precipitation, resources, internal factors also play a role. For humidity, and wind, “climate” constitutes the example, the length and layout of the water-flow summarized and averaged weather situation pathway from the roots to the leaves can limit over a long time. A “long time” is defined by photosynthesis via the effect of hydraulic resis- the World Meteorological Organization as 30 tance and gravity on stomatal conductance (see years or more. In viticulture, we often distin- Chapter 3.3). This influence of a plant’s “plumb- guish three levels of climate: ing” design and length has been put forward as Macroclimate: The climate of a region, which is one of the reasons photosynthesis and vigor tend ordinarily described by data collected at one to decline as plant height and age increase (Ryan or several weather stations. It is sometimes and Yoder, 1997). Alternatively, the decrease in viewed as the mean of all the microclimates gas exchange in tall plants may be a consequence, in a region. It is mainly determined by the rather than the cause, of slower growth because geographic location (i.e., latitude, altitude, growth is more sensitive to water deficit than and distance from large bodies of water) photosynthesis (Ryan et al., 2006). In this case, but is independent of local topography, hydraulics and gravity limit the water potential soil type, and vegetation. The size of the gradient from the xylem to the growing cells region may extend for hundreds and, hence, the turgor necessary for cell expan- of kilometers. sion, which decreases sink strength (see Chapter Mesoclimate: The climate of a site or large 3.1; Bond et al., 2007). In support of this sink limi- vineyard. It is a local variant of the tation hypothesis, taller plants tend to accumulate macroclimate modified by topography (and greater amounts of reserve carbohydrates than do hence also called topoclimate). It may differ shorter plants (Sala and Hoch, 2009). Alterna- from the macroclimate because of altitude or tively, a lower water potential gradient from the elevation from a valley floor. The extent of a xylem to the phloem might curb phloem export particular mesoclimate may be from from the leaves (Barnard and Ryan, 2003). The hundreds of meters to several kilometers. advantage in the latter two scenarios is the same: This is the climate that is relevant for Sugar accumulates in the leaves and curtails pho- vineyard site selection. tosynthesis by feedback inhibition. Whatever its Microclimate: The climate within and direct and indirect consequences, hydraulic limi- immediately surrounding the canopy or tation is probably of little importance in within a vineyard. It may differ from the cultivated grapevines whose size—in contrast mesoclimate because of aspect, slope, and with their tall, tree-climbing wild relatives—is even soil type. Due largely to the presence of strictly limited by trellis design and annual prun- leaves, differences in microclimate may ing practices. occur over as little as a few centimeters or Resource availability is determined by mete- over hundreds of meters. This is the climate orological or climatic (Greek klima ¼ surface of that can be manipulated by vineyard the earth, region) conditions in addition to cultural practices. edaphic (Greek edaphos ¼ ground, soil) factors. The impact of variables related to the soil, such Compared with the situation close to the as water and nutrient availability, is discussed equator, regions at higher latitude experience in Chapters 7.2 and 7.3; this section provides longer periods of summer daylight. Greater 142 5. PARTITIONING OF ASSIMILATES day length enables leaves to be photosyntheti- Greece and Turkey, China’s Beijing region, cally active during a greater portion of each northern California, and near 40SHawke’s day, but this benefit is partially offset by the Bay in New Zealand. During the summer, a lower intensity of solar radiation. This is 50% south-facing slope receives approxi- because from a grapevine’s viewpoint, the sun mately 25% more solar energy than a north- “passes” lower on the horizon—that is, at a facing slope, but the difference is much greater greater zenith angle, whereby the zenith is in the winter because of the lower elevation of directly overhead, at 0, and the horizon is at the sun (Holst et al., 2005). Not only does the 90. Thus, at the summer solstice, or the “lon- disparity in radiation translate into earlier gest” day, the total daily global irradiance is budbreak on south-facing slopes but also, approximately equal between the 30th and the because the energy difference due to vineyard 50th parallel, where most of the world’s grapes aspect increases during the ripening period, are grown. Altitude (elevation) and aspect of a north-facing slopes can be at a serious dis- vineyard site also determine the radiation advantage in marginal climates. Differences received by the vines. The effect of altitude at are also much larger under clear skies; clouds the same latitude is particularly noticeable as filter out the direct radiation from the sun so a strong and predictable decrease in tempera- that during overcast days there is only diffuse ture with increasing elevation (0.5 C for each radiation. Thus, in regions with frequent cloud 100 m), provided the atmosphere is well mixed, cover (few sunshine hours) the incident radia- which occurs courtesy of solar heating and tion is similar regardless of aspect. The leaves wind. Nonetheless, at night and during the (and the roots) perceive differences in solar winter (especially in sheltered valleys), such radiation mainly as differences in tempera- mixing is often insufficient (especially during ture, with more radiation leading to higher clear, calm weather) to prevent the formation daytime air and soil temperature, whereas of so-called temperature inversions. During light intensities above the canopy are similar such inversions, cold air settles in valley floors regardless of aspect, at least during the and local depressions so that the temperature growing season (Holst et al., 2004; Mayer is coldest at ground level and increases et al., 2002). (2.5–3 C per 100 m) up to the inversion top It is clear that annual climate variation and (typically 200–300 m above ground level), short-term weather fluctuations are also impor- above which it begins to decrease normally tant in viticulture. Whereas the long-term cli- (Daly et al., 2008). matic averages are pertinent to site selection A 10% south-facing slope at 45N receives and choice of cultivars and rootstocks when as much radiation, and hence energy, during establishing a new vineyard, climate variation the April–October growing season as a hori- among growing seasons and weather variation zontal plain at 40 N. Grape-growing regions within seasons (see Chapters 7.2, 7.4 and 7.5) near the 45th parallel, the midpoint between often influence management decisions in estab- the equator and the poles, include Bordeaux lished vineyards. Climatic conditions that are or the Rhoˆne Valley in France, Italy’s relevant for individual vines are part of the Piemonte, Croatia, Ukraine’s Crimean Penin- (canopy) microclimate, which is strongly influ- sula, northern China, Oregon’s Willamette enced by the presence of leaves. These condi- Valley, and, in the Southern Hemisphere, tions include light, temperature, wind, and New Zealand’s Otago region. Examples of humidity (Smart, 1985). Leaves alter all of these regions near the 40th parallel include central from the exterior of the canopy to the interior Spain and Portugal, southern Italy, northern and from the top to the bottom. 5.2. CANOPY–ENVIRONMENT INTERACTIONS 143

5.2.1. Light protein and carotenoids (especially xantho- phylls) and up to 50% lower respiration rates Light has a more profound effect on plant than sun leaves (Evans, 1989; Schultz, 1991; development than does any other climatic fac- Seemann et al., 1987). These adaptations tor or signal. Grapevines, like all plants, use enhance light absorption and energy transfer light both as a source of energy (in fact, as their in the shade but make these leaves consider- only energy source) and as a source of informa- ably less efficient at higher light intensity tion. They can accurately perceive fluctuations (Ortoidze and Du¨ ring, 2001; Schultz et al., in quantity (intensity), quality (spectral compo- 1996). Moreover, such leaves are highly light sition), directionality, and periodicity (day sensitive, and when they are suddenly exposed length) of the incoming light (Fankhauser and to the sun, they can suffer from severe oxida- Staiger, 2002). The amount of incident light on tive stress and photoinhibition and may even a grapevine canopy varies with latitude, sea- die in extreme cases (Iacono and Sommer, son, time of day, and cloud cover. Leaves effec- 1996; Triantaphylide`s et al., 2008; see also tively absorb sunlight in the visible and Chapter 7.1). ultraviolet (UV) region of the electromagnetic In complete darkness (i.e., at night), a leaf’s spectrum (Blanke, 1990a). More precisely, the net CO2 assimilation is negative because there epidermis absorbs most of the potentially dam- is only respiration but no photosynthesis (see aging UV light but is transparent to visible Chapter 4.4), and the leaf’s metabolism and light, which penetrates the chloroplast-rich continued sugar export depends on the break- mesophyll cells (see Chapter 1.3). The visible down of starch accumulated during the day. wavelength range from 400 to 700 nm is uti- As the irradiance or light intensity or photon lized in photosynthesis (see Chapter 4.1), and flux (number of photons per unit surface area this light is called photosynthetically active per unit time, expressed in mmol m 2 s 1) radiation (PAR). In other words, plants are increases, photosynthetic CO2 uptake increases. mainly sensitive to the same spectral “win- The light intensity at which the leaf’s net CO2 dow” of light that we see as the colors of the assimilation is zero (i.e., photosynthetic CO2 rainbow. Approximately 85–90% of incident uptake balances respiratory CO2 release) is light in the PAR range falling on a grape leaf called the light compensation point. This point is absorbed by the leaf; the rest is either depends on species, cultivar, and developmen- reflected at its surface (6%) or transmitted tal conditions, but in typical grapevine leaves it through the leaf (4–9%) (Smart, 1985). is reached at an irradiance of approximately Because absorbing an optimal amount of 10–30 mmol m 2 s 1 (Du¨ ring, 1988; Keller and light is so important for leaves, they have Koblet, 1994; Ru¨ hl et al., 1981). A further evolved anatomical and physiological strate- increase in photon flux leads to a concomitant, gies that allow them to adapt to a range of light almost linear increase in photosynthesis until environments. Leaves grown in the shade (e.g., it starts to level off and reaches light saturation. in the canopy interior) are larger but thinner Grapevine leaves generally reach light satura- than leaves grown in full sunlight, mainly tion between 700 and 1200 mmol m 2 s 1 because of their drastically shortened palisade (Figure 5.5), which is well below the photon cells. As a consequence of their larger size, flux of full sunlight (which can exceed shade leaves have fewer stomata per unit leaf 2000 mmol m 2 s 1) but above that under cloud area than do sun leaves. Shade leaves also have covers (100–1000 mmol m 2 s 1, depending on more total chlorophyll per reaction center and cloud type and density). The irradiance at per unit nitrogen, but they have less rubisco which individual leaves reach light saturation 144 5. PARTITIONING OF ASSIMILATES

FIGURE 5.5 Relationship between light intensity and photosynthesis (left) and between stomatal conductance and light-saturated photosynthesis (right) of mature grapevine leaves. Note that the influence of cultivar is insignificant com- pared with that of light and nitrogen status (M. Keller, unpublished data). varies widely, depending again on species, cul- not static due to wind moving shoots and tivar, developmental stage, and nutrient status, leaves, which together with the absorption but it generally reflects the maximum irradi- and reflection of light by leaves and soil creates ance to which a leaf was exposed during its an irregular patchwork of highly variable irra- growth. Moreover, at any given stomatal aper- diance within the canopy. Light incident on ture width (i.e., stomatal conductance), light- individual leaves, or only on sections of a leaf, saturated photosynthesis “runs” faster in vines can fluctuate within milliseconds to minutes, with high nutrient (especially nitrogen) status often resulting in fleeting sunflecks (Kriede- than at low nutrient status (see Figure 5.5). mann et al., 1973; Rascher and Nedbal, 2006). Photosynthesis below light saturation is The contribution of these sunflecks to the daily referred to as light limited (i.e., insufficient photon flux at any given point within a typical light for maximum photochemistry), and that canopy varies from 20 to 90%. Leaves that above saturation is referred to as CO2 limited experience frequent but brief sunflecks benefit (i.e., enzymatic reactions cannot keep pace with most in terms of photosynthesis, but because photochemistry). The fact that less than 10% of shaded leaves can heat up very rapidly upon the PAR falling on a grape leaf will reach the sudden exposure to sunlight, they are prone leaf underneath has implications for canopy to heat damage and wilting from water stress photosynthesis. Only the leaves on the exterior (Pearcy, 1990). of a canopy are exposed to saturating irradi- Depending on trellis and training system, ance, so only these exterior leaves will achieve and on vine vigor, light in the fruiting zone maximum rates of photosynthesis (Smart, can range from less than 1% of ambient (e.g., 1974). The interior leaves receive much less non–shoot-positioned single-curtain trellis) to light (often less than 10 mmol m 2 s 1 inside a approximately 10% (e.g., vertically shoot- dense canopy) that is either transmitted positioned systems) and more than 30% (e.g., through other leaves or passes through gaps double-curtain systems) (Dokoozlian and in the canopy (Dokoozlian and Kliewer, 1995a, Kliewer, 1995b; Gladstone and Dokoozlian, b; Mullins et al., 1992). Most of these gaps are 2003; Williams et al., 1994). Moreover, the 5.2. CANOPY–ENVIRONMENT INTERACTIONS 145 shaded side of a canopy can receive as little for their share of the respiratory costs of the as 3–6% (40–100 mmol m 2 s 1) of the light shoots and roots that support them (Reich intercepted by the sunlit side (Smart, 1985). et al., 2009). Because a decline in photosynthesis As a consequence, whole-canopy photosynthe- is accompanied by a decline in stomatal con- sis is almost never light saturated (Flore and ductance and consequently in transpiration Lakso, 1989; Intrieri et al., 1997; Poni et al., rate, the delivery to these leaves of root-derived 2003). On the other hand, shade leaves inside cytokinins via the transpiration stream also a grapevine canopy have 30–50% lower respira- decreases, which may serve as a signal to initi- tion rates than sun leaves at the canopy surface, ate the early senescence program (Boonman irrespective of temperature and leaf age. et al., 2007, 2009; Buchanan-Wollaston, 1997; Although this adaptation (together with their Pons et al., 2001). The ratio of red:far-red light lower light compensation point) enables many incident on shaded leaves is also lower than shade leaves to maintain a positive daily net at the canopy surface, and this may provide carbon balance, this balance is only approxi- an additional senescence signal (Boonman mately 10–20% of that of sun leaves. Although et al., 2009; Rousseaux et al., 2000). Leaf senes- particle films (e.g., clays such as kaolin) applied cence therefore depends more on a leaf’s posi- to canopies to protect the vines from heat and tion than on its age, and abscission due to water stress decrease light absorption by the canopy shading or during stress may be leaves, they can leave whole-canopy photosyn- viewed as the elimination of surplus leaves that thesis unaffected or even enhance it because do not contribute to canopy photosynthesis the particles also reflect light. This reflection (Hikosaka, 2005). Senescence is accompanied can improve the light distribution within the by remobilization of carbon, proteins, and min- canopy, which tends to compensate for the eral nutrients from these leaves, although decrease in photosynthesis of the exterior approximately half of a leaf’s resources cannot leaves. Similarly, the contribution of interior be recycled and are lost (Bertamini and leaves to whole-canopy photosynthesis is Nedunchezhian, 2001; Hikosaka, 2005). This greater in loose, open canopies than in dense adaptive strategy enables the plants to survive canopies, where the outermost leaves absorb episodes of limited supply of photosynthates almost all of the available light (Smart, 1974). by remobilizing buffer reserves from older, Grapevines also adjust the distribution of sequentially senescing leaves and permanent proteins, chlorophyll, and photosynthetic parts of the plant and translocating them to capacity to canopy density so that well-exposed organs with high sink priority to temporarily leaves have high nitrogen and photosynthetic supply carbon for maintenance and/or growth capacity per unit leaf area (Bowen and Kliewer, processes (Geiger and Servaites, 1991; Hunter 1990; Kriedemann, 1968b; Williams, 1987). et al., 1994). Leaf senescence does not cause When photosynthesis in some leaves drops any change in the direction of phloem transport below a certain threshold level for some time, in the leaf: There is no switch from export to the vine sheds these leaves by initiating the import. There is only a progressive change in processes of senescence and abscission in order the nature of the exported materials from pre- to prevent a wasteful situation in which the dominantly sucrose to mostly amino acids and leaves’ demand for water and nutrients out- other nutrients. In other words, shaded leaves weighs their supply of fixed carbon (Flore and in the interior of dense canopies are not “para- Lakso, 1989; Poorter et al., 2006; Taylor and sitic” on a vine (Koblet, 1975; Quinlan and Whitelaw, 2001). In other words, the leaves Weaver, 1970; Wardlaw, 1990). Such leaves still are dropped when they can no longer “pay” maintain a positive daily carbon balance and 146 5. PARTITIONING OF ASSIMILATES are simply discarded following recycling of and, for fully developed canopies, varies from their accessible resources: A senescing leaf is 30 to 85% of the total leaf area depending on still a source (Reich et al., 2009). Where the trellis design and row spacing. Vines with a recycled compounds end up depends on the small proportion of exposed surface area have relative strength of the various sink organs. many shaded (interior) leaves and may pro- When the shoots are actively growing, remobi- duce less assimilate for export to be available lized nutrients are redistributed to young, bet- for fruit ripening, root growth, nutrient uptake ter exposed leaves, but when shoot growth and assimilation, and replenishment of storage has stopped (e.g., due to water deficit), the fruit reserves for cold hardiness and spring growth. clusters or the permanent structures may be the The fact that photosynthetic CO2 assimila- main recipients of recycled nutrients. tion shows light saturation while the absorp- Grapevines are quite shade tolerant and tion of photons continues to increase with adapt to low light by altering leaf and shoot rising irradiance is a potential source of trou- growth, although this occurs at the cost of ble for leaves. When a leaf absorbs more light greater shoot hydraulic resistance (Schultz and than it can utilize, some of the excess energy Matthews, 1993). When entire shoots or vines must be driven away (dissipated) as heat, or experience low light, such as during overcast else it will cause photoinhibition (see Chapter periods, grapevines produce new leaves, espe- 4.1). Photoinhibition in grapevines occurs fre- cially lateral leaves, rather than maintain the quently around midday, when incident light source capacity of old leaves; however, this and temperature are at a maximum, and leads response is at the expense of fruit production to a temporary reduction in CO2 assimilation and root growth (Keller and Koblet, 1995a). (Chaves et al., 1987; Correia et al., 1990; Such vines tend to have elevated concentra- Du¨ ring, 1999; Iacono and Sommer, 1996). Over tions of tissue nutrients and low amounts of the course of a growing season, this temporary reserve carbohydrates (Keller et al., 1995). depression of photosynthesis, known as Wild vines are very well adapted for maxi- “dynamic” photoinhibition, can result in a mum light capture. Their elaborate branching roughly 10% loss of potential biomass produc- structure with a large number of short shoots tion. Leaves acclimate to high light conditions armed with tendrils enables them to spread by moving the chloroplasts, which are nor- their foliage over tree canopies, which results mally aligned at the abaxial surface of meso- in an enormous increase in sunlight absorption. phyll cells (and thus perpendicular to the This option is not available to cultivated grape- solar rays to maximize light absorption), to vines that are often confined to small trellis sys- anticlinal positions in the cells (and thus paral- tems with foliage concentrated within a more lel to the solar rays). This reversible chloro- or less defined canopy volume; this is espe- plast relocation is regulated by a protein cially true for shoot-positioned vines. In fact, photoreceptor termed phototropin that is acti- less than 20% of the leaves often account for vated by blue light (450 nm) and is an attempt more than 80% of a vine’s total carbon assimila- to avoid photoinhibition (Li et al., 2009; Spalding tion. Therefore, the canopy surface area of a and Folta, 2005). Both low and high temperatures vineyard, rather than its total leaf area, is exacerbate the effect of strong light and photoin- important because the more solar energy that hibition (Gamon and Pearcy, 1990). This can be is intercepted by foliage, the greater is the bio- problematic in cool-climate regions, which mass production and the yield potential. Can- often experience large diurnal temperature opy surface area is referred to in terms of fluctuations so that vines can be exposed to exposed surface area or effective surface area below-optimum temperatures and high light 5.2. CANOPY–ENVIRONMENT INTERACTIONS 147 intensities in the morning (especially on the east radiation are detected by various photorecep- side of the canopy), particularly in spring and fall. tors or photosensors that absorb light and Off-season cold spells (combined with a clear translate the light signals via signaling net- sky) can result in irreversible damage due to deg- works into physiological responses. Perhaps radation of photosynthetic pigments; this condi- the most important photosensor is a pigment tion is called “chronic” photoinhibition (see system called phytochrome that works like a Chapter 7.4). light-regulated switch (Rockwell et al., 2006; Leaf layers alter not only the quantity of Smith, 2000). Like other plants, grapevines light but also its quality. The spectral character- have at least five different phytochrome pro- istics of chlorophyll make leaves strong absor- teins that are abbreviated phyA–phyE, and bers of photons in the blue (400–500 nm) and each exists as a mixture of two reversible red wavebands (600–700 nm) of the solar forms—an inactive form (Pr), which absorbs spectrum (Figure 5.6; see also Chapter 4.1). red light (660–670 nm) and has a half-life of Absorption of green (500–600 nm) and particu- approximately 100 h, and an active form (Pfr), larly far-red (700–800 nm) light is weaker which absorbs far-red light (725–735 nm) and (Blanke, 1990a), and many photons of these has a half-life of only approximately 1 h. When wavelengths are reflected or transmitted (and Pr absorbs red light, it alters its configuration to scattered) in the form of diffuse radiation. become Pfr, which induces other proteins to Thus, light that is reflected from leaves is carry Pfr from the cytosol to the nucleus. Inside enriched in the far-red (FR) region, which lies the nucleus, Pfr interacts with yet other proteins in the infrared portion of the electromagnetic termed transcription factors to modify gene spectrum, whereas light that is transmitted expression (Bae and Choi, 2008; Fankhauser through leaves is depleted in the red (R) region and Staiger, 2002; Franklin, 2008; Smith, 2000). of the spectrum. The outcome in both situations In other words, red light photoactivates Pfr, is identical: The R:FR ratio declines. These which then indirectly switches light-regulated changes in the spectral composition of solar genes on. Conversely, when Pfr absorbs far-red

FIGURE 5.6 Light reflection, absorption, and transmission of a typical leaf (left; reproduced from Taiz and Zeiger, 2006), and interior of a dense grapevine canopy (right, photo by M. Keller). 148 5. PARTITIONING OF ASSIMILATES light, it changes back to Pr, switching the light- Together with blue-light receptors, the phy- regulated genes off, as shown in the following tochrome system enables plants to monitor the diagram (Chory, 1997): quantity and quality of light, as well as its duration and direction. This information allows Red light! ! Pr Pfr Shade - avoidance response them to adjust their development in ways that Farred light optimize the capture of energy for photosyn- Although light quantity during the day var- thesis and synchronize vegetative and repro- ies as much as 10-fold due to variations in ductive development (Whitelam and Devlin, cloud cover, weather conditions, and time of 1998). Generally, red light induces phyto- year, these conditions have virtually no effect chrome-controlled responses, whereas far-red on R:FR, which is remarkably constant at light inhibits these responses. The detection of approximately 1.15 (Holmes and Smith, 1977). a lower R:FR by phytochrome (translated into However, during dawn and dusk R:FR drops less Pfr) provides a clear signal of light trans- temporarily, especially at higher latitudes with mitted through, or reflected from, nearby longer twilight periods, and the magnitude and plants and thus indicates the proximity of duration of this decline inform plants about sea- potential competitors. Plants can detect the sonal changes (Franklin, 2008). The largest vari- presence of neighbors very early, well before ation in R:FR occurs due to sunlight interacting they begin shading each other. To avoid being with leaves. As vines grow and their canopy shaded, they respond to low R:FR signals by size increases, the R:FR of the light reaching several morphological changes, collectively individual leaves (even on the exterior) termed the shade avoidance syndrome (Morelli decreases. When the plant spacing is wide and Ruberti, 2002; Smith and Whitelam, 1997; and/or canopy density low, vines do not shade Whitelam and Devlin, 1998). These changes each other, and the R:FR decreases mainly due mainly include stronger apical dominance (less to the increase in reflected FR. In grapevines lateral shoot growth), accelerated shoot elonga- with a loose canopy and vertical shoots, this tion rates (vigor), decreased leaf size and thick- increase in FR modifies the light environment ness, altered shape and more horizontal of the internodes without greatly affecting the orientation of leaf blades, and more vertical spectral balance of the leaves, which is domi- shoots. Shoots that do grow in the shade tend nated by the contribution of direct sunlight. to be thinner and lighter, have longer inter- Nonetheless, the decrease in R:FR is large nodes and thinner leaves, and accumulate less carbohydrate reserves than shoots that grow enough to be sensed by phytochrome molecules et al located in the shoot tissue and to reduce the pro- in the sun (Buttrose, 1969b; Kliewer ., 1972; May, 1960; Morgan et al., 1985). Such portion of Pfr. Above a grapevine canopy or on its sunlit side, R:FR is similar to the ambient alterations of vegetative growth often occur at value (1.1–1.2), whereas on the shady side, the expense of reproductive growth. Therefore, values of 0.3–0.5 have been recorded. Similarly, prolonged low R:FR signals, which indicate inside a dense canopy R:FR can be reduced to that the competitors cannot be outgrown, also values as low as 0.1 (Dokoozlian and Kliewer, lead to developmental responses, such as early 1995a,b; Smart et al., 1982). In this case, the flowering or even inhibition of flower (inflores- decrease is due to the selective absorption of R cence) initiation, reduced seed and fruit set, by the leaves. This effect may be more important truncated fruit development, and often in juice grape (e.g., Concord) vineyards, which decreased seed viability (Morelli and Ruberti, commonly have considerably denser canopies 2002). The changes are probably brought about than wine grapes. by the interference of phytochrome with the 5.2. CANOPY–ENVIRONMENT INTERACTIONS 149 polar transport of auxin and its distribution in cover. In addition, UVB has been increasing the shoots and roots and also by increased eth- at Earth’s surface due to the depletion of et al ylene production, which may stimulate brassi- stratospheric O3 (Madronich ., 1998; nosteroid and gibberellin action and tissue McKenzie et al., 1999), although this process is sensitivity to gibberellin (Franklin, 2008; thought to be close to its maximum and is Morelli and Ruberti, 2002; Pierik et al., 2004), expected to recover by approximately 2050— leading to cell wall loosening and enhanced cell provided all countries continue to implement elongation in the shade. This regulation of the so-called Montreal Protocol that limits the auxin transport also enables phytochrome to emission of O3-depleting chemicals, such as coordinate shoot and root growth, whose recip- chlorofluorocarbons. The main problem with rocal adjustment allows plants to better utilize UV light is that as the wavelength of electromag- the available light. Thus, low R:FR enhances netic radiation declines, its energy content shoot elongation but reduces root growth, increases. Therefore, although it comprises only whereas high R:FR has the opposite effect. In approximately 0.5% of the total solar irradiance, addition to its involvement in the control of the high energy of UVB makes it damaging to photomorphogenesis and photoperiodism, the membranes, proteins, and DNA; thus, plants phytochrome system also induces changes in have evolved mechanisms to screen out UV chemical composition, such as mineral nutri- radiation at or near the surface of their organs ents, chlorophylls, anthocyanins, and other (Jansen et al., 1998; Rozema et al., 1997). metabolites. Phytochrome seems to play a role Leaf hairs, which are particularly dense on in regulating the activities of certain enzymes, the young, expanding leaves of some cultivars, such as nitrate reductase (important for nitro- scatter and attenuate a large fraction of the UV gen assimilation; Smart et al., 1988; see also radiation (Karabourniotis et al., 1999). The epi- Chapter 5.3), phosphoenolpyruvate carboxyl- cuticular wax, although itself not a strong UV ase (producing oxaloacetate for the TCA cycle; absorber, also reflects and scatters some of the see Chapter 4.4), and phenylalanine ammonia incident UV light (Figure 5.7) (Kerstiens, 1996; lyase and chalcone synthase (two key enzymes Rozema et al., 1997; Shepherd and Griffiths, for the synthesis of phenolic compounds, including anthocyanins and tannins; see Chap- ter 6.2). For example, Pfr may lead to activation of the genes that code for the enzymes that make anthocyanins; thus, a decrease in R:FR leads to a decrease in anthocyanin production, which impairs fruit color. In addition to the effects of visible light, the UV range of the elecromagnetic spectrum, which is commonly divided into UVA (320–400 nm), UVB (280–320 nm), and UVC (<280 nm), is also important. Much of the UVB and all of the UVC are absorbed by the ozone (O3) layer in the stratosphere and never reach the surface of the earth. Just as in the case of visible light, the FIGURE 5.7 Cabernet Sauvignon leaves grown in full UVB radiation a vineyard receives depends sunlight (right) and with the UV portion of the spectrum mainly on the position of the sun (latitude, filtered out (left) display differences in epicuticular wax altitude, season, and time of day) and on cloud and hence in leaf surface glossiness (photo by M. Keller). 150 5. PARTITIONING OF ASSIMILATES

2006). In addition, the epidermis cells of leaves set (see Chapter 2.3), high UV exposure might and fruits accumulate phenolic compounds lead to poor fruit set, especially in combination known as flavonols and cinnamic acids, which with nitrogen deficiency, which further pro- act as an optical filter or “sunscreen” that motes flavonol formation (M. Keller, unpub- absorbs the damaging UVB radiation and, lished data). being strong antioxidants, detoxify the reactive oxygen species generated as a consequence of 5.2.2. Temperature UV-induced damage (Bachmann, 1978; Egger et al., 1976; Herna´ndez et al., 2009; Kolb and Leaf temperature changes rapidly in Pfu¨ ndel, 2005; Kolb et al., 2001; Rozema et al., response to fluctuations in radiation and air 1997; Yamaskai et al., 1997). Additional pheno- turbulence. Exterior leaves and grapes are lics are incorporated into the leaf cell walls heated by the sun, whereas shaded leaves and (Weber et al., 1995). Together these phenolics grapes in the interior of the canopy are gener- effectively screen UV radiation so that virtually ally close to air (ambient) temperature (Vogel, none is transmitted through the leaves. Particu- 2009). The heat load on a leaf exposed to full larly high concentrations of flavonols, in sunlight is so great that the leaf would heat addition to anthocyanins that also absorb UV up by 1 or 2Cs 1 to temperatures that would light, are produced in the unfolding leaves at denature proteins and kill the tissues within the shoot tips, giving the shoot tips of many less than 1 min if no heat was lost to the envi- grape cultivars a pink or red appearance (see ronment. This is indeed a problem under Figure 2.5). This protects the UV-susceptible completely windstill conditions, whereas even and highly sun-exposed young leaves from slow air movement can prevent lethal leaf heat- photo-oxidative damage while the photosyn- ing (Vogel, 2009). Although leaves on the sunlit thetic apparatus is being assembled in their side of a canopy are often approximately 2 or cells. When the leaves senesce at the end of 3C warmer than leaves on the shaded side, the growing season, they again accumulate fla- the leaves are generally less than 5 C warmer vonols and, in some cultivars, anthocyanins to than the surrounding air because of the emis- protect their cells from sun damage, this time sion of longwave radiation, sensible heat loss, during the disassembly of the photosynthetic and evaporative cooling (latent heat loss) due apparatus and retrieval of nutrients from the to transpiration (see Chapter 3.2). Sensible heat leaves. Moreover, grape flowers and berries at loss is the process whereby air circulation the exterior of the canopy are also exposed to around the leaf removes heat from the leaf high intensities of UV radiation, most of which surface, as long as the leaf is warmer than the is absorbed by the berry skin (Blanke, 1990a). air. Evaporative heat loss occurs because water Accumulation of phenolic compounds, espe- evaporation requires energy. Water stress cially flavonols, in the skin is greater in these decreases transpiration and therefore increases grapes than in grapes in the interior of the can- leaf temperature. Accordingly, the leaf temper- opy, which has implications for fruit quality ature of grapevines that are experiencing (see Chapter 6.2). It also has implications for water deficit tends to be higher than that of yield formation (see Chapter 6.1). The need fully irrigated vines, regardless of whether the for sun-exposed flowers to produce flavonols leaves are sunlit or shaded (Grant et al., 2007). for UV protection may interfere with auxin Growing grapevines in a manner that maxi- transport, which can be inhibited by flavonols mizes the canopy surface area for maximum (Peer and Murphy, 2007; Winkel-Shirley, light interception creates a dilemma for the 2002). Because auxin flow is necessary for fruit vines, especially in a warm climate. Although 5.2. CANOPY–ENVIRONMENT INTERACTIONS 151 they appear to be able to survive brief temper- canopy while simultaneously reducing CO2 ature “spikes” up to 60C, grape leaves are assimilation. It is possible that the higher tran- killed at temperatures above approximately spiration rate of Chardonnay compared to 45C due to disintegration of the cell mem- Cabernet Sauvignon at high temperature (see branes, leading to membrane leakage and loss Figure 5.8) is related to the differences in leaf of cell contents, and due to thermal denatur- shape between the two cultivars (see Fig- ation of proteins (see Chapter 7.4). In a move- ures 1.13 and 5.7). Local leaf temperature ment termed paraheliotropism, grapevine increases approximately with the square root leaves change their angle to the sun over the of the distance from the edge, and lobing course of a day and a growing season, aligning improves heat transfer (Vogel, 2009), so Char- the leaves in parallel with the solar rays during donnay’s more-or-less entire leaves may need hot periods, to avoid overheating and keep to evaporate more water to keep them from light intensity on the leaf at or slightly below overheating. light saturation to maintain photosynthesis As would be expected from a process that while avoiding photoinhibition (Gamon and involves biochemical (enzymatic) reactions, Pearcy, 1989). In addition, transpiration rates CO2 assimilation is strongly influenced by tem- increase within a range of less than 10Cto perature (Geiger and Servaites, 1991). More- more than 40C, although stomata usually start over, other photosynthetic processes are also closing at approximately 35C(Figure 5.8). The sensitive to temperature (e.g., electron trans- increase occurs because warmer air can hold port has a pronounced optimum at 30C), more moisture, thereby increasing the leaf-to- especially at high irradiance. Below approxi- air vapor pressure deficit, and protects the leaf mately 15C, photosynthesis is strongly cur- from overheating. The stomatal closure at high tailed by an inhibition of sucrose synthesis, temperature seems to be a response to heat- which leads to accumulation of phosphorylated induced reduction in photosynthesis rather intermediates and prevents the release of phos- than the cause of it. Therefore, high tempera- phate (Pi) for regeneration of ribulose-1,5- tures can result in excessive water loss of the bisphosphate (Hendrickson et al., 2004b; see also Chapter 4.2). This so-called “end-product limitation” or “feedback inhibition” occurs because low temperature restricts cell division more than photosynthesis; the time it takes for a cell to divide increases exponentially with declining temperature (Ko¨rner, 2003). This decrease in sink activity results in surplus sugar accumulating in the leaves and in the perennial organs of the plant. As in the case of very high temperatures, the stomata will partially close in response to the reduced pho- tosynthesis, rather than photosynthesis declin- ing because of lower stomatal conductance. Similar to light, therefore, rising temperature initially stimulates photosynthesis. However, FIGURE 5.8 Relationship between leaf temperature and instead of reaching a saturation point, the tem- transpiration of mature leaves of two grapevine cultivars perature response shows a relatively broad (M. Keller, unpublished data). optimum, with very high temperatures 152 5. PARTITIONING OF ASSIMILATES resulting in a reduction of carbon fixation. In (by approximately 0.5C per 100-m elevation grapevine leaves, there is very little photosyn- gain) and at a given site increases from spring thesis below 10C, the optimum ordinarily falls to summer and declines in autumn. Leaves between 25 and 30C, and photosynthesis typi- developing during the hot summer months cally declines sharply above approximately have a considerably higher temperature 35C (Currle et al., 1983; Downton et al., 1987; optimum for photosynthesis than leaves devel- Gamon and Pearcy, 1990; Hendrickson et al., oping in spring or autumn. However, even 2003; Kriedemann 1968b; Williams et al., 1994). mature leaves can acclimate to changes in Yet sometimes the photosynthetic rate at 45C temperature within 1 or 2 weeks. The seasonal may still approach half the rate at 30C (Mullins shift in temperature optimum occurs in both et al., 1992). The optimum temperature range irrigated vines and vines growing under depends on species, cultivar, light intensity, natural water stress conditions, but water and developmental stage, but it also reflects stress (see Chapter 7.2) can become the domi- the maximum temperature experienced during nant factor determining photosynthetic per- leaf growth. For example, low irradiance leads formance, largely overriding the temperature to a flatter and broader temperature response response. curve so that shaded leaves have a lower and Clouds alter not only the light intensity but less pronounced temperature optimum than also the temperature. Although clouds are usu- exposed leaves (Berry and Bjo¨rkman, 1980; ally associated with cooler days than would be Gamon and Pearcy, 1990). Furthermore, in the case under clear skies, they also prevent north–south-oriented rows, east-facing leaves nighttime inversions and thereby dampen the typically reach lower daily maximum temp- amplitude of the diurnal temperature swings. eratures than west-facing leaves, which can In other words, clouds keep days cooler and limit photosynthesis and growth on cool sites nights warmer. This has implications for the (Hendrickson et al., 2004a). Nevertheless, the daily carbon balance of grapevines. Because west-facing leaves often contribute less photo- canopy net photosynthesis is light limited and synthates than their east-facing counterparts thus lower during cloudy days, whereas because the high vapor pressure deficit (not whole-plant respiration is higher during cloudy the higher temperature) in the afternoon may nights, less carbon is available for growth, fruit lead to partial stomatal closure. On the other production, and ripening. Frequent cloud cov- hand, high temperatures during leaf develop- ers, which are indicated by relatively few sun- ment can shift the optimum to as much as shine hours in climate records, in many cool 35C due to photosynthetic temperature accli- and maritime growing regions are one reason mation (Calvin cycle enzymes and membranes for the marked interannual fluctuations in yield become more heat tolerant). Conversely, low and fruit quality in such areas. temperatures will shift the optimum down- Both the increasing temperature and rising ward (leaves produce more photosynthetic pro- CO2 content of the air ([CO2]) associated with teins). In general, the optimal temperature for global climate change will also influence photo- photosynthesis tends to increase by approxi- synthesis. Models as well as empirical observa- mately 1C for each 2 or 3C increase in growth tions generally show a relatively steep increase temperature—up to the maximum (Berry and in net CO2 assimilation rates above approxi- et al Bjo¨rkman, 1980; Hikosaka ., 2006; Schultz, mately 20 C when [CO2] rises from 300 to 2000). This means that the temperature opti- 600 ppm (current values are 380 ppm) and a mum decreases with increasing vineyard eleva- shift of the optimum temperature for photosyn- tion due to the decrease in average temperature thesis from 25–35 to 35–40C (Sage and Kubien, 5.2. CANOPY–ENVIRONMENT INTERACTIONS 153

2007). Below 20C, there appears to be little all plant parts, not just the leaves. This temper- effect of rising [CO2], and above approximately ature effect also reduces net CO2 assimilation 40C photosynthesis drops rapidly, although because at higher temperatures vines use a at 45C it is still twice the rate (and similar to greater proportion of their daily fixed carbon therateat20C) at 600 ppm CO2 compared for respiration. Therefore, even at modestly with 300 ppm. Although heat effects will high temperatures (25–30C) there may be less certainly be important in a warmer world carbon available for vine growth and with higher [CO2], the influence of climate fruit ripening than at cooler temperatures change will be more important at the lower (15–20C). Incidentally, water-stressed plants Q limits of temperature because current and often have a lower 10 than plants with abun- predicted temperature increases will be dant water supply (Atkin et al., 2005). In other higher at night, at higher latitudes, and in the words, water deficit decreases the temperature winter (Intergovernmental Panel on Climate sensitivity of respiration, although this is often Change, 2007). complicated by an increase in soil temperature Q The temperature response of CO2 assimila- as the soil dries. Because the 10 declines when tion partly reflects a conflict of interests in the temperature increases briefly (Atkin and addition to the dilemma created by the need Tjoelker, 2003), a hot day may stimulate res- for evaporative cooling at high temperatures. piration more in vines growing in cool climates Enzyme activities are stimulated by increasing than in vines growing in warm climates. temperature, and rubisco is no exception. However, respiration acclimates somewhat to Unfortunately, rubisco’s oxygenation rate above- or below-average temperatures within (releasing CO2) increases faster with increasing days to compensate for the change in tempera- temperature than its carboxylation rate (fixing ture. Thus, heat waves lasting several days et al CO2) (Berry and Bjo¨rkman, 1980; Foyer ., decrease the respiration rate, whereas cold 2009; Woodrow and Berry, 1988). This is due waves increase the respiration rate (Atkin and to a temperature-induced increase in both the Tjoelker, 2003). Above approximately 35C, specificity of rubisco for oxygen and the solu- the capacity of the electron transport system bility of oxygen. In addition to its effect on pho- becomes limiting to photosynthesis, and at very torespiration, temperature also influences hot temperatures (above 45C) membranes mitochondrial respiration—that is, glycolysis become increasingly fluid and leaky so that and the TCA cycle. The proportionality factor respiration declines again (Sage and Kubien, Q Q or temperature coefficient ( 10) of respiration 2007). The fact that the 10 is higher when there is approximately 2 or 3; in other words, the is abundant (saturating) sugar for respiration respiration rate doubles or triples for every suggests that a low crop load might stimulate 10 C rise in temperature, although respiration respiratory CO2 release more in a warm climate declines rapidly above approximately 30C than in a cool climate. (Kruse et al., 2008; Mullins et al., 1992; Schultz, The temperature that a leaf experiences dur- 1991; Williams et al., 1994). Because of this ing its growth also affects leaf growth. Meri- exponential increase with rising temperature, stem temperature, through its effect on cell the respiration rate of grapevine leaves at division, is an important determinant of the 10C is close to zero, whereas at 25C it can be rate of leaf appearance and the rate of leaf m 2 1 as high as 2 mol CO2 m s . The tempera- expansion, as long as the temperature causes ture effect on respiration is especially impor- neither chilling stress nor heat shock (see Chap- tant because photosynthesis “rests” at night, ter 7.4). Leaf area development depends on whereas respiration “works” 24 h a day in three separate processes: leaf initiation, leaf 154 5. PARTITIONING OF ASSIMILATES expansion, and outgrowth of lateral shoots. above the ground (Braam, 2005; Niklas and Increasing temperature accelerates all three of Cobb, 2006). Wind during the day seems to these processes so that higher temperature inhibit shoot growth more than at night (Hotta leads to more rapid canopy development, lon- et al., 2007). The reduction in shoot growth is ger shoots, and denser canopies. Indeed, a fun- also more severe on the windward (into the damental response of plants to high wind) side of the canopy than on the leeward temperature appears to be a shift in carbon par- (away from the wind) side and intensifies with titioning (see Chapter 5.1) to favor shoot increasing number of wind perturbations growth at the expense of fruit growth and rip- rather than with increasing wind speed in each ening and, probably, storage reserve accumula- single event (Tarara et al., 2005; Williams et al., tion (Richardson et al., 2004). 1994). Moreover, the shoots are often displaced away from the wind so that the canopy 5.2.3. Wind becomes lopsided, which has consequences for the intensity and duration of fruit exposure Strong wind (>6ms 1) can induce physical to sunlight. Similar thigmomorphogenetic damage in addition to a reduction of shoot responses may also be triggered by other phys- length, leaf size, and stomatal density. How- ical influences, such as repeated bending ever, even if it is not strong enough to induce or touching by passing vineyard workers, visible damage (Figure 5.9), wind mechanically animals, or machinery. disturbs shoot growth, leading to shorter but As discussed in Chapter 3.3, wind decreases thicker shoots. This so-called thigmomorphoge- a leaf’s boundary layer resistance, which netic response intensifies with increasing height increases transpiration and enables evaporative

FIGURE 5.9 Physical wind damage on Merlot canopy from shoot being knocked against a foliage wire (left) and leaf being twisted at the petiole junction (right, photos by M. Keller). 5.2. CANOPY–ENVIRONMENT INTERACTIONS 155 cooling. This is an advantage under warm con- inside a canopy sufficiently to briefly increase ditions because leaves tend to heat up rapidly their light exposure. The resulting sunflecks when the wind speed drops below approxi- on these leaves can account for a temporary mately 0.5 m s 1 (Vogel, 2009). Under other- rise in photosynthesis, improving the overall wise similar conditions, the temperature of carbon balance of the canopy (Kriedemann sun-exposed leaves tends to vary inversely et al., 1973). with wind speed, whereas shaded leaves track the air temperature (Vogel, 2009). To avoid 5.2.4. Humidity excessive water loss and dehydration in stron- ger wind, however, grapevines respond to Intuitively, one would expect transpiration wind speeds greater than 2.5 m s 1 by partly by leaves and berries to increase the humidity closing their stomata (Freeman et al., 1982; Wil- inside the canopy, with subsequent implica- liams et al., 1994). Although this strategy may tions for the development of fungal diseases conserve water, it also reduces photosynthetic (see Chapter 7.5). This is a subject that has been CO2 assimilation and increases leaf tempera- very little studied. Increases in humidity of less ture. This may at least partially explain why than 10% have been recorded, and the signifi- vines growing in areas with frequent strong cance of these increases is not well understood. winds often produce fewer and smaller clusters However, a decrease in relative humidity from and lower fruit-soluble solids (Williams et al., 95 to 50% increases the vapor pressure deficit 1994). Conversely, reduced wind speed and between the leaf and the surrounding atmo- air mixing in sheltered vineyards could also sphere more than 10-fold (see Chapter 3.1). decrease photosynthesis because leaves may Moreover, humidity strongly depends on air deplete the CO2 in the air surrounding the temperature because increasing temperature foliage. Moreover, wind speeds less than also sharply increases the vapor pressure defi- 0.5 ms 1 result in humid canopies, which cit (see Chapter 3.2). Therefore, as the vapor favors disease development. For instance, the pressure deficit increases (e.g., on west-facing powdery mildew fungus E. necator requires leaves in the afternoon), stomatal conductance only 40% relative humidity for germination tends to decline in an attempt to control exces- (see Chapter 7.5); this threshold is easily sive water loss by transpiration, which will exceeded within the leaf boundary layer, where limit photosynthesis. This means that an the fungus resides. increase in relative humidity effectively Wind moving down the rows in a vineyard reduces the transpiration rate and increases creates less turbulence and movement of leaves CO2 assimilation. Due to the modulating effect than wind moving across rows. This may of leaf layers, this benefits exterior leaves more decrease water loss, especially under dry con- than interior leaves. ditions, and reduce the negative effect of sto- Humidity also affects leaf growth. A high matal closure on photosynthesis. In addition, vapor pressure deficit reduces the growth rate wind speed at the center of a canopy is often of leaves by decreasing the rate of cell division less than 20% of the speed at the exterior. and cell expansion, even when there is no soil Although this may not be important in terms water deficit. Therefore, leaves growing in low of stomatal effects on CO2 assimilation (which humidity remain smaller than leaves grown in is light limited), it has implications for drying high humidity. Vines growing in dry climates of leaves and fruits after rain; interior surfaces tend to have more open canopies than vines dry more slowly than exposed surfaces. Never- growing in more humid climates, even when theless, even mild winds can move leaves they are equally well watered. 156 5. PARTITIONING OF ASSIMILATES

In addition to air humidity, even small decreases, the percentage light interception changes in vine water status can have an effect by the canopy increases. However, on canopy microclimate. Water-stressed vines decreasing row width increases the (see Chapter 7.2) often have higher canopy likelihood of one row shading the base of the temperatures than fully irrigated vines because neighboring row. Similarly, for any one row there is less transpirational cooling of water- width, as the height of the canopy increases, stressed leaves and because such vines have a the percentage of light interception by the sparser, more open canopy. canopy increases, but again the potential for cross-row shading also increases. The 1:1 ratio is a compromise between the canopy’s 5.2.5. The “Ideal” Canopy intercepting as much light as possible and avoiding one row shading another. The upper limit on vineyard productivity is • Foliage should be trained vertically to avoid set by the total seasonal amount of PAR inter- shading on one side of the canopy and cepted by the vines planted in the vineyard. promote light interception by leaves and Canopy structure, and especially the spatial grapes (fruit exposure). However, this distribution of leaves, has important conse- requirement may be less important in quences for canopy light interception and regions with high temperature and high hence productivity. As discussed previously, irradiance during the ripening period than the acquisition of energy and carbon by cano- in regions with frequent cloudy and cool pies depends on total leaf area, leaf surface dis- conditions. tribution, canopy structure, and photosynthetic • The canopy surface area should be capacity of individual leaves. A canopy that approximately 21,000 m2 ha 1, and 80–100% has an ideal microclimate in terms of maximum of the leaves should be on the outside of the light interception for vine productivity has the canopy. The larger the surface area, the more following features [modified from Smart light is intercepted, and hence the potential (1985) and Smart et al. (1990)]: for photosynthesis and yield production is • Rows should be oriented from north to south increased. However, if the surface area is too to maximize light interception by both sides large, then the canopy height:row width of the canopy for some part of the day. ratio of 1:1 is not adhered to. However, row direction is of relatively little • The canopy should be approximately importance for wine quality compared to the 30–40 cm wide with 1–1.5 leaf layers. This leaf features described next, and the north–south layer number is the number of leaves from one requirement may well be overridden by side of a canopy to another. Higher values are other considerations (e.g., topography and associated with shading and reduced fruit economic row length). In a high-irradiance quality, whereas lower values are associated environment, it may be beneficial to deviate with incomplete light interception. from the north–south orientation because • There should be approximately 15 shoots per rows shifted somewhat to the northeast– meter of canopy. A higher figure means southwest may protect grape berries from that shoots may be crowded and shading overheating on the vulnerable west side of occurs. A lower figure means that light the canopy. interception is suboptimal. The value varies • The ratio between canopy height and row according to cultivar and vine vigor and can width (distance between rows) should be be considerably higher without detriment in 1:1. For any one canopy height, as row width warm regions with plenty of sunshine 5.2. CANOPY–ENVIRONMENT INTERACTIONS 157

compared with cool regions where light is • The renewal zone, which is the part of the often limiting. Maintaining ideal shoot shoot that will become the fruit-bearing unit spacing is the underlying theme of canopy in the following year, should also be near the management. top of the canopy to promote inflorescence • A total of 20–40% of the canopy should be initiation (i.e., bud fruitfulness). composed of gaps. Too many gaps reduce the • The pruning weight (total weight of canes yield potential, whereas too few gaps indicate pruned off during winter) should be in the that shading in the canopy is likely. range 0.3–0.6 kg m 1 of canopy length with • Shoots should stop growing when they are canes weighing 20–40 g each. Values higher approximately 15 nodes long (approximately than 1 kg m 1 indicate overly vigorous 1 m) to provide sufficient leaf area to ripen vines. the fruit (10–15 cm2 g 1). If shoots grow • The ratio of yield to pruning weight per vine beyond this length and are not supported or should be in the range of 5–10. This is a trimmed, they will fall across each other and measure of the balance between create shade. Excessive trimming, however, reproductive and vegetative growth. Values wastes the vine’s resources and is an higher than 10 are associated with indication of too much vigor. overcropping and delayed ripening, whereas • Lateral shoot growth should be limited to values lower than 5 are associated with low less than 10 lateral nodes per main shoot. For yield and high shoot vigor. example, 10 lateral nodes could be made up of five lateral shoots of 2 nodes each or two Vineyard sites with low to moderate vigor lateral shoots of 5 nodes each. Excessive potential tend to produce canopies that are lateral growth indicates high shoot vigor, close to this “ideotype.” Such sites are often and growing shoot tips compete with the characterized by relatively shallow rootzones fruit for carbohydrates. and well-drained soils with slightly limited • The fruit zone should be near the top or the water and nutrient storage capacity. Provided outside of the canopy so that 50–100% of the vines are not spaced too closely at planting fruit is exposed to the sun. This promotes and are not grafted to high-vigor rootstocks, anthocyanin and tannin production and also they can be easily trained to a vertical shoot improves disease control. High temperature positioning system (especially in cool climates) (>33 C) and the UV radiation of bright or sprawl trained with little or no shoot posi- sunlight can inhibit the spread of powdery tioning (especially in warm climates). Deep mildew colonies, and exposed fruit also and fertile soils with high water storage capac- dries more quickly after rain, which reduces ity are less desirable because they may result in bunch rot infections. overly vigorous vines and dense canopies • Fruit exposure on the east side of north– (Cortell et al., 2005). Although achieving south-oriented rows should be near 100%, balanced vines with ideal canopy characteris- whereas on the west side it should be closer tics is more difficult on such sites, it is not to 50% because of the increased heat load in impossible. Vines can be grafted to low-vigor the afternoon. Excessive fruit exposure can rootstocks (see Chapter 1.2) and spaced at wider result in heat damage and sunburn, and in planting distances that accommodate lighter impaired anthocyanin accumulation, while pruning levels (see Chapter 6.1). In contrast, increasing phenolics other than close spacing often results in shoot crowding anthocyanins beyond desirable levels, and competition among shoots for access especially in white grapes (see Chapter 6.2). to light rather than in decreased growth due to 158 5. PARTITIONING OF ASSIMILATES competition between roots for water and nutri- Nitrogen is generally the fourth most abundant ents, which tends to exacerbate the canopy element in grapevines after hydrogen (H), car- problem (Falcetti and Scienza, 1989). In fact, bon (C), and oxygen (O), although calcium (Ca) high planting density could result in a so-called can exceed N in vines grown on calcerous, high- “tragedy of the commons,” whereby plants that pH soils. Typical concentrations in dry matter compete with each other for the same soil are roughly 60 mmol H g 1,40mmolCg1, resources may increase both root and shoot 30 mmol O g 1, and 1 mmol N g 1. Therefore, growth—thereby keeping the root:shoot ratio N is the mineral nutrient for which plants have constant—at the expense of fruit growth (and the highest demand and the nutrient that most hence crop yield and quality) rather than limit- often limits growth. This makes N the most ing their root production to match resource important mineral nutrient of grapevines. More- availability (O’Brien et al., 2005). Vines growing over, when grapes are harvested, some of the on sites with high vigor potential can also be fixed N is permanently removed from the vine- trained to vertically or horizontally divided yard soil. This loss amounts to 2 or 3 kg t 1 of fruit trellis systems that increase the length of the removed but can be reduced to less than 1 kg t 1 canopy per plant. Such canopy division is if stalks (grape peduncles after destemming) and advisable when the pruning weight exceeds pomace (solid remains of grapes after pressing 1kgm 1 canopy length. This will allow greater of juice or must) are recycled back to the vineyard. numbers of shoots to be spaced more ideally, Any such loss has to be replaced by addition which in turn will improve light interception of organic or mineral fertilizer or by biological and fruit exposure and accommodate higher N fixation using leguminous cover crops. yields without sacrificing fruit quality (see Although N makes up almost 80% of the Chapter 6.2). The impact of shoot number per atmosphere, grapevines, in contrast to legumes unit canopy length may also depend on when whose roots “employ” symbiotic bacteria called the shoot density is established. For example, rhizobia for the task of fixing atmospheric shoot thinning approximately 3 weeks before nitrogen gas (N2), cannot directly utilize this bloom led to compensatory growth of lateral N2. Instead, they generally rely on uptake by shoots, which negated the beneficial influence their roots of nitrogen ions—mostly in the form of lower shoot density on canopy microclimate of nitrate (NO3 )—dissolved in the soil water. (Reynolds et al., 1994a), whereas shoot thinning These nitrate ions are then reduced to ammo- þ at veraison had no such effects (Smart, 1988). nium (NH4 ) and assimilated into amino acids However, delaying shoot thinning can never- in the roots and leaves for translocation as both theless be associated with delayed fruit ripen- nitrate and amino acids throughout the vine to ing in cool climates (Reynolds et al., 2005). be used in growth, metabolism, or storage. Amino acids are N-containing organic acids, the units or “building blocks” from which pro- 5.3. NITROGEN ASSIMILATION tein molecules are manufactured by cellular AND INTERACTION WITH ribosomes. Ribosomes, composed of proteins CARBON METABOLISM and RNA, utilize the DNA’s genetic code to assemble amino acids into proteins, thereby translating the RNA template that is tran- Nitrogen (N) assimilation is the process by scribed from the plant’s genes. Each 1 g protein which inorganic N acquired by the vine is typically contains 0.16 g N, and plants invest incorporated into the organic carbon com- approximately 55% of their total N content in pounds necessary for growth and development. proteins (Niklas, 2006). 5.3. NITROGEN ASSIMILATION AND INTERACTION WITH CARBON METABOLISM 159

5.3.1. Nitrate Uptake and Reduction Leaves and fruit

þ NO − → NH + ↔ Amino acids ↔ Proteins Due to rapid nitrification of NH4 derived 3 4 from organic sources to NO3 by microbes in most aerobic soils, NO3 is the primary source of N for grapevines (Bair et al., 2008; Keller et al., 1995, 2001b). However, Vitis species are − þ et al NO3 Sucrose also capable of taking up NH4 (Jime´nez ., Amino acids

Xylem Amino acids 2007) and, almost certainly, amino acids as Proteins Phloem well, especially in acidic soils that are rich in organic matter (Fischer et al., 1998; Grossman Roots and Takahashi, 2001). In fact, where they are NO − → NH + ↔ Amino acids ↔ Proteins available, roots prefer amino acids over NO3 3 4 for uptake (Miller et al., 2008). Nitrate is dis- solved in the soil water and taken up through the root’s epidermis and cortex (see Chapters − + 1.3 and 3.2). The concentration of NO3 in the NO3 NH4 Soil solution soil water can vary from 10 mM to 100 mM, but FIGURE 5.10 Simplified diagram of nitrogen uptake, it is generally orders of magnitude lower than assimilation, and circulation in grapevines (modified from the concentration inside the xylem stream Keller, 2005; reprinted by permission of AJEV). (Crawford, 1995; Crawford and Glass, 1998; Keller et al., 1995, 2001b; Robinson, 1994). Although the removal of NO3 by nitrogen assimilation and xylem transport should favor Once inside the root cells, nitrate can be passive diffusion into the root, the concentra- moved to the vacuoles for temporary storage tion gradient in the “wrong” direction is far or loaded into the xylem and transported to too great for this to occur under normal condi- the shoots and leaves as shown in Figure 5.10. tions. Therefore, roots absorb NO3 actively by þ Most of the NO , however, is assimilated into means of proton/nitrate (H /NO ) cotran- 3 3 organic N compounds—that is, amino acids sport using an ATP pump, a protein called þ (Loulakakis and Roubelakis-Angelakis, 2001; H -ATPase embedded in the cell membrane Roubelakis-Angelakis and Kliewer, 1992; Tester (Crawford, 1995; Crawford and Glass, 1998). and Leigh, 2001). Nitrogen assimilation in In this process, energy from ATP consumption grapevines occurs in both the roots and the is used to “pump” protons out of the root cells leaves (Keller et al., 1995; Llorens et al., 2002; into the soil water, generating a proton gradi- Perez and Kliewer, 1982; Stoev et al., 1966; ent across the membrane. The protons diffuse Zerihun and Treeby, 2002). The following back into the cells, carrying negatively charged overview of nitrogen assimilation mainly nitrate molecules with them. The micronutrient summarizes aspects of the detailed review boron (B) is essential to keep the ATP pump provided by Loulakakis and Roubelakis- going so that B deficiency results in a strong Angelakis (2001). The first step of this process decrease in the roots’ ability to absorb NO 3 is the reduction of NO to nitrite (NO )by (and potassium) and, consequently, may lead 3 2 the enzyme nitrate reductase (NR) using two to secondary N deficiency and accumulation electrons provided by NADH or NADPH: of sugars and starch in the leaves (Camacho- þ þ ! þ Cristo´bal and Gonza´lez-Fontes, 1999). NO3 2H NO2 H2O 160 5. PARTITIONING OF ASSIMILATES

There are two slightly different forms of generated by the oxidative pentose phosphate nitrate reductase. One is located in the cytosol pathway in the nonphotosynthetic plastids of root epidermis and cortex cells and leaf (Crawford, 1995). Because nitrite is toxic, vines mesophyll cells. The other, which appears to maintain an excess of nitrite reductase when- be restricted to roots, is bound to the outer sur- ever nitrate reductase is present. In the roots, face of the plasma membrane (and thus located nitrate is sufficient to activate nitrite reductase, in the apoplast). The cytosolic NR is active only whereas in the leaves light is required in addi- during the day, whereas the apoplastic NR tion to nitrate. “operates” day and night but peaks during the night. The membrane form of NR also nor- 5.3.2. Ammonium Assimilation mally prefers electrons from succinate over those from NADH. Nitrate reductase is the Like nitrite, ammonium is toxic to plants and main molybdenum (Mo)-containing protein in is either rapidly incorporated into amino acids plants (Schwarz and Mendel, 2006), and one (i.e., assimilated) or stored (in case of excess symptom of Mo deficiency is the accumulation supply) in the cell vacuoles. Ammonium assim- of nitrate due to diminished NR activity (see ilation (Figure 5.11) is normally catalyzed Chapter 7.3). Nitrate reductase is stimulated by the two enzymes glutamine synthetase by the presence of nitrate but requires light (in (GS) and glutamate synthase, also known the leaves, where activation is mediated by as glutamine-2-oxoglutarate aminotransferase phytochrome; see Chapter 5.2) or carbohy- (GOGAT), in a cycle consisting of two sequential drates (mainly sucrose, in the roots) for full reactions: induction. The plant hormone cytokinin pro- þ Glutamate þ NH ! glutamine motes but the amino acid glutamine suppresses 4 NR activity. Glutamine þ 2-oxoglutarate ! 2 glutamate Grapevines can distribute large amounts of nitrate or store it for later use in the cell The first step of the GS/GOGAT cycle vacuoles (which usually occupy more than requires energy provided by ATP and involves 2þ 2þ 2þ 2þ 90% of the volume of mature cells) without del- a divalent cation (Mg ,Mn ,Ca ,orCo ) eterious effects (Roubelakis-Angelakis and as a so-called cofactor of GS (Roubelakis- Kliewer, 1992). This vacuolar pool strongly buf- Angelakis and Kliewer, 1983). There are two fers the nitrate concentration in the cytosol. In main forms (isoenzymes or isozymes) of gluta- contrast, nitrite is a toxic ion that can induce mine synthetase: GS1 is located in the cytosol of mutations in plant tissues. It is therefore imme- all plant organs and in the phloem companion diately transported from the cytosol into plas- cells, and GS2 is located in the plastids of pho- tids (in roots) or chloroplasts (in leaves), tosynthetic tissues and roots (Grossman and where it is reduced to ammonium by the Takahashi, 2001). The cytosolic GS1 is central enzyme nitrite reductase (NiR). This reaction to ammonium assimilation in the roots, and uses six electrons provided by reduced ferre- its activity increases with increasing sugar con- doxin (Fdred), which is oxidized (Fdox) in the tent. In contrast, GS2 predominates in ammo- process: nium assimilation in the leaf mesophyll. The þ þ ! þ þ glutamine produced by GS in the first reaction NO2 8H NH4 2H2O stimulates the activity of glutamate synthase, The protein ferredoxin is produced in the producing two molecules of glutamate, which photosynthetic electron transport chain in the consumes two more electrons. Plants contain chloroplasts (see Chapter 4.1) or by NADPH two types of glutamate synthase: One is called 5.3. NITROGEN ASSIMILATION AND INTERACTION WITH CARBON METABOLISM 161

FIGURE 5.11 Structure and reactions of compounds involved in ammonium metabolism. Reproduced from Taiz and Zeiger (2006).

NADH-GOGAT because it accepts electrons during photorespiration (Stitt, 1999; Stitt et al., from NADH, and the other is termed Fd- 2002). Both glutamine synthetase and gluta- GOGAT because it accepts electrons from ferre- mate synthase are stimulated by light doxin (Temple et al., 1998). The NADH type is and sucrose and inhibited by amino acids located only in plastids of nonphotosynthetic (Grossman and Takahashi, 2001). The carbon tissues such as roots or vascular bundles of “backbone,” 2-oxoglutarate, also known as developing leaves, whereas the ferredoxin a-ketoglutarate, is provided by the TCA cycle type, which contains iron and sulfur, is located (see Chapter 4.4). in both chloroplasts and nonphotosynthetic One of the two glutamate molecules pro- plastids but dominates in leaves and is very duced by GOGAT is used to regenerate the important in the recapture of ammonium cycle, and the other is used to supply amino released during photorespiration (see Chapter acids for general metabolism. In the roots, glu- 4.3) in addition to its role in assimilating tamate can also be transported back to the cyto- ammonium derived from nitrate reduction plasm, where it is converted back to glutamine (so-called primary ammonium assimilation). by a slightly different form of glutamine syn- Up to 90% of the ammonium that flows thetase for export in the xylem to the shoots. through the GS/GOGAT cycle is generated Glutamine provides N groups, either directly 162 5. PARTITIONING OF ASSIMILATES or via glutamate, for the production of all molecules across membranes, and others store organic nitrogenous compounds in the vine. nitrogen for future use. Nitrogen uptake and In an alternative pathway for ammonium assimilation “cost” large amounts of energy to assimilation (see Figure 5.11), the enzyme glu- convert stable, low-energy inorganic com- tamate dehydrogenase (GDH) catalyzes a pounds present at low concentration in the soil reversible reaction that can either form or water into high-energy organic compounds degrade glutamate: with high concentrations inside the plant. þ Nitrate assimilation uses 2.5 times the NADPH 2-oxoglutarate þ NH4 $ glutamate þ H2O required for CO2 assimilation. The reduction of Glutamate dehydrogenase is localized in the one molecule of nitrate to nitrite and then to mitochondria (especially those of phloem com- ammonium requires the transfer of 10 electrons panion cells) of both roots and leaves and also and often accounts for 10–25% of the total in the chloroplasts in leaves. As a stress-related energy expenditures in both roots and shoots. enzyme, it may participate in ammonium This means that a vine may use as much as assimilation (using electrons donated by either one-fourth of its energy to assimilate N, a con- NADH or NADPH) in tissues with excessive stituent that comprises less than 2% of total ammonium concentration and in senescing plant dry weight. (dying) leaves, where it is thought to recycle The distribution of available N in the soil is and thereby detoxify the ammonium that extremely heterogeneous. Nitrate concentra- is released during protein remobilization tions in the soil solution can vary by several (Loulakakis et al., 2002; Masclaux et al., 2000). orders of magnitude, both temporally and spa- Thus, GDH may complement the GS/GOGAT tially, even over short distances (Crawford, cycle, whose activity declines during senes- 1995; Keller et al., 1995, 2001b; Robinson, cence. However, the enzyme can “work” in 1994). Because N is so important to them, the reverse direction, oxidizing glutamate when plants have evolved mechanisms that remove fixed carbon is depleted, for example, as a nitrate from the soil solution as quickly as pos- result of restricted photosynthesis. Under these sible. The root system can react to a patch of conditions, GDH can participate in the remobi- nitrate by rapidly activating nitrate uptake lization of proteins and degradation of amino and, more slowly, initiating lateral root prolif- acids to supply carbon skeletons to the TCA eration within the nitrate-rich zone (Forde, cycle (see Chapter 4.4) for continued energy 2002; Gastal and Lemaire, 2002; Nibau et al., (ATP) regeneration (Aubert et al., 2001; 2008; Scheible et al., 1997). These responses are Miyashita and Good, 2008; Robinson et al., 1991). especially pronounced when vine N status is low (see Chapter 7.3). To support such loca- 5.3.3. From Cells to Plants lized nitrate uptake, those roots in contact with the rich N source also augment water influx, Nitrogen is a structural component of whereas in a compensatory response the roots nucleic acids (i.e., of the organic bases in that happen to grow in N-poor soil patches DNA, RNA, and ATP), chlorophyll, hormones, simultaneously take up less water (Gloser and amino acids. Many amino acids are ulti- et al., 2007; Gorska et al., 2008). It seems obvious mately assembled into proteins such as that this mechanism would improve nitrate enzymes (which catalyze or increase the rates delivery to those roots that are best positioned of biochemical reactions) and are essential to for immediate uptake by accelerating mass plant metabolism and energy generation. flow to the root surface. Accordingly, the rates Other proteins become transporters that carry of nitrate uptake and respiration decline 5.3. NITROGEN ASSIMILATION AND INTERACTION WITH CARBON METABOLISM 163 rapidly as newly formed roots age (Volder takes place in the roots or the leaves depends et al., 2005). on a number of factors, including species, culti- When photosynthetic electron transport gen- var and rootstock, weather conditions, and the erates more energy than is needed by the Cal- amount of nitrate the roots take up (Alleweldt vin cycle (see Chapter 4.2), some of this and Merkt, 1992; Keller et al., 1995, 2001b; energy becomes available for N assimilation. Llorens et al., 2002; Zerihun and Treeby, 2002). Because the production of amino acids also Species native to temperate regions usually rely requires a supply of carbon through the TCA more heavily on root N assimilation than do cycle, the incorporation of inorganic N into species of tropical or subtropical origin. Despite amino acids is a dynamic process that is regu- the variation among cultivars in their capacity lated by both internal (e.g., availability of car- to assimilate nitrate, neither total N nor nitrate bon and N metabolites) and external factors concentrations in the leaf blades vary much (e.g., light and availability of inorganic N). among cultivars, although nitrate does fluctu- When a grapevine takes up nitrate through ate in the petioles (Christensen, 1984). When the roots, this nitrate functions as a signal that environmental factors permit rapid photosyn- induces the plant to switch on the pathway of thesis that is associated with high vine carbon N assimilation and divert carbon away from status, and when nitrate availability in the soil starch production toward the manufacture of water is relatively low, nitrate absorbed by the amino acids and organic acids such as malate roots is rapidly assimilated in the roots and and citrate, which act as a counteranions that transported in the xylem as metabolically active substitute for nitrate (Foyer et al., 2003; Huppe glutamine. As the supply of N increases, so and Turpin, 1994; Jime´nez et al., 2007; Stitt, does root uptake, and the roots’ capacity for 1999; Stitt et al., 2002). Therefore, N assimilation assimilation becomes increasingly saturated so proceeds rapidly in vines with high carbohy- that increasingly more nitrate is transported to drate status and slows as the photosynthetic the shoots in addition to glutamine (Alleweldt sugar supply declines (Perez and Kliewer, and Merkt, 1992; Keller et al., 1995, 2001b; Pate, 1982). A decrease in carbon status can occur 1980). due to poor weather conditions, drought stress, This makes sense for the vine because reduc- loss of leaf area due to hail or insects, or infec- tion and assimilation of one nitrate molecule in tion by pathogens and can result in nitrate the roots costs the equivalent of 12 ATP mole- accumulation in the vine’s tissues (Keller, cules, but it is substantially cheaper in the 2005). Although energy-dependent nitrate leaves. The reason for this is that N assimilation uptake also declines in vines with low carbon in the roots is dependent entirely on sucrose status, uptake is less limited than N assimila- transported in the phloem for both carbon ske- tion (Keller et al., 1995; Oaks and Hirel, 1985; letons and energy (Huppe and Turpin, 1994; Rufty et al., 1989). In contrast, when nitrate Oaks and Hirel, 1985). The capacity of the roots becomes limiting, N metabolism slows, which to assimilate N is directly related to their carbo- leads to accumulation of starch and, ultimately, hydrate status, and the carbon skeletons for suppression of photosynthesis through feed- glutamine production are derived from back inhibition. The increase in carbohydrate recently imported sucrose or from stored starch content in response to N deficiency is often (Emes and Neuhaus, 1997). Therefore, high N greater in roots than in leaves. availability, such as after heavy fertilizer appli- Both roots and shoots of grapevines have the cation or tilling of cover crops (Bair et al., 2008; capacity to assimilate nitrate to amino acids. Mu¨ ller, 1985), can lead to lower starch reserves The relative extent to which nitrate reduction in the vine’s permanent organs, especially 164 5. PARTITIONING OF ASSIMILATES under conditions that limit photosynthesis (see Chapter 5.2). In the roots, on the other (Cheng et al., 2004b; Keller et al., 1995). Because hand, N assimilation proceeds both day and nitrate reduction and subsequent assimilation night. The “primary” amino acids glutamine are more sensitive to sugar supply than are and glutamate can be converted to many nitrate uptake or long-distance transport, low other amino acids by the action of enzymes carbohydrate status leads to export of nitrate called transaminases. These glutamate-utilizing to the shoots. enzymes recycle 2-oxoglutarate so that it can be In contrast to roots, leaves can use excess used again in ammonium assimilation, and the photosynthetic energy for N assimilation reactions can proceed at night. Under favorable (Huppe and Turpin, 1994). Indeed, increasing conditions that are associated with high rates of N supply and leaf N status stimulates light- N assimilation, the vine converts surplus gluta- saturated photosynthesis in leaves (Keller mine (19% N) to arginine. Arginine has the et al., 2001b; see also Figure 5.5), but it also highest N:C ratio of all amino acids (32% N) increases the respiration rate due to the energy and is the major and most efficient N storage demands of N assimilation. The qualifier compound in grapevines, both as soluble “light-saturated” is important because light amino acid and incorporated in proteins intensities below saturation limit photosynthe- (Kliewer, 1967; Kliewer and Cook, 1971; Schal- sis directly (see Chapters 4.1 and 5.2), and N ler et al., 1989; Xia and Cheng, 2004). The has no effect. Moreover, the total amount of reserves stored in the woody parts of the vine carbon fixed per day saturates at high N levels are available as a buffer during periods of low so that there is an optimal leaf N content that N supply and to support new growth in spring. maximizes carbon gain. The optimal N concen- In fact, reserve N may be at least as important tration is higher in leaves grown at high irradi- as reserve carbohydrates in supporting spring ance than in those grown under cloudy growth of grapevines (Cheng et al., 2004b; conditions or in the shade (Hikosaka et al., Keller and Koblet, 1995a; Schaefer, 1981). Up to 2006). Excessive N supply reduces the amount half of the N demand by the developing can- of sucrose available for export, worsening the opy is supplied from such reserves. Because situation for the roots. Leaves also gradually of the rapid shoot growth in spring, a vine’s acquire an increased capacity for N assimila- nutrient demand is greatest between budbreak tion as they unfold and mature along with an and bloom, even though most of the N uptake increase in photosynthesis. Accordingly, the from the soil occurs after bloom (Conradie, amount and activities of GS and GOGAT initi- 1986; Lo¨hnertz et al., 1989; Peacock et al., ally increase with leaf age but decrease in 1989), provided there is sufficient soil moisture senescing leaves. This is especially true for (Keller, 2005). Limited availability of N reserves GS2, whereas GDH follows the opposite trend. due to inadequate refilling in the previous þ At least some of the NH4 that forms by the growing season can restrict early shoot growth action of GDH during senescence may be vola- and canopy development (Celette et al. 2009; tilized to the atmosphere (Farquhar et al., 1979). Keller and Koblet, 1995a) and lead to poor fruit As long as sufficient photosynthate and set (Keller et al., 2001a). The reserve pool is energy are available, vines can assimilate most depleted during and after budbreak, reaching of the absorbed nitrate. Because it depends on a minimum by bloom time or, sometimes, as photosynthetic energy, the production of gluta- late as veraison, and it is replenished later in mine and glutamate in the leaves occurs mainly the season (Eifert et al., 1961; Kliewer, 1967; during the day, and all the necessary enzymes Lo¨hnertz et al., 1989; Schaller et al., 1989; Schrei- are induced by the active form of phytochrome ner et al., 2006; Zapata et al., 2004). Insufficient 5.3. NITROGEN ASSIMILATION AND INTERACTION WITH CARBON METABOLISM 165

N availability at this time—for example, parts of the vine for storage (Conradie, 1986; because of late-season growth of a competing Dintscheff et al., 1964; Kliewer, 1967; Loulakakis cover crop—may not allow the vine to fully et al., 2002). This process is also called resorp- replenish its N reserves (Celette et al., 2009). tion and is associated with a decline in photo- Accumulation of storage reserves occurs synthesis and a transition from nutrient mostly when other plant requirements, such assimilation to nutrient remobilization (Hoch as growth and fruit production, have been et al., 2003; Lim et al., 2007). As much as 90% satisfied—that is, when supply of resources of leaf N may be recycled during senescence exceeds demand (Lemaire and Millard, 1999; (Lo¨hnertz et al., 1989), and the GS1 form of glu- see also Chapter 5.1). In warm climates, where tamine synthetase produces glutamine for leaves remain photosynthetically active for sev- export from the leaves. Some of the remobilized eral weeks or even months after harvest, it protein is not broken down completely and appears that much of the N taken up by the may be exported by the phloem in the form of roots after harvest is directly incorporated into small peptides two or three amino acid units the reserve pool (Conradie, 1986). However, in length. Remobilization also occurs from even in cool climates, late-season foliar applica- shaded leaves, and from flower clusters prior tion of N fertilizer can markedly increase the to fruit set, in the interior of dense canopies reserve N pool, albeit at the expense of carbo- (see Chapter 5.2). The recovered N is recycled hydrate reserves (Xia and Cheng, 2004). to unshaded leaves and to the shoot tips for Clearly, then, late-season fertilizer application production of new leaves that are better or cover crop tillage can be used to increase exposed to sunlight (Hikosaka, 2005). the amount of reserve N for the following The ability of GDH to oxidize glutamate spring. Increasing the size of the reserve during periods of carbon starvation may have pool—for instance, by N fertilizer application— implications for yield formation in grapevines. will increase the N content of the emerging When adverse environmental conditions, such leaves and early season growth, which enhances as overcast skies or unseasonably cool weather, photosynthesis and may lead to greater restrict photosynthesis (see Chapter 7.4), vines seasonal demand for N due to the larger can experience severe carbon shortage. canopy (Cheng et al., 2004b; Keller and Koblet, Meanwhile, N assimilation is reduced when 1995a; Treeby and Wheatley, 2006). photosynthesis declines, which leads to an Much of the N that continuously arrives, inhibition of amino acid production. Conse- mainly in the form of glutamine and nitrate, quently, plants may become starved for both via the xylem in the leaves is redistributed as carbon and N (Stitt et al., 2002). This may be glutamine via the phloem to the shoot tips, fruit especially critical during bloom, when storage clusters, and woody tissues for further use in reserves in the permanent organs are at their growth and metabolism or storage. Because lowest. In addition, local carbon starvation can nitrate cannot be transported in the phloem, it also occur inside dense canopies or due to has to be assimilated to glutamine once the stunted shoot growth limiting leaf area. In such xylem has delivered it to the leaves or stored situations, GDH participates in the resorption as nitrate in the leaf vacuoles to buffer tempo- of proteins from relatively unimportant sinks rary shortages in N supply. In addition, nitrog- that may subsequently be discarded. It is possi- enous compounds, as well as other mineral ble that this mechanism may account for the ions, are remobilized from proteins and nucleic observed accumulation of ammonium in grape acids in senescing leaves at the end of the flowers during the development of inflores- growing season and exported to the perennial cence necrosis. This syndrome is also called 166 5. PARTITIONING OF ASSIMILATES early bunch stem necrosis, and it is an extreme apparatus via oxidative stress, the leaves form of poor fruit set that culminates in abscis- decrease their chlorophyll content (i.e., turn sion of portions of or whole inflorescences and pale green or even yellow) and activate their is probably induced by carbon starvation energy-dissipation and antioxidant systems (Jackson, 1991; Jackson and Coombe, 1988; (Chen and Cheng, 2003a; Keller, 2005). In addi- Keller and Koblet, 1994; see also Chapter 6.1). tion, they decrease the angle between the leaf In other words, the buildup of toxic ammo- blade and the petiole, which further reduces nium likely is a consequence, not a cause, of the number of light photons captured by a leaf the senescence process associated with organ (see Chapter 7.3). abscission. Such abscission and associated Warm conditions stimulate organ growth nutrient recycling can be viewed as a “sacri- (e.g., leaf expansion) more than N assimilation fice” by the vine of comparatively less impor- and use (e.g., protein synthesis), so the N:C ratio tant tissues or organs that will permit more in the vine’s organs decreases compared to important sinks to survive episodes of stress- cool conditions. Low temperature also reduces induced carbon starvation. For instance, if the protein production and enzyme activity so that incident light is insufficient to sustain growth amino acids and nitrate accumulate in the leaves of all vine organs, leaf growth, especially on because nitrate uptake can continue under lateral shoots, may be enhanced to capture low temperature (Lawlor, 2002). Cell division more light at the expense of inflorescence appears to be particularly sensitive to N status, survival (Keller and Koblet, 1994, 1995a, 2001a). and inadequate N supply generally suppresses The rate of photosynthesis in grapevine growth more than photosynthesis so that leaves increases as the supply of N in the xylem carbohydrates accumulate in the leaves and the increases (Keller, 2005). The leaves’ photosyn- N:C ratio is low (Gastal and Lemaire, 2002). thetic capacity—that is, the light-saturated rate However, under the elevated nitrate concentra- of photosynthesis—also seems to increase line- tion in the soil water that occurs after fertilizer arly with increasing leaf N content, mainly applications (Mu¨ ller, 1985), root absorption can because rubisco, the enzyme responsible for exceed the capacity of a vine for assimilation CO2 fixation, comprises approximately one- because nitrate reduction and assimilation are third of a leaf’s protein and up to 25% of its N among the most energy-intensive reactions. This (Evans, 1989; Seemann et al., 1987; see also is especially true under conditions that favor Chapter 4.2). Thus, the optimal leaf N content rapid transpiration, such as warm and sunny required to maximize photosynthesis increases days, and therefore enhance nutrient uptake. with increasing light intensity. Plants therefore At high light intensity, glutamine can be tend to distribute leaf N so that it closely fol- transported back from the leaves to the roots in lows the light distribution within a canopy in the phloem as a signal to limit nitrate uptake order to maximize whole-canopy photosynthe- (Gessler et al., 1998; Lemaire and Millard, 1999). sis (Gastal and Lemaire, 2002). This nonuni- This feedback regulation normally coordinates form distribution of N has to be taken into nitrate uptake with the vine’s demand for account when leaves are sampled for nutrient N. However, when grapevines have access to analysis to determine vineyard fertilizer large amounts of soil nitrate during periods of requirements. On the other hand, when N low solar radiation, nitrate assimilation cannot becomes limiting, rubisco activity declines keep pace with uptake because of a sucrose more than electron transport capacity (see and energy shortage, and nitrate accumulates Chapter 4.1). To avoid excessive light absorp- in the leaves and other organs once available tion that would damage the photosynthetic starch reserves have been exhausted (Keller 5.3. NITROGEN ASSIMILATION AND INTERACTION WITH CARBON METABOLISM 167 et al., 1995, 2001b; Perez and Kliewer, 1982; samples collected in the vineyard to estimate fer- Scheible et al., 1997; Zerihun and Treeby, 2002). tilizer requirements: If samples are collected High leaf nitrate reduces the amount of sucrose during cool or cloudy periods, the vine N status available for export, depleting root starch may appear higher than it is in reality. reserves and inhibiting root growth, which During drought or salt stress, and possibly strongly decreases the root:shoot ratio (Keller also with N deficiency, a larger proportion of and Koblet, 1995a; Keller et al., 1995; Scheible glutamate appears to be converted to the amino et al., 1997). This has special implications during acid proline. Much of this proline synthesis the establishment phase of a vineyard. Heavily results from glutamine produced by the GS1 fertilizing and irrigating young vines in order form of glutamine synthetase in the phloem’s to harvest a crop in the year after planting may companion cells. Proline is highly soluble and curb root development, which might be detri- can accumulate in cells to high levels without mental to vine performance in the long term. disrupting their metabolism. This allows plants The increase in leaf nitrate under cool or overcast to lower their tissue water potential while conditions also has implications for the inter- maintaining turgor pressure during periods of pretation of the results from analysis of leaf drought or salinity. CHAPTER 6

Developmental Physiology

OUTLINE

6.1. Yield Formation 169 6.2.6. Lipids and Volatiles 205 6.1.1. Yield Potential and Its Realization 172 6.3. Sources of Variation in Fruit 6.2. Grape Composition and Fruit Composition 209 Quality 178 6.3.1. Fruit Maturity 211 6.2.1. Water 181 6.3.2. Light 212 6.2.2. Sugars 183 6.3.3. Temperature 214 6.2.3. Acids 186 6.3.4. Water Status 218 6.2.4. Nitrogenous Compounds and 6.3.5. Nutrient Status 220 Mineral Nutrients 190 6.3.6. Crop Load 223 6.2.5. Phenolics 193

6.1. YIELD FORMATION (sometimes the number of clusters) per The amount of fruit production in a given shoot or per unit of canopy length, the “crop year and over the lifetime of grapevines deter- size” is the yield per vine or per unit of land mines both their reproductive success as a area, and the “crop load” is the crop size rela- species and their agronomic trait of yield tive to vine size (estimated as pruning weight potential. Viticultural yields are determined or leaf area) and is a measure of the sink: by the amount of carbohydrate (sugar) parti- source ratio. The terms crop level and crop tioned to the fruit rather than to other organs. size are often used synonymously, and “over- Yield formation is often referred to as crop- cropping” refers to the production of ping, with the crop being the amount of fruit more crop than a vine can bring to acceptable borne on a vine or produced by a vineyard. maturity by normal harvest time (see The “crop level” is the amount of fruit Chapter 6.2).

The Science of Grapevines 169 Copyright # 2010 Markus Keller. Published by Elsevier Inc. All rights reserved. 170 6. DEVELOPMENTAL PHYSIOLOGY

Grapevine yield is made up of a number of (pruning, canopy management, irrigation, different components. Yield components are nutrition, and pest and disease management), those factors in grapevine reproduction that, as well as legal aspects and market-driven multiplied together, total the yield obtained actions (wine style, winery demand, and yield from a single vine or an entire vineyard. For regulation). a single vine, this can be written as follows Although interactions between genotype (Coombe and Dry, 2001): and environment lead to tremendous spatial and temporal variation in grape yield, not all buds shoots clusters Yield ¼ components of yield respond equally to envi- vine bud shoot ronmental conditions. The total number of berries shoots growing on a vine is determined pri- berry weight cluster marily by planting density, trellis and training system, and pruning level (i.e., the number of The vineyard yield is the sum of the yield buds retained at pruning). For a given planting of all individual vines and depends on the density and pruning level, the berry weight is number of vines per unit land area (i.e., planting relatively highly conserved, and the number density) and the trellis and training system of clusters per shoot ordinarily varies far less (e.g., single vs. divided canopy), which together than the number of berries per cluster (Currle with the pruning method (e.g., spur, cane, or et al., 1983). This is true for both the variation minimal) define the size of each vine. The upper between cultivars and the variation between limit on the number of fruitful shoots per vine is growing seasons for the same cultivar. largely determined by the number of buds left Vine growth and maintenance (i.e., sur- after winter pruning (i.e., pruning severity), vival), on the one hand, and reproduction, on but the maximum number of fruit clusters the other hand, both require resources that the available for harvest is limited by the number environment provides in limited supply. There- of inflorescences initiated in those buds during fore, there is an optimal balance between a the previous growing season. The weight of vine’s vegetative and reproductive growth that each cluster depends on the number of berries is the outcome of a trade-off between survival on the cluster and their final weight. The berry and reproduction. The interaction between number, in turn, is determined by the number these two goals is related to the interdepen- of flowers that set fruit. dence of vine capacity and vigor (Huglin and The discussion of the reproductive cycle in Schneider, 1998; Winkler et al., 1974). Capacity Chapter 2.3 is mainly concerned with anatomi- is defined by the total annual growth of the cal and temporal aspects involved in the initia- vine, including its production of fruit, leaves, tion and differentiation of grape clusters and shoots, and roots in one growing season. their subsequent development to fruit maturity. Capacity is thus a measure of the net resource However, yield formation also depends on a gain from the environment, analogous to profit number of both internal and external factors in economic theory (Bloom et al., 1985). It can and the interactions among them. They include be estimated from a vine’s total weight of fruit the genetic makeup (genotype) of the plant and shoots at the end of the season—that is, (species, cultivar, clone, and rootstock), the for manually pruned vines, yield plus pruning vineyard site (soil, water and nutrient avail- weight. In practice, pruning weight is often ability, and climate), seasonal weather patterns used as the sole indicator of vine capacity. (light, temperature, rainfall, and humidity), the Pruning weight, however, is not an appropriate trellis and training system, cultural practices measure of vine capacity for mechanically or 6.1. YIELD FORMATION 171 minimally pruned vines because insufficient leaves per vine, delays canopy development amounts of cane material are pruned off to per- (delay in the realization of maximum leaf mit accurate estimates. Vine capacity increases area), and reduces the total amount of fruit as the size of the root system, shoot number, produced. Vines typically respond to more and leaf area increase. In fact, this total annual severe pruning with increased vigor and growth is roughly proportional to the vine’s production of more total 1-year-old wood. ability to intercept sunlight, which depends on • The production of fruit tends to depress vine its total and sun-exposed leaf area. Capacity is capacity by reducing vigor and decreasing also thought to be approximately proportional the amount of stored carbohydrates, which to the three-fourths power of total plant dry can lead to lower capacity during the mass (C m3/4), which simply states that following year. although large plants are able to grow more • The vigor of a shoot is inversely proportional than small plants, the extra gain diminishes as to the number of shoots per vine and to plant size increases further (Niklas, 2006; crop load. Thus, an increase in shoot number Niklas and Cobb, 2006). Vigor, on the other and an increase in crop load both lead to a hand, is defined as the rate of shoot growth decrease in vigor. and can be measured by the change in shoot • The direction of shoot growth influences length over time. Vigor and capacity are inter- vigor; that is, shoots growing upward are related and are ultimately determined by vine most vigorous, and shoots growing size, pruning level, the amount of stored downward are least vigorous. reserves present at the beginning of the grow- • Fruitfulness increases with shoot vigor to a ing season, seasonal weather patterns, and maximum and then levels off, and it may availability of water and nutrients in the root- even decline as vigor increases further due zone. Thus, a young vine may grow vigorously to deterioration in canopy microclimate. but have a low capacity, whereas a mature vine • Vines can self-regulate by adjusting budbreak with many buds can give rise to shoots of low to bud number. Severe pruning stimulates vigor but can have a high capacity. According budbreak of buds not deliberately retained to Winkler et al. (1974) and Coombe and Dry at pruning (noncount buds) and thus results (2001), the main factors involved in the regula- in a large number of double shoots, water tion and interdependence of capacity, vigor, shoots, and suckers. Greater than 100% and crop load (i.e., vine balance) are as follows: budbreak (i.e., more than one shoot per retained bud) is an indication that the vine • Grapevines have a limited capacity because has the capacity to support more growth. of the limitations imposed by the amount of resources available. Thus, vines can only Siquidem luxuriosa vitis nisi fructu compescitur, male support a certain number of shoots and deflorescit, et in materiam frondemque effunditur; infirma ripen a certain amount of fruit in any one rursus, cum onerata est, affligitur. season. (Indeed, a vigorous vine, unless restrained by crop- • Vine capacity is proportional to total ping, aborts its flowers and teems with vegetative growth; a weak vine, on the other hand, declines potential growth. Thus, older and/or larger when burdened with fruit.) vines (with greater capacity) can support —Columella: De Re Rustica more shoots and more fruit than younger and/or smaller vines. The relationships have important implica- • Pruning tends to depress vine capacity tions for trellis design and training system, as because it reduces the number of shoots and well as pruning and other cultural practices. 172 6. DEVELOPMENTAL PHYSIOLOGY

For example, the balance-pruning concept is achieved on vines of medium vigor because based on the assumption that larger vines excessive vigor tends to impair bud fruitful- can support more shoots with a larger crop. ness, whereas very low vigor is generally a sign This concept was obviously known to the of limitation by some stress. A grapevine bud is Romans, as the preceding quotation from Col- said to be fruitful if it contains at least one umella (4–ca. 70 AD) shows, and it eventually inflorescence primordium that develops into a led to the recommendation to retain approxi- cluster. Such clusters “in the making” can be mately 30 buds for each kilogram of canes seen (using appropriate magnification) in lon- removed during winter pruning in order to gitudinal cuts of winter buds, and this enables make optimal use of the capacity of most wine growers to conduct an initial assessment of a grape cultivars. In addition, irrigation and vineyard’s yield potential prior to winter prun- nutrition can also modulate the relationship ing. Based on the observed average number of among vine balance, vigor, and capacity clusters per shoot, the pruning strategy can be because vegetative growth often reacts more adapted to compensate for annual changes in strongly to water or nutrient supply than bud fruitfulness and bud injury. Bud fruitful- reproductive growth (especially after fruit ness can also be estimated retroactively by set; see Chapters 7.2 and 7.3). counting the number of clusters per shoot Grapevines have an inherent propensity to during the growing season or at harvest, but self-regulate the balance between the growth this ignores clusters that have been abscised of roots, shoots, and fruit. For example, effects due to environmental stress. Fruitfulness is an of high planting density are partly offset by inherited characteristic that is modulated production of fewer buds per vine and (some- by environmental factors at the time of inflo- times) fewer shoots per bud. Similarly, leaving rescence initiation, which occurs before bloom, twice as many buds per vine during winter and subsequent differentiation, beginning aro- pruning will normally not double the yield und bloom (see Chapter 2.3). Yield per node is because the number of berries and their aver- different from bud fruitfulness because it is age size will decrease. Generally, as the number a function of the number of shoots per node, of clusters per vine increases, the number of the number of clusters per shoot, the number berries per cluster and the average weight of of berries per cluster, and the size of these these berries decrease. Consequently, the yield berries. component compensation principle states that Once the maximum number of potentially changing the level of one yield component fruitful shoots has been determined by the will induce a compensatory change of other number of buds retained at winter pruning yield components, resulting in a subproportional (pruning level), bud fruitfulness sets the vine’s change in yield. yield potential for the following growing sea- son. Warm, sunny conditions with adequate 6.1.1. Yield Potential and Its Realization soil moisture and abundant nutrient supply, along with a sufficiently large, actively photo- The yield potential of a crop plant can be synthesizing leaf area, are crucial for the forma- roughly defined as the yield of a cultivar when tion of the maximum number of inflorescence grown in environments to which it is adapted, primordia. The extent to which this initial yield with external resources nonlimiting, and with potential is realized by the vine depends on pests, diseases, weeds, and other stresses effec- how many flowers develop on each inflores- tively controlled. Taking all external factors cence and how many of these flowers set fruit into account, the largest yield potential is often and develop into berries (Alleweldt, 1958). 6.1. YIELD FORMATION 173

Long days ostensibly favor the induction of Seedless and Muscat of Alexandria) appear to a maximum number of inflorescence primordia be unfruitful below 20C. Severe loss of leaf in American Vitis species and their cultivars area (e.g., due to pest or disease infection or (Mullins et al., 1992) and in some V. vinifera cul- hail) can reduce inflorescence initiation and tivars (e.g., Riesling and Muscat of Alexandria). differentiation (i.e., branching), resulting in However, the influence of photoperiod (day fewer and smaller inflorescences being formed length) on bud fruitfulness is far less important in each winter bud (Bennett et al., 2005; than the impact of irradiance (Srinivasan and Candolfi-Vasconcelos and Koblet, 1990; Lebon Mullins, 1981). The effect of light intensity on et al., 2008; May et al., 1969). Because of the fruitfulness is independent of the effect of tem- importance of functional leaf area for inflo- perature, although the two are usually linked: rescence development, the number and size of Cloudy or shady conditions are often asso- the future inflorescences decrease as the sever- ciated with cool temperatures. Low light on ity of leaf destruction increases and the earlier the leaves above and below a bud reduces the loss occurs, although a negative effect still bud fruitfulness; thus, buds located in the inte- persists if loss of leaf area occurs as late as rior of dense canopies generally give rise to 2 months after bloom. fewer clusters than buds at the canopy exterior Water stress normally reduces bud fruitful- (Buttrose, 1974a; Dry, 2000; Sa´nchez and ness (Buttrose, 1974b; Srinivasan and Mullins, Dokoozlian, 2005; Sommer et al., 2000). This 1981), but this is not always the case. A mild may be part of the shade avoidance syndrome water deficit reducing canopy density can actu- that is mediated by phytochromes (Morelli ally increase fruitfulness due to the improved and Ruberti, 2002; see also Chapter 5.2). More- canopy microclimate and thus better light over, overcast conditions during the bloom– exposure (Keller, 2005; see also Chapter 7.2). fruit set period not only reduce fruit set (and Mineral nutrition also influences bud fruitful- thus the number of berries per cluster) but also ness, but little is known about what these decrease bud fruitfulness for the next growing effects are. It is likely that they also involve season (Keller and Koblet, 1995a; May and changes of canopy microclimate. Soil nitrogen Antcliff, 1963). A marked reduction in fruitful- (N) availability during bloom is important ness occurs when daily total photosynthetic because the response of cluster initiation to vine photon flux is reduced to one-third or less of N status shows a clear optimum, with both N that reached under clear skies (which can have deficiency and N excess resulting in a reduced >2000 mmol m 2 s 1). The primary reason for number of inflorescences per shoot (Alleweldt this pronounced light effect is probably the and Ilter, 1969; Currle et al., 1983; Keller and influence of light on leaf photosynthesis; low Koblet, 1995a; Srinivasan and Mullins, 1981). light limits the amount of assimilates produced Although the number of flowers per inflo- by the leaves and thus the amount supplied to rescence of a particular cultivar can vary from the developing buds. In addition to high light less than 10 to more than 1000 in the same vine- intensity, relatively high temperatures are also yard, the environmental causes of this variation required for maximum inflorescence initia- are mostly unknown. It seems clear, however, tion and development (Buttrose, 1969a, 1970b, that much of this variation may be introduced 1974a; Sommer et al., 2000; Srinivasan and during the period of inflorescence differentia- Mullins, 1981). Fruitfulness increases as the tion in the buds. In addition, high temperatures temperature during the cluster initiation during the budswell and budbreak period are phase in the buds increases from 10 to almost associated with fewer flowers per inflorescence 35 C, although some cultivars (e.g., Thompson (Petrie and Clingeleffer, 2005; Pouget, 1981), 174 6. DEVELOPMENTAL PHYSIOLOGY perhaps because with the faster development at period to more than 1 month, and the abscised higher temperature there is more competition calyptra may remain on the flower. This does from the emerging shoots for remobilized not prevent pollination, but it can interfere storage reserves (Bowen and Kliewer, 1990). with fertilization by reducing pollen viability High temperature and warm, moist soil condi- or germination rate, leading to excessive tions, on the other hand, accelerate the rate of abscission of flowers (shatter) and poor fruit reserve mobilization in spring and favor both set (Koblet, 1966). shoot growth and flower development. Flower The proportion of flowers that set fruit is differentiation and early development during often between 20 and 50% (Currle et al., 1983; and after budbreak are entirely dependent on Mullins et al., 1992). The final fruit yield is pri- the availability of stored nutrient reserves and marily dependent on the photosynthetically the rate of their remobilization because the active leaf area early in the growing season. unfolding leaves act as sinks during this Loss of leaf area (defoliation) during this period. The more shoots that are growing on a period generally leads to poor fruit set due to vine, the more their demand for stored nutri- low pollen viability resulting from carbon ents may compete with the formation of flow- starvation of the flowers, which is a conse- ers. The amount of reserves stored during the quence of their low sink strength before fruit previous growing season is therefore critical set (Candolfi-Vasconcelos and Koblet, 1990; for maximum flower formation. Overcropping, Coombe, 1959; Winkler, 1929; see also Chapter excessively cool or hot seasons, untimely loss of 5.1). Although the total requirement for assimi- active leaf area, and poor water and nutrient lates by flowers and small berries is quite low, management all contribute to limiting (and the developing flowers cannot compete with sometimes depleting) reserves in the vine’s the growing shoot tips if resources become lim- permanent organs and thus interfere with iting and are very susceptible to environmental flower formation. In other words, the number stress. Ideal conditions during bloom, leading of flowers formed per inflorescence and per to maximum fruit set, are practically identical vine increases as vine reserve status increases to those required for maximum inflorescence (Bennett et al., 2005). Adequate reserve status initiation. The optimum temperature range for may be particularly important to sustain pro- bloom and fruit set in grapevines appears to ductivity of high-yielding vineyards, where be 20–30 C, and both high (>35 C) and low functional leaves seem to be required after (<15 C) temperatures reduce fruit set (Currle fruit harvest to replenish the reserve pool et al., 1983; Kliewer, 1977a). Overcast, cool or (Holzapfel et al., 2006). hot weather, and water or nutrient deficits limit As discussed in Chapter 2.3, bloom occurs the amount of photosynthate available to the within 5–10 weeks after budbreak. The actual flower clusters and can lead to embryo abortion timing depends on the cultivar and weather and hence poor fruit set (Keller, 2005; Lebon conditions. Anthesis typically does not occur et al., 2008; Mu¨ ller-Thurgau, 1883). Water defi- until the mean daily temperature exceeds cit and other stresses that lead to low sugar approximately 18 C. Under favorable condi- supply to and starch accumulation by the tions (high light and high temperature), an developing pollen before anthesis also interfere inflorescence can complete flowering within a with meiosis and result in male sterility, which few days, and bloom lasts approximately 7–10 also curtails fruit set (Dorion et al., 1996; Goetz days for individual vines (Currle et al., 1983). et al., 2001; Saini, 1997). Even moderate water Unfavorable environmental conditions (e.g., deficit at this time can significantly reduce fruit low temperature or rain) can extend the bloom set and final yield (see Chapter 7.2). Fruit set 6.1. YIELD FORMATION 175 can also suffer in the interior of dense canopies, vine. Removing the shoot tips, especially those which might be associated with the shade of vigorously growing shoots, or cane or trunk avoidance syndrome that is mediated by phy- girdling during bloom can sometimes amelio- tochromes (Morelli and Ruberti, 2002; see also rate the impact of such adverse conditions by Chapter 5.2). temporarily eliminating strong competitors for Unfavorable conditions may also result in a assimilates, whereas removal of leaves before high number of so-called “shot berries” (ovar- bloom accentuates fruit loss (Coombe, 1959, ies that fail to develop properly) or, in extreme 1962; Koblet, 1969; Mu¨ ller-Thurgau, 1883; cases, a syndrome termed inflorescence necro- Vasconcelos and Castagnoli, 2000). Thus, the sis or early bunch stem necrosis, whereby number of berries per cluster is most suscep- whole inflorescences or portions of them die tible to environmental stress at or just before and are abscised (Figure 6.1) (Jackson, 1991; anthesis. Jackson and Coombe, 1988; Keller and Koblet, In addition to its effect on fruit set, bloom- 1994; Keller et al., 2001a; Winkler, 1929). The time temperature might also somehow influ- term “necrosis” (Greek nekro´s ¼ dead) may be ence bunch stem necrosis, a physiological dis- unfortunate because it implies an unpro- order that is associated with necrotic lesions grammed, chaotic cell death without nutrient on the rachis (see Chapter 7.3). The lesions recycling (Mu¨ ntz, 2007), whereas the death develop only after the berries begin to ripen, and abscission of portions of an inflorescence apparently often beginning around stomata or is likely to be organized and genetically pro- dead bracts at branching points or beneath the grammed and to involve mobilization and epidermis, and may interrupt assimilate supply recycling of nutrients to other sinks. Deliberate to the berries distal to a lesion, which interferes shedding of reproductive organs allows the with ripening and may lead to abortion of vine to adjust its reproductive output accor- the afflicted cluster portion (Currle et al., 1983; ding to the prevailing external conditions. Hifny and Alleweldt, 1972; Theiler, 1970). Such self-thinning avoids a situation whereby However, although a negative correlation has resources are (over)invested in reproduction at been found in some circumstances between the expense of the long-term survival of the the average daily maximum temperature

FIGURE 6.1 Inflorescence necrosis on Mu¨ller-Thurgau following a cool and overcast bloom period (left) and on nitrogen-deficient Cabernet Sauvignon following water deficit during bloom, with normal fruit set on nitrogen-sufficient vines (right). Photos by M. Keller. 176 6. DEVELOPMENTAL PHYSIOLOGY during the bloom period and the incidence (Currle et al., 1983; Williams and Matthews, of bunch stem necrosis (Theiler and Mu¨ ller, 1990). Whereas gibberellin ostensibly stimu- 1986, 1987), no satisfactory explanation for this lates the berries’ sink strength (Weaver et al., apparent phenomenon has been given. 1969), bark removal, called girdling, eliminates The final size of grape berries that set after phloem flow to the perennial parts of the vine bloom is determined by the number of cell so that more sugar is available to the develop- divisions before and after bloom, the extent of ing fruit clusters (Roper and Williams, 1989). expansion of these cells, and the degree of However, girdling typically constitutes only a weight loss (shrinkage or shrivel) prior to har- temporary interruption of phloem flow because vest. Because there are many more cycles of cell callus, ostensibly formed by nearby phloem division (doublings of cell number) before cells, heals the wound (Sidlowski et al., 1971). bloom than after bloom [according to Harris Cell enlargement is generally more sensitive et al. (1968), the ovary contains approximately to temperature than is cell division. Neverthe- 200,000 cells compared with 600,000 in the ripe less, maximum cell division occurs at approxi- berry], there should be ample opportunity for mately 20–25 C, and temperatures below early season environmental factors to influence 15C or above 35C lead to a marked reduc- berry size (Coombe, 1976). Because cell division tion in the rate of cell division, resulting in is dependent on carbon supply, stress factors smaller berries (Kliewer, 1977a). The decrease that diminish assimilate supply will also in cell division due to both cold and heat stress limit the cell number (Van Volkenburgh, 1999). may be caused by the thermosensitivity of cyto- The initial postbloom rates of cell division and kinin production and transport. It is believed cell expansion are controlled by the number that cytokinins are inactivated at low tempera- of seeds that have been fertilized, perhaps tures by conversion of the active form to a stor- because more seeds make more auxin and age form (Mok and Mok, 2001), whereas during stimulate more gibberellin production, making a heat period, the seeds ostensibly produce less the berry a stronger sink (Cawthon and Morris, cytokinin and the xylem transports less cytoki- 1982; Scienza et al., 1978). Thus, the final berry nin from the roots (Banowetz et al., 1999). It is volume increases with increasing number of possible that the need for leaf cooling causing seeds per berry (Gillaspy et al., 1993; Winkler rapid transpiration rates in leaves during heat and Williams, 1935). Although this assertion stress (see Chapter 5.2) reduces the availability has not been tested, it is possible that a loss of of cytokinin for fruit development. Heat also ovule viability at temperatures above appro- decreases cell expansion; however, whereas ximately 35C might be associated with fewer high temperature before the berry enters the seeds per berry and thereby constrain berry lag phase of growth leads to an irreversible size (Williams et al., 1994). The effect of seed reduction in berry size, the temperature experi- number is most pronounced at the lower end enced after veraison does not seem to have a so that the increase in volume when seed num- noticeable effect on berry size (Hale and ber increases from three to four is only small. Buttrose, 1974; Kliewer, 1973). Because heat The absence of seeds in many table grape culti- has a negative effect on both fruit set and berry vars limits berry growth, which conflicts with growth, poor set and small berries are typical the general demand for large table grapes. of years that experience a “heat wave” during Growers therefore often stimulate berry expan- the bloom–fruit set period. The temperature sion by applying gibberellin sprays at bloom sensitivity of berry cell division and expansion and fruit set and/or by removing a strip of also has implications for vineyard manage- bark around the canes or trunk at fruit set ment: Removing leaves in the cluster zone early 6.1. YIELD FORMATION 177 during berry development may result in smal- Moreover, the epidermis of the developing ler berries due to solar heating of the sun- berry has a few stomata (approximately 1 or exposed fruit. Warm temperatures in the range 2mm 2, compared with 100–400 mm 2 in leaves) of 25–32C between bloom and the lag phase for gas exchange. As the berry expands, the of berry growth are furthermore thought to stomatal density on the berry surface decreases promote the capacity of seeds to germinate because no new stomata are produced after (Currle et al., 1983). anthesis, and the stomata are progressively In addition to their effect on berry size, both occluded by epicuticular wax, suberin, and other cool and hot temperatures also delay fruit components so that they become nonfunctional development, especially by prolonging the lag by veraison (Bessis, 1972b; Currle et al., 1983; phase of berry growth (Hale and Buttrose, Swift et al., 1973). Only early in its development 1974). Water stress or nutrient stress during does the berry fix an appreciable amount of the early stages of fruit development after fruit carbon, which it can use for growth and meta- set normally limit potential berry size by reduc- bolism, and photosynthetic enzymes may be ing the rate of cell division. If the stress occurs degraded after veraison (Famiani et al., 2000). later, then it will limit cell expansion, which Consequently, although berries may be able to also leads to somewhat smaller berries. How- recycle approximately 20–30% of the respiratory ever, in contrast to the weak sink strength of carbon loss via photosynthesis, the photo- the flowers prior to fruit set, the developing synthetic rate declines to approximately 10% of seeds and berries are stronger sinks than the the respiration rate during ripening (Koch and shoots and, especially, the roots. Therefore, Alleweldt, 1978; Ollat et al., 2002). Therefore, although stress does reduce seed and berry size, grape berries rely heavily on continued import the decrease is less than would be expected of sugar for proper development and metabolism. from the reduction in photosynthate avai- Any limitation in assimilate supply (e.g., due lability. This is probably due to remobilization to loss of active leaf area, poor weather condi- of stored reserves in both the leaves and the tions, and water or nutrient stress) during permanent parts of the vine. pollination and early berry growth can lead to Although the berries and their seeds are abortion of embryos. Such early abortion of predominantly sink organs, it should be some fruits may enable the survival of others; remembered that they are green—at least that is, by limiting its investment in the number before veraison—and their skin absorbs much of reproductive organs, the vine has a greater of the incident light (Blanke, 1990a). The cells chance to support the remaining fruits to seed of both the seeds of preveraison berries and maturity. Thus, early stress also leads to fewer the berry pericarp contain chloroplasts with seeds per berry and strongly reduces the size of functional (and shade-adapted) photosynthetic mesocarp cells but does not impair fruit ripening machinery that participates in berry and seed (Ollat et al., 2002). The amount of photosynthates metabolism (Blanke and Leyhe, 1989b; Famiani that are partitioned to the fruit, especially early et al., 2000; Goes da Silva et al., 2005; Kriedemann, during fruit development, is critical for final 1968a). Berry and seed photosynthesis may refix fruit size (Candolfi-Vasconcelos and Koblet, et al CO2 that would otherwise be lost in respiration 1990; May ., 1969). Insufficient photosyn- (Koch and Alleweldt, 1978; Palliotti and thate supply limits both cell division and cell Cartechini, 2001) and may produce some of the expansion, with very little compensation later NADPH and ATP required for energy-intensive in the season so that final berry size is irrever- metabolism in addition to the accumulation of sibly reduced. Therefore, berry size at harvest malate as a by-product of CO2 refixation. also tends to decline as the number of berries 178 6. DEVELOPMENTAL PHYSIOLOGY per vine increases (Keller et al., 2008). Con- to keep in mind the yield component compen- versely, limiting assimilate supply late in the sation principle when deciding on strategies growing season (i.e., after veraison) has little to aimed at optimizing yield and fruit quality. no effect on berry size but restricts ripening. In addition, because berry growth is most sen- Water or nutrient deficits typically reduce sitive to stress factors during the early stages yield, particularly if the deficit occurs early in of development and because final berry size the growing season, when the inflorescences is largely predetermined by veraison, yield are not competitive in comparison with the predictions are generally much more accurate shoot tips (Williams and Matthews, 1990). after this stage than before it. Inadequate water or nutrient supply before bloom can lead to abortion of entire inflores- cences (limiting cluster number), stress during 6.2. GRAPE COMPOSITION AND bloom results in poor fruit set (limiting berry FRUIT QUALITY number), and stress during the period of cell division restricts berry enlargement (limiting berry size). Although water deficit may not The grape berries’ physical and chemical inhibit cell division (but the stress may never- composition at harvest is responsible for the theless slow the rate of cell division), it does fruit quality characteristics and, consequently, limit cell volume (see Chapter 7.2). Grape the quality attributes of the wine or grape juice berries grow mostly at night, and they often produced from the fruit. Much of what we call lose some weight during the day, especially “quality” in grapes, juice, and wine has to do during periods of water deficit (Greenspan with our perception of taste and flavor, which et al., 1994, 1996; Keller et al., 2006). However, is the outcome of the processing in the brain whereas such daytime berry shrinkage is com- of stimuli received by taste receptors in the mon before veraison, it only seems to occur tongue and olfactory receptors in the nose. under severe water deficit after veraison. The Compounds ranging from sugars to acids and same extent of water deficit occurring before phenolics to volatile chemicals all contribute the lag phase of berry growth normally curtails interactively to the overall taste and flavor berry size more than if it occurs during or after perception (i.e., sensory quality). As berries that phase. Moreover, applying more water ripen, they undergo a multitude of both physi- later in the season cannot compensate for the cal and chemical changes, although many decrease in berry size due to early season defi- changes and processes important to fruit qual- cit. This has implications for type, timing, rate, ity also occur long before ripening begins and duration of irrigation. (Brummell, 2006; Conde et al., 2007; Coombe, At every step during the yield formation 1992; Deytieux et al., 2007; Hrazdina et al., process there are management tools available 1984; Kanellis and Roubelakis-Angelakis, 1993; to maximize or optimize final yield according Sarry et al., 2004). These developmental chan- to the desired specifications. For example, the ges are in turn coordinated by the interplay timing and rate of water or nutrient application and cooperation of hundreds or thousands of may be varied according to the intended end genes (Boss et al., 1996; Castellarin and Di use of the grapes produced in a particular Gaspero, 2007; Deluc et al., 2007; Goes da Silva vineyard block. If and when these cultural et al., 2005; Pilati et al., 2007; Robinson and practices are applied often depends on seasonal Davies, 2000; Terrier et al., 2005). A general over- weather patterns, as well as economic and view of the changes during berry development legal considerations. Growers also always have is shown in Figure 6.2. 6.2. GRAPE COMPOSITION AND FRUIT QUALITY 179

FIGURE 6.2 Diagrammatic representation of the relative size and color of grape berries at 10-day intervals after bloom and of the major changes occurring during berry development. Reproduced from Coombe (2001).

The beginning of grape ripening (veraison) (see Chapter 1.2); they owe their skin color is easily recognized by the change of skin color to a combination of chlorophylls (diminishing and the sudden softening of the berry (see during ripening), carotenoids, and the pale Chapter 2.3). The change in color occurs due to yellow, but barely visible, flavonols. A minor the degradation of the green chlorophylls portion of the chlorophylls may be converted in (Hardie et al., 1996a), which unmasks the the skin and, to a lesser extent, in the pulp, into previously invisible yellow to orange-red colorless tetrapyrroles, which are identical to carotenoids, and due to the simultaneous accu- those produced in senescing leaves and are mulation of red, purple, or blue anthocyanin strong antioxidants (Mu¨ ller et al., 2007). pigments in dark-skinned cultivars. White Actually, softening, which can be measured grapes, of course, do not make anthocyanins as deformability, coincides with the beginning 180 6. DEVELOPMENTAL PHYSIOLOGY of sugar accumulation but precedes by several be activated in the skin and, to a lesser extent, days the change in skin pigmentation and in the mesocarp at veraison (Caldero´n et al., resumption of berry growth (Coombe, 1992; 1994; Ros Barcelo´ et al., 2003). Coombe and Bishop, 1980; Terrier et al., 2005; Cell wall loosening in the pulp seems to be Wada et al., 2009). Softening occurs due to the responsible for berry softening, and soon after- gradual disassembly of the mesocarp cell walls wards cell wall loosening in both the pulp and (Brummell, 2006; Huang and Huang, 2001) and the skin enables berry expansion (Huang and the decline in mesocarp cell turgor during the Huang, 2001; Huang et al., 2005a), which thus preveraison lag phase of berry growth (Thomas occurs despite the more than 10-fold decline in et al., 2008). Subsequent alterations include mesocarp turgor pressure just before veraison expansion (and later shrinkage) of the berry (Thomas et al., 2006). This turgor loss occurs due volume; structural changes in the skin, pulp, to the accumulation of sugars and other solutes and vascular tissues; a switch in metabolic in the mesocarp apoplast, which starts slightly pathways; and rapid accumulation of solutes before veraison (Wada et al., 2008, 2009) due to leading to dry mass gain, decrease in acidity, the switch from symplastic to apoplastic phloem and increase in pH. The loosening of the skin unloading (Zhang et al., 2006). Although berry cell walls (aided by release of calcium and volume increases during ripening, it cannot do modifications in polysaccharides such as cellu- so indefinitely because the skin sets a limit to lose, hemicellulose, and pectin) and incorpora- mesocarp expansion (Matthews et al., 1987). tion of proteins (e.g., hydroxyproline-rich Following an initial increase at veraison, the extensins for added tensile strength) enables extensibility of the skin later decreases (i.e., its the skin cells to become tangentially stretched stiffness increases) in parallel with the stiffening while the pulp cells resume expansion after of the cuticle. These changes might be caused by et al the lag phase (Chervin ., 2008; Huang a decrease in the unsaturated and elastic C18 cutin et al., 2005a; Nunan et al., 1998; Ortega-Regules monomers and a concomitant increase in the et al et al ., 2008). Cell wall-loosening proteins such saturated and rigid C16 monomers (Bargel ., as expansin, whose activity peaks at the end 2006; Marga et al., 2001). Furthermore, additional of the lag phase, and various enzymes modify polysaccharides and phenolics such as hydro- the cell wall polysaccharide components by xycinnamic acids and flavonoids, including “unlocking” the hydrogen bonds between the tannins, may be deposited in the skin cell walls stiff cellulose microfibrils and the hemicellulose (Amrani Joutei et al., 1994; Lecas and Brillouet, matrix just before and during ripening to 1994; Schlosser et al., 2008). The phenolics are enable the berry to soften (Deluc et al., 2007; bound to cell wall polysaccharides and proteins Ishimaru and Kobayashi, 2002; Nunan et al., by peroxidase, which further stiffens the cell 2001; Pilati et al., 2007; Rose and Bennett, 1999; walls and limits cell expansion and thus Rose et al., 1997, 1998; Schlosser et al., 2008). In counteracts the initial wall-loosening properties addition to this enzyme action, the hydroxyl of peroxidase (Bargel et al., 2006; Potters et al., radical (•OH), produced from other reactive 2009). Note that because many environmental oxygen species with the help of apoplastic per- stresses increase the amount of phenolics in the oxidase enzymes, is thought to sever the bonds apoplast, one way by which such stresses often between cell wall polysaccharides nonenzyma- limit berry size might be due to the influence of tically, which may enhance the cell wall loosen- phenolics on cell wall stiffness. By the same logic, ing and degradation process required for cell the bonding of phenolics to cell wall polymers expansion and fruit softening (Mller et al., would presumably diminish their extractability 2007; Rose et al., 2004). Peroxidase appears to during fermentation. 6.2. GRAPE COMPOSITION AND FRUIT QUALITY 181

At the same time, protein incorporation and these vacuoles may occupy 99% of the mesocarp polysaccharide modifications also occur in cell volume (Diakou and Carde, 2001; Storey, the mesocarp cell walls (Ortega-Regules et al., 1987), grape juice essentially consists of vacuolar 2008). Peroxidase-mediated inclusion of soluble sap of mesocarp cells. Thus, water is by far the proteins, especially extensin glycoproteins dominant chemical component of the ripening (proteins attached to sugar molecules), rein- berry. Nonetheless, due to the accumulation of forces the cell walls (Nunan et al., 1998; sugars and other solutes during ripening, the Ros Barcelo´ et al., 2003). Despite these massive relative water content is highest before veraison changes, the thickness of the epidermis, hypo- (90%), declining to approximately 75–80% at dermis, and mesocarp cell walls remains essen- maturity and sometimes to less than 70% in tially constant during ripening (Hardie et al., overripe grapes (>30 Brix). Consequently, 1 kg 1996b; Nunan et al., 1998). Moreover, the ultra- of grapes yields approximately 0.6–0.8 L of juice structure, cell walls, and membranes of the berry or wine, depending on species, cultivar, berry cells remain intact (Diakou and Carde, 2001; size, maturity, and extent of pressing (Viala Fouge`re-Rifot et al., 1995; Hardie et al., 1996b); and Vermorel, 1909). However, the water cell compartmentation is retained, mitochondria content can decrease to as little as 15% when remain functional, and plasmodesmata main- grapes are dried to produce raisins; thus, 1 kg tain their cell-to-cell connections (except the of grapes yields less than 250 g of raisins. ones in the phloem membranes, which are Xylem sap is the main source of water for plugged to facilitate apoplastic phloem unload- the berry before veraison; it is thought to ing; see Chapter 5.1). Only the locule area account for approximately 75% of the total around the seeds appears to be subject to water influx (Ollat et al., 2002). Xylem flow into some degradation, especially toward the end of the berry declines at veraison, and phloem sap the ripening phase (Krasnow et al., 2008). In the concomitantly becomes the primary or only remainder of the pericarp, senescence and loss source of berry water (Greenspan et al., 1994; of cell compartmentation only seem to set in Keller et al., 2006) because excess water is once berries have attained their maximum delivered to the berry in the phloem along with weight, at least in some cultivars, and are assimilates such as sucrose and amino acids. sometimes associated with subsequent berry The rate of carbon import is thought to increase shrinkage (Hardie et al., 1996a; Tilbrook and 3.5-fold at veraison (Ollat et al., 2002). Inciden- Tyerman, 2008). Nonetheless, shrinkage due to tally, a similar shift from the xylem to the dehydration occurs only slowly, which is why phloem as the main water source during ripen- the production of raisin grapes is essentially ing also occurs in apples and tomatoes. Berry restricted to hot climates. transpiration represents the bulk of water loss from the berry, especially before veraison, but 6.2.1. Water transpiration rates of grape berries are 10–50 times lower than those of leaves and decline The final size of the berries of a particular during berry development (Blanke and Leyhe, grape variety is mainly influenced by cell 1987; Du¨ ring and Oggionni, 1986; Rogiers enlargement because cell division determining et al., 2004a). Indeed, it appears that grape the number of cells is comparatively little berries are designed to minimize evaporative affected by environmental conditions. The rapid water loss. First, they are typically round or growth of the berry during ripening is mostly nearly so, which geometry translates into the due to water import and retention by the least amount of surface area relative to volume; mesocarp vacuoles. Because by mid-ripening this in turn translates into a large water storage 182 6. DEVELOPMENTAL PHYSIOLOGY capacity compared with the evaporative sur- preveraison berries shrink and expand readily face. Second, they have far fewer stomata than with fluctuating vine water status, postveraison leaves, which moreover become nonfunctional berries are much less subject to such fluctua- by veraison (Blanke et al., 1999; Swift et al., tions. If the results obtained with algae are 1973). Finally, their cuticle is impregnated with applicable to grape berries, then the increase approximately 10 times more wax than the leaf in solute concentration, leading to “osmotic cuticle (Possingham et al., 1967; Radler, 1965). dehydration,” should lead to closure of aqua- However, in addition to evaporating from the porins, which would decrease membrane water berry surface, water can also flow back through permeability (Ye et al., 2004). This not only the xylem from the berry to the shoot (Keller could contribute to the high resistance to dehy- et al., 2006; Lang and Thorpe, 1989). Due to dration of ripening berries but also may sup- the low transpiration rate of the berry, much port the changes in skin elasticity in limiting of the water received by a preveraison berry berry growth. Although the berries will still via the phloem may be recycled back to the shrink during prolonged episodes of water C et al vine, especially in the afternoon, when leaf stress (Keller ., 2006), shrinkage becomes C C may be lower than berry. The decreasing berry limited because phloem water supply is much after veraison due to sugar import changes the less prone to changes in vine water status than water potential gradient (DC) in favor of the is xylem water supply. On the other hand, berry berry so that water that has already entered expansion becomes limited because the increas- the berry cell vacuoles becomes increasingly ing cutinization of the thick epidermal cell protected from being “pulled back” by the walls and the increasing stiffness of the cuticle leaves. Instead, surplus phloem water, which decrease the skin’s elasticity as the fruit has moved to the berry cell walls osmotically matures (Bargel and Neinhuis, 2005; Bargel during apoplastic phloem unloading (see et al., 2006; Matthews et al., 1987). Cuticle stiff- Chapter 5.1), may be recycled out of the berry ening is at least partly due to incorporation of via the xylem before entering the berry cells phenolics and polysaccharides into the cutin (Keller et al., 2006). It appears that cultivars matrix (Domı´nguez et al., 2009). differ in the extent of such xylem backflow, A rigid (i.e., unelastic) skin may be necessary perhaps due to variation in the hydraulic resis- for grapes to accumulate sugars and other tance of the pedicel or berry xylem (Tilbrook solutes to high concentrations (i.e., high p) with and Tyerman, 2009; Tyerman et al., 2004). limited cell expansion, whereas recycling of The (usually) reversed xylem flow in the phloem water in the xylem may maintain the postveraison berry provides some indepen- low mesocarp turgor. However, the increase dence from the fluctuations in C experienced in surface area of the expanding berry causes by the leaves due to transpiration. Although mechanical stress due to strain and tension in the berry is not hydraulically isolated from the skin tissues that also bear the turgor pres- the vine (i.e., the berry xylem remains func- sure that is transmitted to them from the inte- tional after veraison; Bondada et al., 2005; Cha- rior mesocarp cells (Considine and Brown, telet et al., 2008b), the sensitivity of berry water 1981; Considine and Knox, 1981). Thus, the status to soil and plant water status declines stiffening of the cuticle and decrease in skin greatly after veraison, and the daily cycle of extensibility seem to occur at the cost of berry shrinkage during the day and expansion increased susceptibility to cracking (splitting), at night becomes much less pronounced which can occur during humid or rainy condi- (Greenspan et al., 1994, 1996; Matthews tions when water may be absorbed through and Shackel, 2005). Accordingly, whereas the skin (Considine and Kriedemann, 1972; 6.2. GRAPE COMPOSITION AND FRUIT QUALITY 183

Lang and Thorpe, 1989). Cracking can ruin the The slow transpiration rate of grape berries quality of table and juice grapes and increase strongly limits transpirational cooling, which the incidence of bunch rot (see Chapter 7.5). It results in marked day–night fluctuations in is also a common problem in cherry production berry temperature and can increase the tem- (Sekse, 1995), where the resistance to osmotic perature of sun-exposed berries by more than water uptake through the fruit skin is dozens 15 C above ambient temperature (Smart and of times lower than that to transpirational Sinclair, 1976; Spayd et al., 2002). The post- water loss and declines as fruit sugar concen- veraison decrease in berry transpiration also tration increases (Beyer and Knoche, 2002). slows the drying rate of raisin grapes, espe- Warm and humid conditions may favor crack- cially if the grapes are harvested late in the ing because the water permeability of the cuti- season, when temperatures and vapor pres- cle increases with rising temperature (Beyer sure deficits decline. To facilitate drying and and Knoche, 2002; Scho¨nherr et al., 1979). The mechanical harvesting, growers often cut off permeability also increases with increasing the fruiting canes with clusters still attached relative humidity of the atmosphere, probably totheshootsinlatesummer,apracticeknown because absorbed water molecules lead to swel- as “drying on the vine” or “harvest pruning.” ling of the cuticle (Scho¨nherr, 2006; Schreiber, Yet such drying takes much longer than 2005). The uptake of surface water by moist drying manually harvested clusters spread on grape berries, or even berries in a very humid the ground because the temperature in the atmosphere, is one reason why removal of canopy is lower than that on the ground, so leaves around the fruit clusters is a key late-ripening cultivars such as Thompson canopy management practice, especially in Seedless are not well suited to this practice. regions that typically experience rainfall during Because severing canes eliminates photo- the ripening phase. The increased air flow and, synthesizing leaf area at a time when inflores- once sunshine returns, higher berry surface cence primordia may still be differentiating, temperature are conducive to rapid drying of this practice can sometimes come at the cost the clusters after a rainfall event, which limits of some reduction in yield potential for the the amount of water that can be absorbed subsequent year (May, 2004; Scholefield et al., through the skin and the extent of dilution of 1977a,b, 1978). berry solutes. However, cracking may also ensue under conditions of root pressure (e.g., 6.2.2. Sugars when irrigation suddenly relieves a drought episode) that may prohibit recycling of phloem- Sugars, especially glucose and fructose, are derived water out of the berry via the xylem among the principal grape ingredients deter- (Keller et al., 2006). Either way, the pressure mining fruit and wine quality because they are generated inside the berry by the incompressible responsible for the sweet taste of the fruit water is transmitted from the mesocarp cells (and thus of grape juice and wine); decrease (i.e., (whose thin cell walls are elastic—that is, exten- mask) the perception of sourness, bitterness, sible) to the epidermal cells (whose stiff cell and astringency (which is the main reason many walls are easily fractured). Berries probably people add sugar to tea and coffee); and enhance crack when the mesocarp cell pressure exceeds the “mouthfeel” (aka “texture”), “body,” or the cell wall resistance of the skin cells, a “balance” of wines (Hufnagel and Hofmann, relationship that is cultivar specific (Considine, 2008b). Most important for wine, (anaerobic) 1981; Lang and Du¨ ring, 1990; Lustig and fermentation converts hexose (C6H12O6) sugars Bernstein, 1985). into alcohol (ethanol: CH3CH2OH) and CO2 184 6. DEVELOPMENTAL PHYSIOLOGY

(two molecules each for each molecule of more than 90% of the soluble solids in mature hexose sugar): berries, with much of the remainder being ! þ organic acids. In berries of most Vitis cultivars, C6H12O6 2CH3CH2OH 2CO2 95–99% of these sugars are present in the form This process is accomplished by various of the hexoses glucose and fructose, with the species of yeast (but mostly by Saccharomyces cer- remainder predominantly made up of sucrose evisiae), which live on the surface of grapes (Hawker et al., 1976; Hrazdina et al., 1984; Liu (Belin, 1972). Ethanol is important for wine qual- et al., 2006; Lott and Barrett, 1967). The two ity partly because most aroma volatiles are more hexoses have been estimated to be responsible soluble in ethanol than in water so that their for more than 80% of the osmotic pressure (p) aroma impact declines with increasing ethanol in the mesocarp and almost 60% in the exocarp content and partly because it can suppress (Storey, 1987). In contrast, sucrose may account the perception of a wine’s overall “fruity” for up to 30% of the sugar content in some Mus- character (Escudero et al., 2007). In addition to cadinia berries (Carroll et al., 1971). With regard ethanol and CO2, yeasts convert a portion of to the taste of sweetness, fructose > sucrose > the sugars to glycerol as a by-product of fermen- glucose. The fact that fructose is almost twice tation, which also adds to the sweetness, as sweet as sucrose may explain why inexpen- mouthfeel, and body of wines (Hufnagel and sive high-fructose corn syrup is so widely used Hofmann, 2008b; Ribe´reau-Gayon et al., 2006; by the food industry. At low temperatures Noble and Bursick, 1984). Note under the (10 C), preveraison V. vinifera berries contain aerobic (i.e., oxygen-rich) conditions of a vine- approximately equal amounts of glucose and yard, yeasts meet their energy requirements by fructose, but the glucose:fructose ratio rises as (aerobic) respiration rather than fermentation the temperature increases, and at 35C it can so that the sugars present in unharvested grapes reach 3–10 (Currle et al., 1983; Findlay et al., are turned into CO2 and water. The little ethanol 1987; Harris et al., 1968; Kliewer, 1964, 1965a). that is being produced is further respired Irrespective of temperature, however, the glu- (i.e., oxidized) to acetic acid (CH3COOH, and cose:fructose ratio declines to approximately eventually to CO2) and water by aerobic bacteria 1 around veraison and remains more or less of the genus Acetobacter: constant thereafter, whereas the proportion of sucrose increases at low temperatures (Kliewer, CH3CH2OH þ O2 ! CH3COOH þ H2O 1964, 1965a; Viala and Vermorel, 1909). At or just before veraison, hexose sugars Employing the same process in the presence of begin to accumulate in the cell vacuoles of the oxygen, these bacteria can also turn wine into mesocarp and exocarp, although the former vinegar (French vin ¼ wine, aigre ¼ sour), espe- precedes the latter (Coombe, 1987). Sugar accu- cially at high temperature and high pH. mulation is dependent on import of sucrose Although grape flowers and young berries from photosynthesizing leaves or woody stor- may contain some starch due to their photosyn- age organs and is thought to proceed more thetic activity (Fouge`re-Rifot et al., 1995; Hardie rapidly in berries that transpire more rapidly et al., 1996a; Kriedemann, 1968a; Swift et al., (Rebucci et al., 1997). Up until veraison, most of 1973), sugar is imported into the developing the imported sucrose is used for energy berries as sucrose via the phloem. The seed generation (respiration; see Chapter 4.4) and endosperm accumulates starch that serves as a production of other organic compounds but source of energy and carbon during and after does not enter the vacuole. By the time they seed germination. Sugars typically make up begin changing color, the berries generally have 6.2. GRAPE COMPOSITION AND FRUIT QUALITY 185 reached a soluble solids concentration of 9 or approximately 15% (v/v) in wine made from 10Brix (g/100 g of liquid, or % w/w soluble such grapes, which also happens to be close solids, as estimated by refractometry at a stan- to the upper limit of survival for the most dard temperature of 20C). During ripening, ethanol-tolerant yeasts. Berries at 25Brix have there is a rapid increase in hexose sugars due a total solute concentration of approximately to import of sucrose and its subsequent 2 M, which is equivalent to an osmotic pressure breakdown by invertases (see Chapter 5.1); it is of 5 MPa. Although import of sugar ceases thought that the activation of sugar storage around this stage, the sugar concentration can triggers malate efflux from the vacuoles (Sarry increase further due to berry dehydration and et al., 2004). Moreover, the respiratory carbon shrinkage (Figure 6.3). Thus, the final sugar flux through glycolysis (see Chapter 4.4) also concentration at harvest varies widely; table declines at veraison, at least in the mesocarp grapes and grapes destined for juice produc- (Giribaldi et al., 2007; Harris et al., 1971), whereas tion may be harvested at 15–17Brix, whereas the rate of glycolysis may actually increase overripe, late-harvest wine grapes may reach during ripening in the skin (Negri et al., 2008). twice that sugar concentration, and dried raisin In the mesocarp, malate, rather than sucrose, grapes are much more concentrated still. becomes the substrate for berry respiration, Sugars are carbohydrates, a class of chemicals which further enhances sugar accumulation. that also includes the mostly insoluble polysac- Thus, hexose storage and malate breakdown charides contained in the cell walls. The bulk of are activated simultaneously at veraison, and the cell wall polysaccharides in grape berries is sugar accumulation can continue for some time composed of cellulose and pectins with smaller after berry expansion ceases. Depending on amounts of hemicelluloses, with most of the latter species and cultivar, the natural sugar content made up of xyloglucans (Nunan et al., 1997; Vidal in grape berries reaches a maximum at appro- et al., 2001). Hundreds to thousands of glucose ximately 25Brix, when the berries contain units are assembled in the long cellulose chains, approximately 0.7 M each of glucose and fruc- whereas pectins and hemicelluloses also contain tose. This results in an ethanol concentration of other sugar “building blocks,” such as arabinose,

FIGURE 6.3 Syrah berries reach a maximum amount of sugar when their sugar concentration approaches 25Brix, although the concentration may continue to increase as berries shrink due to water loss; notice the large variation in sugar content due to variation in berry size (left, M. Keller, unpublished data) and mature Cabernet Sauvignon berries shrinking from dehydration (right, photo by M. Keller). 186 6. DEVELOPMENTAL PHYSIOLOGY xylose, galactose, mannose, and rhamnose in do not taste sweet, but they are nonetheless addition to glucose, as well as their oxidized important for fruit and wine quality. The cell forms, or sugar acids, such as glucuronic acid wall matrix, especially the shorter pectin frag- and galacturonic acid (Brummell, 2006; Doco ments that result from cell wall disassembly, et al., 2003; Vidal et al., 2001). The latter forms may bind other berry ingredients, such as tan- the “backbone” of pectin that is interspersed with nins, during fruit ripening and postharvest pro- rhamnose to which other sugars are attached as cessing (Kennedy et al., 2001). This binding side chains. In contrast, the highly branched capacity arises from the multitude of negative hemicellulose does not have a backbone proper charges of pectins and their degradation and consists of a mixture of different sugar units. products, which confer on these cell wall mate- As the berries begin to soften, b-galactosidases, rials considerable cation exchange capacity aided by other enzymes, pry a portion of the (Sattelmacher, 2001). The binding capacity pro- pectin away from its bondage in the cell wall bably exerts its major influence after berries are and cut its sugar chains into shorter pieces, damaged and their cell compartmentation is making them water-soluble; this depolymeriza- broken to release the juice from the vacuoles tion process intensifies due to the activity of poly- during the juicing and winemaking processes. galacturonases and pectate lyases in the In addition, the binding of tannins to pectin late stages of fruit ripening (Nunan et al., 2001; fragments might be responsible for the Robertson et al., 1980; Rose et al., 1998). Cell decrease in the perception of astringency in rip- expansion during the early ripening phase is ening fruit because such binding may make accompanied by production of additional pectin tannins less available to bind to salivary pro- so that the total amount of pectin increases while teins when berries are chewed (Macheix et al., the concentration declines (Silacci and Morrison, 1990). Thus, alterations in cell wall polysacchar- 1990). At the same time, the xyloglucans are also ides may account for some of the changes in shortened (depolymerized), and their content extractability of quality-relevant compounds decreases under the influence of enzymes that observed during ripening that are discussed degrade and modify the cell walls (Brummell, later. Moreover, yeast enzymes can degrade 2006; Deytieux et al., 2007; Doco et al., 2003; and extract some of the noncellulose cell wall Ishimaru and Kobayashi, 2002; Rose and Bennett, polysaccharides, especially pectins, during fer- 1999; Schlosser et al., 2008). Due to the very large mentation. Because most white grapes are fer- volume and thin walls of the mesocarp cells, most mented after pressing—that is, after the skins of the cell wall material is in the berry skin, where and seeds have been removed—they result in xyloglucans tightly bind the cellulose microfibrils wine with much lower polysaccharide content and pectins, forming an extensive and tough net- than red wines, which are typically fermented work of polysaccharides (Doco et al., 2003). In “on the skins” (O’Neill et al., 2004). addition to their cross-linking function, xyloglu- cans may also keep the cellulose fibrils properly 6.2.3. Acids spaced and porous (McCann et al., 1990). Thus, hemicellulose depolymerization and pectin dis- Acidity plays an important part in the per- assembly enable berries to soften at veraison. ception of fruit and wine quality because it Although the mesocarp accounts for approx- affects not only tartness (sour taste) but also imately 75% of the fresh weight of a mature sweetness by masking the taste of sugars and berry, 75% of the berry’s total pectin is located thus gives grapes, juice, and wine their fresh, in the skin (Vidal et al., 2001). In contrast to crisp taste. In addition, acids (especially the simple sugars, cell wall polysaccharides malate) also taste astringent and increase the 6.2. GRAPE COMPOSITION AND FRUIT QUALITY 187 overall perception of astringency (Hufnagel influence of organic acids by substituting for þ and Hofmann, 2008b), which is why acidity in H and thereby raising the juice pH (Boulton, wine is sometimes difficult to distinguish from 1980a,b). Although the pH is generally inversely astringency due to tannins. Wines with high related to acid concentration (Figure 6.4), there is acidity (and low pH) are often perceived as no simple relationship between titratable “sour” (unless the acidity is masked by residual acidity and pH nor (especially) between “total sugar), whereas wines with low acidity (and acidity” and pH (Smith and Raven, 1979). high pH) are described as “flat,” because the In fact, the organic acids tend to buffer and tongue functions essentially as a pH meter, hence stabilize the pH (Shiratake and Martinoia, detecting sour taste as the concentration of 2007). þ hydronium ions (H3O ). Similarly, the acidity Although plants generally regulate their of grape juice is estimated by titration (hence cell’s cytoplasmic pH within quite narrow lim- the term titratable acidity) against an alkaline its, the pH inside the vacuoles can vary consid- solution (usually sodium hydroxide) that, more erably. Because grape juice consists mostly of accurately, measures the concentration of titrat- the sap from mesocarp vacuoles, the pH of þ able hydrogen ions or protons (H ). The high the juice is somewhat influenced by changes concentration of organic acids in grape berries in soil conditions, vine nutrition, and solute and the active transfer into the fruit vacuoles transport (Conradie and Saayman, 1989). Nev- þ of protons by H pumps (which, in turn, drives ertheless, juice from mature grapes harvested the membrane-bound pumps for many other from vines grown on a soil with pH 8.5 usually components including acids) are responsible has a pH of approximately 3.5, much like that for the low pH of grape juice. In other words, from vines grown on a soil with pH 5.5, þ þ the pH falls as the H concentration rises as although the H concentration in the two soils ¼ þ defined by the relationship pH log10[H ] differs by a factor of 1000. The pH increases þ þ (where [H ] is the H concentration in mol during fruit ripening due to both a decrease þ L 1). Metal cations, especially potassium (K ) in organic acids and an increase in metal þ and sodium (Na ), tend to counter the cations. The latter effect becomes dominant ) − 1 Titratable acidity (g L Titratable

Titratable acidity (g L−1)

FIGURE 6.4 The acidity declines as the concentration of sugars (expressed as soluble solids) increases (left) and the pH rises as the acidity decreases (right) during ripening of Cabernet Sauvignon berries over 3 years; year 2 had the coolest and year 3 the warmest ripening period (M. Keller, unpublished data). 188 6. DEVELOPMENTAL PHYSIOLOGY toward the end of the ripening phase because produce succinate) and is converted to the the rate of malate respiration decreases (most much milder-tasting lactate (and CO2) by lactic malate has already been respired, and the tem- acid bacteria (e.g., Oenococcus oeni) during perature typically declines in autumn), whereas (anaerobic) malolactic fermentation. In contrast, þ K may continue to move into the berries until tartrate is rather metabolically and microbially phloem influx ceases. Consequently, juice stable, which is why it is often added in expressed from harvested fruit typically has a winemaking to adjust the juice and wine pH. pH between 3.0 and 3.5, but it can sometimes Nonetheless, the fungus Botrytis cinerea (see exceed 4.0 in overripe fruit. Values of pH in Chapter 7.5) and some lactic acid bacteria are excess of approximately 3.6 are undesirable able to degrade tartrate, especially at high pH because they lead to decreased color intensity (>3.5). Also, when iron or copper molecules and microbial stability (i.e., increased potential are present in wine, tartrate can be oxidized to for spoilage) and increased susceptibility to glyoxylate (Monagas et al., 2006). oxidation in wine and other grape products. Most organic acids are accumulated early in Juice from mature grapes typically contains berry development and stored in the vacuoles between 5 and 10 g L 1 of organic acids. A of the mesocarp and skin cells from synthesis titratable acidity concentration of 6.5–8.5 g L 1 inside the berry (Hale, 1962; Ruffner, 1982a,b), is considered optimal for the production of although malate can be transported in both well-balanced wines (Conde et al., 2007). the xylem (Hardy, 1969) and the phloem. Tar- Although acidity is commonly expressed as tar- trate and oxalate are both products of the taric acid equivalents, the organic acids are breakdown of ascorbate (vitamin C), which present as a mixture of several free acids (e.g., occurs at least partly in the cell walls (DeBolt malic acid and tartaric acid) and their salts, et al., 2004, 2006; Green and Fry, 2005; Saito which are usually ionized (e.g., malate and et al., 1997). Ascorbate can be produced by oxi- tartrate) (Smith and Raven, 1979). The relative dation of glucose in the berries or imported proportions of the various forms are dependent from the leaves via the phloem; some may even on the pH: Ionization increases with rising pH. be produced within the phloem (DeBolt et al., In most fruit species, malate and citrate are the 2007). After veraison, the tartrate concentration dominant organic (carboxylic) acids. Grapes decreases somewhat due to dilution from water are unusual because in addition to malate, import, whereas the amount of tartrate per tartrate is much more important than citrate. berry generally remains fairly constant (Possner In fact, tartrate and malate are the major solu- and Kliewer, 1985). Some tartrate turnover ble organic components that accumulate in the or respiration does, however, seem to occur preveraison grape berry and together comprise (Drawert and Steffan, 1966a; Takimoto et al., 70–90% of the berry’s total acids (Jackson, 2008; 1976). Contrary to widespread opinion, tartrate Kliewer, 1966; Ruffner, 1982a). Other organic does not form crystals (and hence become þ þ acids mainly include citrate, oxalate, succinate, insoluble and precipitate) with K or Ca2 in and fumarate, as well as phenolic, amino, and intact grape berries. Despite the continued fatty acids. Citrate is an important flavor pre- influx of K in the phloem, crystals are formed cursor in grapes because it can be converted between oxalate (not tartrate) and Ca rather during malolactic fermentation of wines to than K (DeBolt et al., 2004; Fouge`re-Rifot et al., diacetyl, a compound imparting a “buttery” or 1995). Of course, once the cell compartmen- “butterscotch” flavor. The tart malate, on the tation breaks down when grapes are crushed, other hand, can be produced or degraded tartrate will crystallize with both K and Ca. by various yeasts (which also independently In fact, the discovery of calcium tatrate in an 6.2. GRAPE COMPOSITION AND FRUIT QUALITY 189 ancient jar was used as evidence that Neolithic Hawker, 1977), whereas there is very little people made wine at least 7000 years ago acid synthesis after veraison, although this (McGovern et al., 1996). The insoluble products “production stop” seems to be somewhat of this biomineralization are deposited as delayed in the skin (Gutie´rrez-Granda and bundles of needle-like Ca-oxalate crystals Morrison, 1992; Iland and Coombe, 1988). Fruit (raphides) in the vacuole of hypodermis cells acidity therefore peaks immediately before ver- called idioblasts that specialize in Ca-oxalate aison, a fact that is exploited in the production production (Franceschi and Nakata, 2005; of verjuice or verjus (French vert ¼ green, Loewus, 1999). The idioblasts probably serve unripe; jus ¼ juice). Verjuice has been used as as a storage compartment for surplus Ca and an acidifying cooking ingredient and substitute can release Ca on demand, which is important for vinegar since ancient times and is made by after veraison, when Ca influx into the berry pressing unripe (and unfermented) grapes through the xylem has mostly ceased. The harvested around veraison for this purpose or granular Ca-oxalate crystals (druses) accumu- collected during cluster thinning for crop load lated in the endocarp, on the other hand, may adjustment. The amount of malate per berry serve to store and later release oxalate. Whereas declines after veraison due to inhibition of gly- the production of tartrate and malate ceases by colysis (see Chapter 4.4), re-metabolism of veraison, oxalate continues to accumulate stored malate for respiration, and gluconeogen- throughout ripening. Its (concurrent) break- esis (Drawert and Steffan, 1966b; Ruffner, down releases H2O2 (and CO2) after veraison, 1982b; Ruffner and Hawker, 1977; Ruffner which in turn (and aided by the enzymes et al., 1975; Steffan and Rapp, 1979). It appears polyphenol oxidase and peroxidase) oxidizes that the activation of hexose storage in the the phenolics in the seed coat so that the seeds vacuoles triggers efflux from the vacuoles and gradually turn brown. Raphides are thought breakdown of malate in the mesocarp (Sarry to have an antifeeding role by discouraging et al., 2004). Thus, the berry switches its carbon insects and mammals from eating unripe supply for respiration from glucose to malate at grapes (or leaves). Raphides are also a cause veraison so that the juice acidity declines as the of contact dermatitis among people harvesting sugar content rises (see Figure 6.4). In contrast agave for tequila production, Ca-oxalate is a with the situation in the mesocarp, and consis- major component of kidney and gall stones, tent with the persistence of glycolysis in the and both oxalate and tartrate are believed to skin (Negri et al., 2008), there seems to be little, be the dominant pain-inducing toxins of if any, decrease in malate in the skin after stinging nettles. veraison (Gutie´rrez-Granda and Morrison, Malate is produced from phosphoenol- 1992; Possner and Kliewer, 1985; Storey, 1987). pyruvate as a by-product of glycolysis and as Gluconeogenesis is the formation of glucose a by-product of the refixation of CO2 released through a reversal of the glycolytic pathway. during berry respiration or, in a reversible reac- Such a reversal can occur if the cells’ energy tion, from oxaloacetate in the TCA cycle and is level exceeds demand so that the carbon of initially stored in the berry’s vacuoles (Ruffner, pyruvate can be recycled back to glucose. 1982b). Preveraison malate accumulation is Gluconeogenesis has a temperature optimum greatest at temperatures between 20 and 25 C at approximately 20 C and decreases to half and declines sharply above 38C (Kliewer, the maximum rate at both 10 and 30C 1964; Lakso and Kliewer, 1975, 1978). Like (Ruffner et al., 1975). Respiratory malate degra- tartrate, most malate is formed during the pre- dation increases with increasing temperature veraison period (Kliewer, 1965b; Ruffner and up to 50C, whereas gluconeogenesis is more 190 6. DEVELOPMENTAL PHYSIOLOGY important at lower temperatures, when respira- seed germination. Moreover, proteins are also tion, energy demands, and sugar import are embedded in the pectin matrix of mesocarp low. Nonetheless, the contribution of gluconeo- and skin cell walls (Lecas and Brillouet, 1994; genesis to total sugar accumulation is minor Saulnier and Brillouet, 1989; Vidal et al., 2001). and seems unlikely to exceed approximately Import of nitrogenous compounds into the 5% (Ruffner and Hawker, 1977). berry before veraison can occur in both the xylem, predominantly in the form of glutamine 6.2.4. Nitrogenous Compounds and and nitrate, and the phloem, mostly as gluta- Mineral Nutrients mine, but after veraison it becomes essentially restricted to the phloem. Nevertheless, more Nitrogen-containing compounds are impor- than half of a berry’s total nitrogen is normally tant sources of food for yeast during fer- imported after veraison, and nitrogen accumu- mentation, and the nitrogen content of grape lation may continue throughout ripening must greatly impacts fermentation rate. Musts (Conradie, 1986; Kliewer, 1970; Lo¨hnertz et al., with less than approximately 150 mg yeast- 1989; Rodriguez-Lovelle and Gaudille`re, 2002; assimilable N L 1 tend to result in sluggish or Schaller et al., 1990). Although grape berries stuck fermentations and associated problems can assimilate nitrate (Schaller et al., 1985), with hydrogen sulfide (H2S) production (Bell glutamine is the major nitrogen transport and Henschke, 2005). H2S is liberated from compound (see Chapter 5.3); enzymes called proteins that are broken down by the starving aminotransferases convert glutamine in the yeast cells and has a “rotten egg” odor. Taste berry into other amino acids, such as arginine intensity and aroma quality of wines therefore and proline, which together account for tend to increase as the nitrogen content of the 60–70% of the amino acids in mature grape grapes increases (Rapp and Versini, 1996). berries (Bell and Henschke, 2005; Goes da Silva Grape musts are therefore often supplemented et al., 2005; Roubelakis-Angelakis and Kliewer, et al et al with diammonium phosphate ((NH4)2HPO4) 1992; Stines ., 2000; van Heeswijck ., to enhance yeast nutrition. 2000). However, the concentration of arginine Although the nitrogen content of grape and proline may vary by an order of magnitude berries is extremely variable, they may contain among grape cultivars grown under similar approximately half the total canopy nitrogen conditions (Kliewer, 1970). Amino acids, espe- at maturity (Wermelinger and Koblet, 1990). cially arginine, may also be altered by the root- The majority (50–90%) of the nitrogen is present stock; for example, Ruggeri 140 and 101–14 Mgt in the form of free amino acids (Roubelakis- may sometimes lead to considerably lower Angelakis and Kliewer, 1992). The remainder is amino acid concentrations in the fruit of their made up mostly of proteins, ammonium, and grafting partner than some other rootstocks nitrate. The various berry tissues contain and, particularly, own-rooted vines (Treeby hundreds of different proteins, most of which et al., 1998). Arginine can be accumulated in are enzymes that are involved in the construc- the skin, pulp, and seeds throughout fruit tion of the broad range of berry metabolites and development, although in some cultivars (e.g., in respiration that provides the energy required Chardonnay and Cabernet Sauvignon) accu- for these activities (Goes da Silva et al., 2005; mulation appears to cease at veraison, whereas Sarry et al., 2004). The seeds accumulate large in yet others (e.g., Mu¨ ller-Thurgau) it may not amounts of storage proteins (up to 90% of the begin until veraison (Kliewer, 1970; Schaller total seed protein) that serve as a source of and Lo¨hnertz, 1991). In contrast, most of the energy, nitrogen, and sulfur during and after proline is accumulated after veraison, and 6.2. GRAPE COMPOSITION AND FRUIT QUALITY 191 accumulation seemingly continues throughout amino acid, whereas proline cannot be readily ripening (Coombe and Monk, 1979; Kliewer, used by yeast (Bell and Henschke, 2005). Some 1968; Lafon-Lafourcade and Guimberteau, of the “fermentation aroma” of wines—that is, 1962; Miguel et al., 1985; Stines et al., 2000). the bouquet caused by flavor and aroma Proline often serves to protect cells from exces- compounds generated during fermentation— sive osmotic stress; thus, proline accumulation stems from the conversion by yeasts of amino during ripening could be related to the osmotic acids to so-called higher alcohols, which are pressure caused by the accumulating hexose defined as alcohol molecules with more than sugars (van Heeswijck et al., 2001). Proline the two carbon atoms of ethanol. For example, accumulation might also contribute to the rise leucine yields isoamyl alcohol with a “harsh,” in juice pH because its production from gluta- “burnt,” or “whiskey” odor, and valine yields mate may involve OH release (Smith and ethyl isobutyrate with an aroma of “strawberry.” Raven, 1979). Arginine and other amino acids Several minor amino acids in grapes have and protein residues, but not proline, can react implications for fruit quality and human health. with sugars in the so-called Maillard reaction to The key position of phenylalanine as a chemi- produce brown polymers termed melanoidins, cal precursor for phenolic compounds is which leads to slow nonoxidative, nonenzy- discussed in Section 6.2.5. Phenylalanine is an matic browning during grape processing—for aromatic amino acid, in the chemical rather than instance, during drying for raisin production the sensorial sense, produced by the shikimate and subsequent storage (Frank et al., 2004). pathway. Its aromatic sibling tryptophan not Melanoidins can have pleasant (malty, bread only is one of the precursors for the plant crust-like, caramel, and coffee) or unpleasant hormone auxin but also can be degraded to the aromas (burnt, onion, solvent, rancid, sweaty, aroma volatile methyl anthranilate (Wang and and cabbage) and may taste bitter. Note that De Luca, 2005) or be further metabolized to the beer, coffee, chocolate, toasted bread, and neurotransmitter serotonin and the human hor- caramel owe much of their color and aroma to mone and antioxidant melatonin (Iriti et al., melanoidins. 2006). Tyrosine, another shikimate offshoot, can At very high concentrations, proline and be converted by lactic acid bacteria during malo- some other amino acids (threonine, glycine, lactic fermentation into the neurotransmitter serine, alanine, and methionine) impart a sweet tyramine, which may lead to increased heart rate taste, whereas arginine and other amino acids and higher blood pressure, although the (lysine, histidine, phenylalanine, valine, etc.) amounts of tyramine found in wine are rarely appear to taste bitter, and still others (glutamine, high enough to cause problems. Histidine is glutamate, asparagines, and aspartate) have important because wine spoilage bacteria can an umami (Japanese umami ¼ savory) taste convert it to histamine, a biogenic amine that at (Hufnagel and Hofmann, 2008b). However, with high concentrations is suspected to trigger head- the exception of proline and, perhaps occasion- aches (including migraine) or asthma attacks. ally, glutamate, amino acids are present in wine Unfortunately, ethanol inhibits histamine degra- at concentrations that are far too low to have dation in the body, which tends to exacerbate the any sensory impact (Hufnagel and Hofmann, reaction. Nonetheless, the scientific evidence for 2008b). Moreover, the amino acid profile of a a link between biogenic amines and adverse wine differs considerably from that of its grape reactions in humans appears to be inconclusive constituents and may reflect metabolic activi- (Jansen et al., 2003). ties of yeasts and other microorganisms. For In addition to proline, certain proteins also instance, arginine is the major yeast-assimilable accumulate after veraison and continue to 192 6. DEVELOPMENTAL PHYSIOLOGY

þ accumulate throughout ripening, probably in Although K can be transported in both xylem response to the osmotic stress arising from and phloem, its concentration in the xylem is increasing sugar concentration, and they occupy orders of magnitude lower than that in the an increasing fraction of the total berry proteins phloem, and import into the berry probably (Giribaldi et al., 2007; Monteiro et al., 2007; Negri occurs predominantly in the phloem (see et al., 2008; Salzman et al., 1998). These are the Chapter 5.1). Import occurs throughout berry chitinases and thaumatin-like proteins, which development, but it may sometimes increase þ are members of the so-called pathogenesis- markedly at veraison. Most of the K is stored related (PR) protein families (see Chapter 7.5) in the vacuoles of the inner hypodermis and þ and can make up half or more of the total soluble the mesocarp. It is possible that K contributes protein in mature grape berries, with roughly to berry cell expansion by lowering the cells’ equal amounts in the skin and the pulp. Unfortu- osmotic potential, at least before sugars assume nately, these PR proteins seem to be responsible this role at veraison (Davies et al., 2006; for haze formation or clouding in wine made Mpelasoka et al., 2003; Rogiers et al., 2006). þ from white grapes, so they may contribute to Free K ions, liberated when the cell mem- protein instability in wine (Tattersall et al., branes are ruptured during grape processing, 2001). Due to the rise in protein content, the can form crystals with tartrate, and the resulting potential for protein instability increases with precipitation of the poorly soluble K-tartrate advancing grape maturity. This is exacerbated (termed potassium bitartrate, potassium hydro- by the concomitant increase in mesocarp pH, gen tartrate, or cream of tartar) will decrease which makes PR proteins progressively easier juice and wine acidity and may increase the pH þ þ to extract. In addition, protein extraction into (although the fact that K substitutes H on the þ the juice from damaged mechanically harvested tartrate molecule, so H remains in solution, grapes is much greater than that from hand- could result in an increase in juice pH upon harvested grapes, especially if the grapes are left K-tartrate precipitation). Extraction from the standing for prolonged periods or transported skins during fermentation amplifies this effect over great distances before processing (Pocock in red wines (Mpelasoka et al., 2003). Note that et al., 1998). Red wines, however, are not cream of tartar (along with other components normally subject to this protein haze problem such as anthocyanins and other phenolics) is because the tannins extracted from the grape also sometimes extracted industrially from skins during fermentation precipitate these and pomace remaining after pressing of juice or þ other proteins. must. Free calcium ions (Ca2 ) also combine Macronutrients other than nitrogen also with tartrate to form insoluble calcium tartrate þ þ accumulate in the berry. Potassium is by far the in juice and wine. In contrast to K ,Ca2 import most important of these and is the major cation is largely restricted to the xylem. Consequently, þ in grapes; its concentration in expressed juice 75–100% of the berry’s final Ca2 content is often exceeds 1 g L 1 and can be substantially accumulated before veraison. Magnesium þ higher in the skin (Coombe, 1987; Done`che and (Mg2 ), on the other hand, can be transported Chardonnet, 1992; Hrazdina et al., 1984; in both the xylem and the phloem (see Chapter Mpelasoka et al., 2003; Possner and Kliewer, 5.1), and approximately half the Mg is 1985; Rogiers et al., 2006; Storey, 1987). Because imported into the berry after veraison (Rogiers þ þ þ it can substitute for protons in grape juice, K et al., 2006). The accumulation of K ,Mg2 , and þ þ impacts the juice pH: A 10% increase in K especially Ca2 is higher in berries that have concentration is associated with a rise in pH higher transpiration rates, even after veraison by approximately 0.1 units (Boulton, 1980a). (Du¨ ring and Oggionni, 1986). Windy conditions 6.2. GRAPE COMPOSITION AND FRUIT QUALITY 193 and other factors that favor berry transpiration enzymatic oxidation of phenolics. In addition, could therefore be associated with higher fruit fungicide-derived Cu may lead to an increase mineral nutrient content. in H2S formation by yeast during fermentation Both Ca and Mg are important components (Eschenbruch and Kleynhans, 1974). of cell walls, and Ca also accumulates to high Minute quantities of rare earth elements concentrations in the cell vacuoles in the form have also been detected in grape berries of Ca-oxalate crystals (see Chapter 7.3). The (Bertoldi et al., 2009). Most of these belong to highest concentrations of these ions are found the lanthanoid series and are though to have in the seeds, which is also true for phosphorus biological properties that resemble those of (P) and sulfur (S). However, due to its much Ca, with whose effects they may interfere. In larger volume, the mesocarp can sometimes grapes, the rare earth elements are dominated be an equally important storage site for Mg, P, by cerium, neodymium, and lanthanum, and and S in terms of total amount per berry most of them are present in the skin, with (Rogiers et al., 2006). slightly smaller amounts in the mesocarp and Micronutrients, or trace metals, such as iron only traces in the seeds (Bertoldi et al., 2009). (Fe), copper (Cu), manganese (Mn), and zinc (Zn), are mostly concentrated in the seeds, 6.2.5. Phenolics whereas boron (B) also accumulates in the skin (Rogiers et al., 2006), which probably reflects Compounds other than water, sugars, acids, the importance of B as a cell wall component and minerals account for only a small propor- (see Chapter 7.3). However, similar to macro- tion of the berry weight but contribute signifi- nutrients, the mesocarp and, in some instances, cantly to fruit and wine quality. Among these, even the skin may also contribute substantial the class of phenolic compounds is one of the amounts on a per berry basis. The amount of most important contributors. All phenolic com- most of these ions generally increases through- pounds are made up of an “aromatic ring” con- out berry development and ripening, but most sisting of six carbon atoms with one or more of the Mn and Zn is accumulated before verai- hydroxyl (OH) groups or derivatives of this son (Rogiers et al., 2006). Import in the phloem basic structure (Table 6.1). They are very of Cu and B may be so strong that not only important for the color and astringency of red their amount per berry but also their concentra- wine, contribute to grape and wine flavor and tion continue to increase during ripening, aroma, and are the main substrates for juice and whereas the concentration of other micro- wine oxidation (Macheix et al., 1991; Singleton, nutrients tends to decline. In contrast to the 1992). Their susceptibility to oxidation, which pericarp, however, the seeds essentially stop they owe to their hydroxyl groups and un- accumulating phloem-mobile nutrient ions saturated double bonds, is what makes other than K at veraison (Rogiers et al., 2006). phenolics such good antioxidants (Rice-Evans Trace metals may be important in altering the et al., 1997). color hue of grape juice and wine. For instance, Phenolics are produced inside the berry by a small increase in Fe concentration can lead to several different routes (Figure 6.5) and thus an increase in the blue tint and a corresponding form a diverse group from a metabolic stand- decrease in red. Moreover, dark pigments can point, but they are usually classified into non- be formed through interactions of proline flavonoids that accumulate mainly in the and other amino acids with phenolics in the berry pulp and flavonoids that accumulate presence of Fe, whereas Cu, as a component mainly in the skin, seeds, and stem. The two of polyphenoloxidase, is involved in the main “assembly lines” are the shikimate 194 6. DEVELOPMENTAL PHYSIOLOGY

TABLE 6.1 The Main Classes of Phenolic Compounds in Grapes

Class Name Basic Chemical Structure Examples

Flavan-3-ols Catechin (left) Epicatechin (right)

Tannins Hypothetical proanthocyanidin tetramer with (from top to bottom) epigallocatechin, epicatechin, catechin, and epicatechin gallate (note that the subunits shown are a combination of those found in both seeds and skins)

¼ ¼ Anthocyanins Cyanidin-3-GLU (R1 OH, R2 H) ¼ ¼ Delphinidin-3-GLU (R1 OH, R2 OH) ¼ ¼ Peonidin-3-GLU (R1 OCH3,R2 H) ¼ ¼ Petunidin-3-GLU (R1 OCH3,R2 OH) ¼ ¼ Malvidin-3-GLU (R1 OCH3,R2 OCH3) ¼ ¼ Flavonols Ka¨mpferol-3-GLU (R1 H, R2 H) ¼ ¼ Quercetin-3-GLU (R1 OH, R2 H) ¼ ¼ Myricetin-3-GLU (R1 OH, R2 OH) ¼ ¼ Isorhamnetin-3-GLU (R1 OCH3,R2 H) ¼ ¼ Laricitrin-3-GLU (R1 OCH3,R2 OH) ¼ ¼ Syringetin-3-GLU (R1 OCH3,R2 OCH3) ¼ ¼ Xydroxycinnamic Cinnamic acid (R1 H, R2 H) acids ¼ ¼ Coumaric acid (R1 H, R2 OH) ¼ ¼ Caffeic acid (R1 OH, R2 OH) ¼ ¼ Ferulic acid (R1 OCH3,R2 OH) Continued 6.2. GRAPE COMPOSITION AND FRUIT QUALITY 195

TABLE 6.1 The Main Classes of Phenolic Compounds in Grapes—Cont’d

Class Name Basic Chemical Structure Examples

¼ ¼ Hydroxybenzoic Protocatechuic acid (R1 H, R2 OH) acids ¼ ¼ Gallic acid (R1 OH, R2 OH) ¼ ¼ Syringic acid (R1 OCH3,R2 OCH3)

¼ ¼ Stilbenes Resveratrol (R1 OH, R2 OH) ¼ ¼ Pterostilbene (R1 OCH3,R2 OCH3) ¼ ¼ Piceid (R1 OH, R2 GLU) Viniferins (resveratrol polymers)

GLU, glucose. Chemical structures courtesy of J. Harbertson. pathway (named after one of its intermediates) and Chapple, 2002), the ammonium released by and the malonate pathway. The shikimate PAL is continuously recycled via the nitrogen- pathway is responsible for the synthesis of most assimilating GS/GOGAT cycle (see Chapter 5.3) plant phenolics, whereas the malonate pathway to regenerate more phenylalanine, according to is less important (although it is essential in tissue-specific demands (Weaver and Herrmann, fungi and bacteria). The former operates within 1997). This recycling mechanism ensures the sus- chloroplasts and converts simple carbohydrate tained production of phenolics from an amino precursors derived from glycolysis (phosphoenol- acid even in the face of nitrogen deficiency. It is pyruvate) and the pentose phosphate pathway also possible that additional phenylalanine might (erythrose-4-phosphate) into aromatic amino be derived from the breakdown of proteins under acids (phenylalanine) while releasing phosphate such conditions (Margna et al., 1989). (Weaver and Herrmann, 1997). The well-known Cinnamic acid is further modified and acti- broad-spectrum herbicide glyphosate kills plants vated by the addition of a coenzyme A thioe- by blocking a step in this reaction sequence ster (S-CoA, itself assembled from ATP (Holla¨nder and Amrhein, 1980). The subsequent and pantothenate, aka vitamin B5) to yield 4- phenylpropanoid pathway converts phenylala- coumaryl-CoA. The slightly volatile cinnamic nine into cinnamic acid by the deaminating acid has a “floral” and “honey” odor and forms enzyme phenylalanine ammonia lyase (PAL), part of the flavor of cinnamon. The first step the pathway’s key enzyme. Sitting at a branch specific to flavonoid biosynthesis and hence point between protein and phenolic meta- to the flavonoid pathway, catalyzed by the bolism, PAL exerts a kind of metabolic traffic enzyme chalcone synthase (CHS), is the con- control by routing phenylalanine away from the densation of 4-coumaryl-CoA with three mole- manufacture of amino acids and proteins toward cules of malonyl-CoA, which is derived from the phenolics assembly line. Whereas 30–40% of acetyl-CoA produced during respiration (Yu a plant’s fixed carbon flows down the phenylpro- and Jez, 2008). The product of this reaction, panoid pathway (mainly because this pathway chalcone, is rapidly isomerized to the flavanone also produces the phenolic building blocks of the naringenin, which is the precursor of a large cell wall structural polymer lignin; Humphreys number of flavonoids with a basic chemical 196 6. DEVELOPMENTAL PHYSIOLOGY

FIGURE 6.5 Biosynthetic pathways for the production of phenolics (A) and terpenoids (B) in grapevines. Reproduced from Velasco et al. (2007). 6.2. GRAPE COMPOSITION AND FRUIT QUALITY 197 structure of C6-C3-C6 (see Table 6.1). The flavo- compound that seals wine bottles in the form noids are the most important and most exten- of cork derived from the bark of the cork oak, sive group of phenolics. Alternatively, stilbene Quercus suber L., after which the chemical is synthase—using the same substrates with the named). Nonetheless, hydroxycinnamic acids same stoichiometry as CHS, from which it are the major phenolic constituents of free-run may have evolved—catalyzes the synthesis of grape juice and white wines and, at high resveratrol (Schro¨der, 1997; Tropf et al., 1994), concentrations, seem to contribute some astrin- which is used for the formation of the various gency to grape juice and wine (Hufnagel and stilbene derivatives, such as piceid (resvera- Hofmann, 2008a,b; Ong and Nagel, 1978). Free trol-glucoside), pterostilbene, and the poly- hydroxycinnamic acids may also contribute to meric viniferins (Jeandet et al., 2002; Monagas the heart disease-decreasing properties of et al., 2006; Romero-Pe´rez et al., 1999; Schmidlin grapes and grape products, but their esterifica- et al., 2008). These nonflavonoids are produced tion with ethanol may render hydroxycinna- in the skin in response to injury (e.g., wound- mates somewhat bitter (Hufnagel and ing or pathogen infection) and are important Hofmann, 2008a,b). However, their main components of the vine’s defense against importance may lie in the fact that Brettano- attacking pathogens (see Chapter 7.5). They myces and some other yeasts can convert them have also been implicated in numerous benefi- during fermentation to volatile phenols such cial effects on human health. For instance, as ethyl or vinyl guaiacol and eugenol, which besides its antimicrobial properties, resveratrol are odor active and at low concentrations smell appears to be capable (albeit in some cases at “smoky,” “woody,” “leathery,” or “peppery” very high doses) to reduce heart disease, (Chatonnet et al., 1992; Rapp and Versini, prevent many types of cancer (by acting as an 1996). Incidentally, guaiacol (“smoky”), euge- antioxidant and antimutagen among other nol (“clove”), and related compounds are also activities) and memory loss, delay (type I) extracted into wine from toasted oak barrels diabetes, and even delay aging and diseases as breakdown products of lignin (Chatonnet related to aging (Baur and Sinclair, 2006; and Dubourdieu, 1998), and eugenol and German and Walzem, 2000; Pezzuto, 2008). other phenylpropanoids are important aroma In addition to the stilbenes, nonflavonoid components of basil leaves. However, at phenolics also include the phenolic acids higher concentrations the odor of volatile phe- (hydroxybenzoic and hydroxycinnamic acids). nols becomes unpleasantly “pharmaceutical,” High concentrations of tartrate esters of hydro- “medicinal,” or “burnt” at the expense of varie- xycinnamic acids (e.g., caffeic, coumaric, and tal fruit aroma. This poses a problem not only ferulic acids) are accumulated in the vacuoles following contamination with Brettanomyces of the berry mesocarp and exocarp during and during the winemaking process but also when after bloom, but their concentration often grapes have been exposed during ripening to decreases during berry development (Monagas smoke derived from wildfires, which leads to et al., 2006; Singleton et al., 1986). They may, in accumulation of glycosylated phenols in the part, be used to manufacture lignin for the ber- skin and their subsequent release (turning them ry’s vascular bundles and the seed coat (Currle into volatiles) during alcoholic and malolactic et al., 1983; Humphreys and Chapple, 2002). fermentation (Kennison et al., 2008). In addi- Some ferulic acid also becomes linked to the tion, rapid oxidation of such “simple” pheno- glycerol/fatty acid polyester suberin (Franke lics during grape drying or processing results and Schreiber, 2007) that plugs the berry’s sto- in browning of both raisins and grape juice, mata before ripening begins (and is the same including the grapes used for the sweet, 198 6. DEVELOPMENTAL PHYSIOLOGY fortified Spanish dessert wine Pedro Ximenez among other benefits. Indeed, they have impor- (Sapis et al., 1983; Serratosa et al., 2008). Such tant pharmacological properties, reducing oxi- (enzymatic) oxidation becomes possible when dative stress by acting as antioxidants that are physical disruption of cell compartmentation more powerful than ascorbate and a-tocopherol (due to mechanical damage before or after har- (aka vitamins C and E; Rice-Evans et al., 1997), vest or bunch rot infection) brings the phenolics which is one of the supposed main reasons in contact with oxygen and two groups of why eating grapes and drinking wine and enzymes termed polyphenoloxidases and per- grape juice may be good for our health (Dixon oxidases that convert phenolics to quinones et al., 2005; German and Walzem, 2000). None- (Herna´ndez et al., 2009; Macheix et al., 1991; theless, human physiologists have noted that Pourcel et al., 2007; Singleton, 1992), which can our bodies normally balance reactive oxygen then polymerize to form brown pigments species (see Chapter 7.1) and antioxidants so called melanins. This reaction is particularly carefully that consuming antioxidants may not dominant if the grapes contain plenty of hydro- decrease oxidative damage (Halliwell, 2006, xycinnamic acids and little glutathione that 2007). would otherwise protect the phenolics or at Among enologically important phenolic least the quinones from oxidation by binding compounds, tannins are the most abundant to them (Monagas et al., 2006). class; the lignin formed in the seed coat from Most other phenolics are glycosylated—that cinnamic acids is enologically unimportant is, they are attached to a sugar molecule—to because it is not extracted during fermentation. increase their solubility, prevent their free Tannins are divided into two classes: hydroly- diffusion across membranes, and decrease their sable and condensed (nonhydrolysable) tan- reactivity toward other cellular components nins. The major subunits of the hydrolysable (glycosylation also decreases their antioxidant tannins are gallic and ellagic acids, with small activity), which permits accumulation and amounts of hydroxycinnamic acids, bound to storage in the cell vacuoles (Bowles et al., 2006; glucose. The much longer condensed tannins Rice-Evans et al., 1997). Glucose, following its are composed of flavan-3-ols such as catechin activation by uridine diphosphate to the so- and epicatechin, both of which taste bitter called UDP-glucose, is usually the sugar of when they occur as monomers (Dixon et al., choice for attachment to an oxygen atom (hence 2005). Grapes contain only condensed tannins; the term O-glucoside) of the phenolics by a the hydrolysable tannins present in some wines family of enzymes called glycosyltransferases. are extracted from oak wood, for example, dur- These phenolics include the flavonoids such ing barrel aging. Condensed tannins are also as tannins, anthocyanins (glucosides of the called proanthocyanidins because they break anthocyanidins malvidin, peonidin, delphini- down to anthocyanidins when heated in acids. din, petunidin, and cyanidin), and flavonols Indeed, grape berries manufacture tannins via (glycosides of quercetin, ka¨mpferol, myricetin, a branch in the anthocyanin-forming pathway isorhamnetin, syringetin, and laricitrin) (see by enzymes called leucoanthocyanidin reduc- Table 6.1). Note that although pelargonidin- tase and anthocyanidin reductase, which syn- glucoside, the principal anthocyanin of straw- thesize catechin from the cyanidin precursor berries, does not seem to occur in grapes, the leucocyanidin—epicatechin directly from cya- major flavonols are the same in the two species. nidin and epigallocatechin from delphinidin Similar to other phenolics, flavonoids have also (Bogs et al., 2005; Dixon et al., 2005; Xie et al., been implicated in the protective activity of 2003). In other words, the same anthocyanidins grapes and their products against heart disease can be converted either to tannins (by 6.2. GRAPE COMPOSITION AND FRUIT QUALITY 199 reduction) or to anthocyanins (by glucose addi- rises. Nevertheless, although the total amount tion). Upon extraction from the grapes, tannins of tannins present in a wine is certainly some- can polymerize with anthocyanins and among what related to the wine’s astringency, small themselves and thus are important for color (and mostly unknown) chemical changes, stability in grape juice and wine. However, especially during wine aging, may account for mostly they are responsible for the bitter taste the perception of tannins as “fine,” “soft,” and the astringent tactile sensation—or “dry- “green,” or “harsh.” For instance, the incor- ing,” rough feeling on the tongue, which is also poration of galloylated procyanidins extracted called “texture”—of grapes and wines because from grape seeds also seems to increase astrin- they bind to and precipitate proteins that are gency (Brossaud et al., 2001). The perception of much larger than the tannins (Gawel, 1998; astringency, but not bitterness, also increases Robichaud and Noble, 1990). Precipitation of with increasing acidity, whereas ethanol has salivary proteins increases the friction of the the opposite effect. tongue gliding over the inner surfaces of the Although the amount of tannins contained mouth, a property that gave rise to the term in the seeds is typically several times higher mouthfeel. In fact, the name tannin derives than that in the berry skin, grape cultivars from these molecules’ ability to tan leather— differ in the amount of both seed and skin that is, to convert animal hide into leather by tannins they produce. For example, the seeds interacting with the hide’s proteins. The color- of Cabernet Sauvignon, Merlot, and Syrah seem less tannins are assembled from simple subu- to contain much lower tannin concentrations nits in the vacuoles of the seed coat and, to a than those of Cabernet franc, Pinot noir, much lesser extent, the skin cells of the berries Grenache, or Tempranillo. On the other hand of both red and white cultivars (Amrani Joutei (and perhaps surprisingly), not only Tempra- et al., 1994; Souquet et al., 1996; Thorngate and nillo and Nebbiolo but also many table grape Singleton, 1994). In addition, tannins are also cultivars (e.g., Red Globe, Flame Seedless, and found in the rachis tissues (Souquet et al., Ruby Seedless) seem to have far higher skin 2000), but this source normally contributes tannin concentrations than Cabernet Sauvignon, only a minor portion of the tannins extracted Merlot, Syrah, Pinot, and even Tannat, which during winemaking because grapes are typi- are all relatively similar. Barbera and Malbec cally destemmed prior to fermentation. skins, along with those of more table grapes Tannins are the true polyphenols of grapes (e.g., Muscat Hamburg), are at the low end of and can be composed of chains of almost iden- the spectrum. Although they lack the ability to tical subunits, varying in length by more than produce anthocyanins, white grapes accumu- two orders of magnitude. The shortest of these late the same amount of tannins as their dark- flavonoid polymers taste bitter but are usually skinned siblings (De Freitas and Glories, 1999; confined to the seeds. Skin tannins on average Rodrı´guez Montealegre et al., 2006; Viala and are longer (25 to more than 100 subunits) than Vermorel, 1909). seed tannins (4–20 subunits) and are thought Catechin seems to be the major flavan-3-ol in to provide a better mouthfeel (Kennedy et al., grape skins, with epicatechin and epigallocate- 2001; Monagas et al., 2006; Souquet et al., 1996). chin providing most of the extension subunits, Overall, bitterness decreases and astringency whereas epicatechin dominates in the seeds increases with increasing degree of polymeri- (Bogs et al., 2005; Hanlin and Downey, 2009; zation—that is, with increasing molecule size Prieur et al., 1994; Souquet et al., 1996). In the or chain length (Cheynier et al., 2006). Astrin- seeds, but generally not in the skin, catechin gency also increases as the tannin concentration and epicatechin are often esterified with gallic 200 6. DEVELOPMENTAL PHYSIOLOGY acid; this so-called galloylation increases their At the same time, tannin polymerization, which astringency (Brossaud et al., 2001; Dixon et al., is presumed to be an antioxidant mechanism 2005; Vidal et al., 2003). Thus, epicatechin gal- and increases astringency, may continue during late is usually not found in the skin, whereas ripening (Bachmann and Blaich, 1979; Herna´ndez in a twist of confusing terminology, epigalloca- et al., 2009; Kennedy et al., 2001). In addition, techin is normally absent in the seed (Mattivi a portion of the anthocyanins produced from et al., 2009). Because the genes responsible for veraison onwards appears to be incorporated seed tannin biosynthesis are switched on soon into tannins, or nonglycosylated anthocyanidins after fertilization (Dixon et al., 2005), seed may be converted to tannins. Such changes tannins accumulate from fruit set through may account for most of any apparent tannin veraison or soon thereafter, and then their con- accumulation in the skin after veraison. centration, or at least their extractability, With structural differences, such as different declines (Bogs et al., 2005; Downey et al., 2006; chain length, charge, and stereochemistry, Kennedy et al., 2000). They also become come differences in functional properties increasingly polymerized (and thus less bitter) among tannins and hence in their reactivity during ripening and seed desiccation. Poly- toward other molecules (Kraus et al., 2003). merization is accompanied by oxidation, which This may be one reason why a considerable is responsible for the browning of the seed coat fraction of the skin tannins can be bound to after veraison (Adams, 2006; Kennedy et al., the insoluble matrix, consisting of cell wall pec- 2000; Ristic and Iland, 2005). Oxidized tannins tins and glycans, of grape berries (Amrani bind strongly to cell walls, providing a measure Joutei et al., 1994; Kennedy et al., 2001; Lecas of “stress-proofing” to the seed coat (Pourcel and Brillouet, 1994; Pinelo et al., 2006). The et al., 2007) and limiting the extractability of binding capacity of cell walls, which reduces seed tannins. Moreover, the cuticle of the seed tannin extractability into wine, seems initially coat also strongly limits tannin extraction dur- to increase dramatically after veraison and then ing winemaking. Consequently, the amount of to decrease as the fruit approaches maturity seed tannins per berry and the amount and cell walls are dismantled and pectins extracted into wine depend mainly on the num- degraded (Robertson et al., 1980). Other studies, ber of seeds (rather than the amount per seed) however, suggested a general decrease in extra- and fruit maturity but are relatively unaffected ctability (Downey et al., 2006), and yet others by environmental factors. found no change in tannin or anthocyanin The tannin subunits in the skin are mostly extractability during grape ripening (Fournand produced and assembled in the developing et al., 2006). Nonetheless, it would be surprising flowers and berries up to, or somewhat before, if variations in functional properties among veraison (Adams, 2006; Bogs et al., 2005; the tannins of different cultivars or arising Downey et al., 2003a; Hanlin and Downey, during fruit ripening would not account for at 2009; Harbertson et al., 2002; Kennedy et al., least some of the differences observed in tannin 2001). In other words, tannin production extractability, subsequent reactions during occurs during the warmest part of the growing winemaking, and, ultimately, astringency. season and before anthocyanin accumulation Depending on maturity and binding capacity, begins, a developmental “switch” that grapes between 25 and 75% of the berry tannin may apparently share with strawberries (Fait et al., be extracted during winemaking, 50–80% of 2008). As a result, the concentration, although which is derived from the skins (Cerpa- not the absolute amount, of tannins declines Caldero´n and Kennedy, 2008). Such a wide during the postveraison berry expansion phase. range of extractability means that there is 6.2. GRAPE COMPOSITION AND FRUIT QUALITY 201 virtually no relationship between the tannin grape juice and wine produced when such skins content of grapes and that of the resulting are extracted. Anthocyanin accumulation starts wine. Indeed, the tannin concentration in red at veraison and is probably triggered by the wines of the same cultivar varies more than increasing hexose sugar concentration in the berry two orders of magnitude (Harbertson et al., skin; the threshold for switching on the genes 2008), although the variation in skin tannins is involved in anthocyanin production seems to within less than one order of magnitude (Har- be approximately 9 or 10 Brix (Figure 6.6) bertson et al., 2002; Seddon and Downey, (Castellarin et al., 2007a,b; Keller and Hrazdina, 2008). Due to this incomplete extraction, which 1998; Larronde et al., 1998; Pirie and Mullins, also applies to other phenolic compounds, 1977). In other words, accumulation of antho- organic acids, and other ingredients as well, cyanins begins after the onset of sugar accumu- grape pomace (i.e., the solid remains left after lation and berry softening (Pirie and Mullins, fermentation) contains considerable amounts of 1980) and after tannin accumulation in the skin nutritionally valuable organic and inorganic has mostly ceased. Note that although usually compounds (Arvanitoyannis et al., 2006; used to describe the beginning of ripening, the Kammerer et al., 2004; Lu and Foo, 1999; Mazza term veraison refers to a population of berries in and Miniati, 1993; Monagas et al., 2006). These a cluster or vineyard (e.g., meaning that 50% of chemicals can be further extracted by distillation the berries have changed color) rather than to an (i.e., production of brandy, such as marc or individual berry. At veraison, each cluster can grappa) and pressing (e.g., grape seed oil). Also, have berries ranging from green to blue, often they can be used for the production of dietary spanning a threefold range in sugar concentration supplements or phytochemicals, for compost- (see Figure 6.6), which implies that the different ing, or recycled directly to the vineyard. berries of a cluster are at different developmental Anthocyanins (Greek anthos ¼ flower, kyanos ¼ stages and that the profound changes in the blue) are the second class of phenolics of major expression of ripening-related genes, whereby sensory and enological importance after the many genes are being “switched on” while others tannins. They are responsible for the red, purple, are being “switched off,” occur very rapidly but or blue coloration of dark-skinned grapes and of asynchronously in these berries.

FIGURE 6.6 Change in the pigmentation of Merlot (left and center) and Pinot noir (right) grape skins due to anthocya- nin accumulation with increasing sugar concentration at veraison (M. Biondi and M. Keller, unpublished data; photos by M. Keller). 202 6. DEVELOPMENTAL PHYSIOLOGY

Anthocyanins are generally confined to the of a hydroxyl group at the R position (see vacuoles of the outer hypodermis, except in Table 6.1) results in a red color, whereas hydroxyl teinturier (French teinturier ¼ dyer) cultivars groups at both the R and R0 positions turn whose pulp is red (e.g., Alicante Bouschet or anthocyanins blue (Lillo et al., 2008; Tanaka syn. Garnacha Tintorera, Gamay Fre´aux, et al., 2008). The genes encoding the enzymes that Dunkelfelder, and Rubired) and in overripe are responsible for adding these hydroxyl grapes with senescing skin cells leaking vacuo- groups, the flavonoid hydroxylases, therefore lar components into the pulp (Moskowitz and determine the color of anthocyanins. Hrazdina, 1981; Viala and Vermorel, 1909). Anthocyanins are probably involved in Storage inside the acidic cell vacuoles protects photoprotection, and the skin of red cultivars anthocyanins from oxidation, courtesy of the absorbs much more visible and, especially, vacuoles’ low pH, and ensures that they function ultraviolet (UV) light than the skin of white cul- as red pigments; at the neutral pH of the cytosol tivars (Blanke, 1990a). A dark (red, purple, they would be mostly colorless or blue and blue, or black) berry skin is the “default” ver- unstable. More accurately, however, at least sion for grapes of all Vitis species (Cadle- some anthocyanins accumulate within special, Davidson and Owens, 2008; This et al., 2007; membraneless intravacuolar protein structures Viala and Vermorel, 1909), and all so-called called anthocyanic vacuolar inclusions (formerly white (green or yellow) grapes have evolved called anthocyanoplasts) that trap and stabilize from a dark-skinned relative by two mutations the pigments (Conn et al., 2003; Markham et al., that prevent the activation of the gene that pre- 2000). Anthocyanidins of V. vinifera cultivars scribes glucosyl transferase, an enzyme that are always bound to one glucose molecule (so- attaches a glucose molecule to the anthocyani- called 3-glucosides), whereas those of most dins (Kobayashi et al., 2004; Walker et al., American Vitis species are usually bound to two 2007). The same skin color mutation is also glucose molecules (3,5-diglucosides). Hybrids responsible for the conversion of Pinot noir into (e.g., Concord) contain both the 3-glucosides Pinot blanc. In dark grapes, the pigments based and the 3,5-diglucosides (Hrazdina, 1975; on malvidin are normally more abundant than Mazza and Miniati, 1993). The glucose stabilizes those based on other anthocyanidins, and this the metabolically unstable anthocyanidins, dominance tends to increase during fruit ripen- enhances their solubility, and turns them into ing (Keller and Hrazdina, 1998; Pomar et al., anthocyanins (Bowles et al., 2006) because 2005; Wenzel et al., 1987). Nonetheless, the rela- grapes cannot accumulate anthocyanidins. The tive proportion of the different anthocyanins glucose, in turn, can bind to acetic, coumaric, or varies by cultivar (Cantos et al., 2002; Carren˜o caffeic acid to form acylated anthocyanins, but et al., 1997; Mattivi et al., 2006; Mazza and Min- these may be less stable. Thus, the stability in iati, 1993; Pomar et al., 2005). For instance, solutions (e.g., grape juice or wine) is highest malvidin derivatives dominate in Syrah, Pinot for the 3,5-diglucosides and decreases for the noir, and many red table grapes; peonidin deri- 3-glucosides, then the acylated 3,5-diglucosides, vatives hold the majority in Nebbiolo; cyanidin and, finally, the acylated 3-glucosides. In addi- and/or peonidin derivatives prevail in Pino- tion, anthocyanin stability is also influenced tage and many table grapes; and cyanidin by the type and number of functional groups: and delphinidin derivatives are dominant in More methoxy groups ( OCH3) enhance stabil- Concord. The distribution is more “balanced” in ity, whereas more hydroxyl groups (OH) Merlot, Mourve`dre, or Sangiovese. Moreover, reduce stability. Functional groups are moreover Pinot noir ostensibly lacks the acylated forms important for the color of anthocyanins: Addition of anthocyanins that most other cultivars 6.2. GRAPE COMPOSITION AND FRUIT QUALITY 203 contain; the pigment profile of Pinot gris is very quercetin and myricetin, with small amounts similar to that of Pinot noir, albeit on a much of ka¨mpferol, laricitrin, isorhamnetin, and syr- lower level (Castellarin and Di Gaspero, 2007). ingetin. White grapes, however, produce only Thus, the dark-skinned Vitis cultivars typically quercetin, ka¨mpferol, and traces of isorhamne- have between 5 and 20 anthocyanins (Mazza tin (Mattivi et al., 2006), which indicates that and Miniati, 1993). Anthocyanins are further- an entire branch of the flavonol pathway is more responsible for the reddish or bluish switched off in white grapes. Intriguingly, berry skin of many Muscat cultivars and despite the dominance of malvidin-based Gewu¨ rztraminer, although it is possible that compounds in the anthocyanin profile of many the bronzelike color of Pinot gris and Gewu¨ rz- dark-skinned cultivars, the structurally cor- traminer arises from an interaction between responding flavonol, syringetin, usually occurs anthocyanins and carotenoids (Forkmann, only in trace amounts. Conversely, quercetin 1991). Among the Muscats, cyanidin-glucoside derivatives typically dominate the flavonol often dominates in the more lightly colored culti- profile (although in some cultivars myricetin vars (as it does in Gewu¨ rztraminer), whereas is more prominent than quercetin), whereas malvidin- and peonidin-based pigments tend the “matching” cyanidin often contributes to be more abundant in the blue/black-skinned little to the anthocyanin pool (Adams, 2006; cultivars (Cravero et al., 1994). The berries of Downey and Rochfort, 2008; Mattivi et al., some dark-skinned cultivars (e.g., Syrah, Durif, 2006). However, cyanidin is the preferred Petit Verdot, and Tempranillo) accumulate pig- substrate for anthocyanidin reductase and, ments to much higher concentrations than those thus, for tannin formation (Dixon et al., 2005). of other cultivars (e.g., Malbec, Pinot noir, Neb- Crushing the grapes during or after harvest biolo, and many table grapes), with still others brings anthocyanins in contact with other (e.g., Cabernet Sauvignon, Merlot, and Barbera) juice components with which they react to form being intermediate. Nevertheless, there does polymeric pigments (Monagas et al., 2006). not seem to be a close relationship between the Between 50 and 90% of the total anthocyanins amounts of anthocyanins and tannins in the present in the skins are extracted during fer- skin. mentation. The large variation in anthocyanin Flavonols, which are present at much lower extraction is partly caused by differences in concentrations than tannins and anthocyanins, fruit maturity (extractability seems to decline are produced in the epidermis throughout as grapes mature) and amount and composi- flower and berry development and act as “sun- tion of cell wall material (e.g., cellulose content screen” protecting the berry from harmful UVB and degree of pectin methylation), which are radiation (Downey et al., 2003b; Keller and themselves influenced by maturity and proba- Hrazdina, 1998; Keller et al., 2003b; Kolb et al., bly sun (UV) exposure (McLeod et al., 2008; 2003; Ribe´reau-Gayon, 1964). This is why sun- Ortega-Regules et al., 2006b). It is also partly exposed grapes contain far higher amounts of due to differences in skin contact time, fer- flavonols than shaded grapes, and the sun- mentation temperature, and additions such as exposed side of a berry can have a much higher macerating enzymes. Despite the incomplete flavonol concentration than its shaded side extraction during winemaking, the variation (Downey et al., 2004; Lenk et al., 2007; Price in wine color is preset by the phenolic composi- et al., 1995). Flavonol glycosides may contribute tion of the grapes at harvest, and winemaking some astringency to wines (Hufnagel and practices can do little to change this variation. Hofmann, 2008a,b). Grape cultivars with dark- All phenolic compounds extracted into wine skinned fruit contain flavonols based on are subject to a variety of enzymatic (mainly 204 6. DEVELOPMENTAL PHYSIOLOGY oxidation processes that dominate during the produced during fermentation to form orange- early stages) and chemical (increasingly domi- red pyranoanthocyanins confers additional sta- nant during later stages) reactions that modify bility. Yet red wine owes most of the gradual wine color and decrease astringency during change in color from red-purple to tawny dur- aging (Cheynier et al., 2006; Fulcrand et al., ing aging to conversion of anthocyanins to 2006; Jackson, 2008; Monagas et al., 2006; Ribe´r- polymeric pigments that form by condensation eau-Gayon, 1973; Somers, 1971; Waterhouse between anthocyanins and tannins and other and Laurie, 2006). The type and quantities of components (Monagas et al., 2006; Ribe´reau- the different pigments influence intensity, hue, Gayon, 1973). Contrary to popular opinion, and stability of wine color. Moreover, antho- however, polymerization does not always cyanins come in five main molecular forms; increase color stability. Thus, aged red wine they coexist in a dynamic equilibrium, but most color is determined by the grapes’ anthocyanin of them are actually colorless at juice or wine and tannin composition, and both of these phe- pH (Jackson, 2008). Above pH 3, less than nolic classes can limit wine color. In other 30% exists as the red-colored flavylium cation, words, if grapes are rich in anthocyanins but and its proportion declines further with poor in tannins, wine color will be no better increasing pH to almost nothing at pH 4. Even than if the reverse is the case. Tannins also more important, sulfur dioxide (SO2), used polymerize with other tannins, and there is no in winemaking to control microbial growth, upper limit to this polymerization (Jackson, effectively bleaches anthocyanins. As a conse- 2008). Even long polymeric tannins, as long as quence, the colorless forms account for 70 subunits, remain in solution; tannins precip- 75–95% of the anthocyanins in wine. itate only when they bind to proteins in wine. Once extracted from the grapes into juice or Moreover, polymerization is a dynamic process wine, flavonols can be partially hydrolyzed in in wine; polymeric tannins are susceptible to wine, whereby their glucose “tail” breaks away cleavage so that their degree of polymerization (Monagas et al., 2006). The resulting flavonol can increase or decrease during aging. aglycones, together with cinnamic acids and The grapes’ anthocyanin, flavonol, and tan- catechin, can serve as so-called cofactors, which nin profile determines the color potential of stack together with anthocyanins in wine. This the wine made from these grapes, and color is association of anthocyanin pigments with col- a main driver of perceived red wine quality. orless phenolics is termed copigmentation and Color hue and intensity are obviously impor- shifts the color toward purple or blue and tant for quality in their own right, and they also intensifies it (Scheffeldt and Hrazdina, 1978; influence the (subjective) perception of quality Tanaka et al., 2008). Copigmentation is respon- in other ways. Intriguingly, people associate sible for the temporary purpleness and color certain flavors with specific colors, and the per- stability (i.e., protection from SO2 bleaching ception of taste and/or flavor intensity and and high pH) of young red wines and is said complexity tends to increase as the color inten- to account for 30–50% of their color (Boulton, sity increases. In other words, if two otherwise 2001). Indeed, the color of young wine seems identical grape juices or wines differ in (red) to be more limited by cofactors than by antho- color, the more deeply colored one is perceived cyanins, although anthocyanins can also join as being more complex and as having more together with other anthocyanins in so-called “body” and more dark-fruit aromas (Delwiche, self-association, which also stabilizes color. 2003; Morrot et al., 2001). In the brain, it is The reaction of anthocyanins with acetalde- clearly all about perception. In contrast, pheno- hyde or pyruvate extracted from the grapes or lic compounds are far less important for white 6.2. GRAPE COMPOSITION AND FRUIT QUALITY 205 grape and wine quality, which is determined caused by high light (Du¨ring and Davtyan, mainly by the grapes’ flavor and aroma poten- 2002). Although they have pharmacological tial. In white wines, phenolics may even con- properties as antioxidants and provitamins tribute to undesirable astringency and (Bartley and Scolnik, 1995; Della Penna and bitterness—traits that are particularly unattrac- Pogson, 2006) and contribute to the yellow to tive in sparkling wines. reddish skin color (especially in so-called white cultivars) when the chlorophylls are degraded 6.2.6. Lipids and Volatiles during and after veraison, the concentration of most carotenoids declines during fruit ripen- Lipids—consisting primarily of epicuticular ing. This decrease is at least partly due to and intracuticular waxes, fatty acids, mem- degradation of carotenoid pigments into volatile brane lipids, and seed oils—are present in the norisoprenoids (C13 ketones) that are potent grape berry throughout development. Contrary grape and wine aroma and flavor components to widespread belief, plant membranes also (Baumes et al., 2002; Razungles et al., 1996). When contain cholesterol in addition to many other such volatiles escape into the air, they can be sterols and their derivatives, called steroids detected by the olfactory receptors of the nose so (Fujioka and Yokota, 2003), although its contri- that we can smell them as an odor, in addition bution to the total lipids is approximately two to their sometimes imparting taste as well orders of magnitude less than in animals. (Schwab et al., 2008). Carotenoid cleavage dioxy- Grape seeds contain up to 17% (fresh weight genases are the enzymes that (oxidatively) “slice” basis) polyunsaturated fatty acids (mainly lino- the various carotenoids, thus converting them leic acid) in addition to oleic acid and the into norisoprenoids that contribute to floral saturated fatty acids palmitic and stearic acids and fruity aroma attributes (Mathieu et al., 2005; (Currle et al., 1983). When seeds are damaged Winterhalter and Schreier, 1994). during crushing, linoleic acid can be oxidized Because grape cultivars differ in their fruit to hexanol, which is converted by yeast to carotenoid composition, they also differ in their hexyl acetate (odor of “fruit,” “apples,” or bouquet of flavor and aroma compounds “herb”). However, the most important group produced from these carotenoids or their of lipids in terms of fruit and wine quality are breakdown products. In other words, part of the carotenoids, especially b-carotene (provita- the “varietal aroma” may be derived directly min A) and lutein in addition to minor com- from variations in skin color, which arise from pounds, such as the xanthophylls neoxanthin, the amounts and relative proportions of differ- violaxanthin, and zeaxanthin. Carotenoids be- ent carotenoids (Schwab et al., 2008). Examples long to the class of isoprenoids, which are also of norisoprenoids (apart from the hormone called terpenoids or terpenes. Most carotenoids abscisic acid) include b-damascenone (“rose,” are C40 compounds; that is, they contain a “dried fruit,” “exotic fruit,” or “tropical flow- “backbone” of 40 carbon atoms and are thus ers” aroma) and b-ionone (“violet” or “rasp- tetraterpenes assembled from isopentenyl pyro- berry” odor contributing to “floral” or “fruity” phosphate (Della Penna and Pogson, 2006; aromas) in Chardonnay, Pinot noir, Riesling, Hirschberg, 2001; Tanaka et al., 2008). They are Cabernet Sauvignon, and Concord berries. hydrophobic and, similar to their role in leaves In addition, both b-damascenone and b-ionone (see Chapter 4.1), are accumulated in (skin) seem to strongly enhance the overall fruity plastids of young grape berries as part of the character and reduce vegetative notes of wines photosynthetic machinery to protect berry (Escudero et al., 2007; Pineau et al., 2007). tissues from oxidative stress, especially stress b-Damascenone is also an important aroma 206 6. DEVELOPMENTAL PHYSIOLOGY component of beer, tobacco, and tea leaves, compounds (Gholami et al., 1995; Luan and whereas b-ionone is one of the most important Wu¨ st, 2002). The production of geraniol volatiles contributing to tomato fruit flavor and nerol is mainly restricted to the berry skin, and also to the fragrance of many flowers. The whereas linalool and rose oxide synthesis occurs production of these so-called apocarotenoids in both the skin and the mesocarp, especially in appears to be directly dependent on the amount the Muscats (Gunata et al., 1985; Luan and Wu¨ st, of their respective carotenoid precursor formed 2002; Luan et al., 2005; Park et al., 1991; Wilson during fruit development. However, although et al., 1986). The sesquiterpene rotundone, which the carotenoids are largely accumulated before is the key aroma compound of both black veraison, the production of the resulting apo- and white pepper, also imparts a “peppery” carotenoids does not peak until late in ripening. and “spicy” aroma to grapes (and the resulting Moreover, the bulk of these norisoprenoids wines) of Syrah, Mourve`dre, Durif, and, to are glycosylated (Mathieu et al., 2005), and a lesser extent, Cabernet Sauvignon (Wood only their release from the sugar molecule et al., 2008). during fermentation and wine storage leads to All volatile grape terpenoids are mono-, ses- volatilization and “smellability.” qui-, or norisoprenoid terpenes (the diter- Other volatile isoprenoid compounds such as pernoid hormone gibberellin is not volatile) the highly aromatic monoterpenoids (C10;e.g., produced from the simple isoprene building linalool, geraniol, nerol, and citronellol and block isopentenyl pyrophosphate (McGarvey its derivative rose oxide), which are reminiscent and Croteau, 1995; Tanaka et al., 2008). Two of “rose,” “lilac,” “pine,” or “citrus,” accumulate biochemical assembly lines operate in parallel in postveraison grapes of Muscat cultivars, with some interaction to produce a wide vari- Gewu¨rztraminer, and, to a lesser extent, Riesling, ety of terpenoids (Schwab et al., 2008): the Viognier, and Chenin blanc (Gunata et al., 1985; mevalonate pathway (probably limited to Guth, 1997a,b; Ribe´reau-Gayon et al., 1975; the production of sesquiterpenes and a few Wilson et al., 1984;). Although monoterpenes other compounds) in the cytosol and the are important in Riesling, the typical Riesling fla- deoxy-xylulose 5-phosphate/methyl-erythritol vors may be dominated by the norisoprenoids 4-phosphate (DOXP/MEP) pathway in the vitispirane, trimethyl-dihydronaphthalene (TDN), plastids (see Figure 6.5). Grape berries make and b-damascenone (Skinkis et al., 2008; monoterpenoids mainly through the mevalo- Strauss et al. 1987). Many if not all other cultivars nate-independent DOXP/MEP pathway (Luan (e.g., Sauvignon blanc, Syrah, and the Pinots) and Wu¨ st, 2002), which also produces carote- also produce monoterpenes, but at much lower noids, the chlorophyll “backbone,” the hor- concentrations than these aromatic cultivars. mones gibberellin and abscisic acid (ABA), The “muscat” aroma is sought after in table and other compounds. The berries of different grapes, and the same (and other) monoter- cultivars manufacture specific bouquets of penoids also contribute to the characteristic monoterpenes from the original geraniol, and fragrances (and varietal differences) of many much of this interconversion occurs only late flowers (e.g., rose, lilac, and jasmine), mint during the ripening phase (Luan et al., 2005, leaves, and conifer needles, whereas citrus fruits 2006). Most isoprenoids (e.g., 90% of the ter- and basil leaves owe their aroma predominantly penes) found in grapes are glycosylated (usu- to mono- and sesquiterpenoids (C15). Although ally bound to glucose) and may be regarded grape leaves are capable of producing as potential aroma or flavor precursors that monoterpenes, the berries appear to manufac- can be released enzymatically or chemically ture their own complement of these aroma during winemaking. Many such glycosylated 6.2. GRAPE COMPOSITION AND FRUIT QUALITY 207 precursors continue to release aroma volatiles to volatiles present in grapes (Conde et al., 2007; in aging wines until they are exhausted. Jackson, 2008). It is thought that the more Among the more than 20 red-colored Muscat diverse the mix of volatiles, the less important cultivars, the amount of terpenes (especially will be the role of the individual components linalool) tends to vary inversely with the antho- and the more complex will be the overall cyanin content (Cravero et al., 1994). In other aroma and flavor. words, the more deeply colored cultivars tend Like the phenolics and isoprenoids, these to be less aromatic. Indeed, it appears that the components are normally accumulated in gly- production of terpenoids (i.e., the terpenoid cosylated form, which prevents these otherwise pathway) and the accumulation of phenolics volatile compounds (as aglycones) from prema- (i.e., the shikimate/phenylpropanoid pathway) turely escaping to the atmosphere. However, are in competition for access to carbon sub- although terpene glycosides may add a bitter strates (Xie et al., 2008), although the carbon taste (Noble et al., 1988), it is the volatile com- itself is abundant due to the accumulation of ponents that are central to the distinctive fruity hexoses during ripening. This means not only aroma (and flavor), and many of these are that cultivars that generate many terpenoids slowly released during the winemaking and may tend to produce lower amounts of pheno- aging processes in part due to the activities lics and vice versa but also that stimulating the of glycosidase and other metabolic enzymes of production of one class of compounds might yeast and lactic acid bacteria. For example, come at the cost of lower production of the yeast can transform geraniol into rose oxide other. (although some transformation also occurs in Both fruit and wines contain hundreds of grape berries). In other words, the majority of different volatiles in addition to the isopre- potentially important wine flavor and aroma noids. Although these belong to many different components cannot be perceived in the fruit classes of chemicals, including alcohols, esters because human saliva lacks the glycosidase and aldehydes, ketones, acids, lactones, and enzymes necessary to break away the sugar furanones, the majority of them (the sugar- molecules (Kennison et al., 2008). Although derived furanones are one exception) are pro- grape berries do contain glycosidase, their high duced by oxidation of fatty acids by enzymes glucose content greatly slows the enzyme’s such as lipoxygenase, isomerases, alcohol activity (Aryan et al., 1987). This is why the dehydrogenase (which converts aldehydes into nonvolatile glycosylated compounds (glyco- alcohols), and alcohol acyl transferases (which sides) are odorless and are generally called convert alcohols into esters) (Jackson, 2008; aroma (flavor) precursors or potential aroma Schwab et al., 2008). Smaller portions of the (flavor) compounds. Therefore, when harvest same chemical classes are also contributed by decisions are based on tasting fruit in the vine- the catabolism of amino acids—for instance, yard, they often (consciously or unconsciously) the leucine-derived methylbutanal and methyl- become decisions on the basis of the perception butanol, which are also important flavor com- of sweetness and sourness in addition to the pounds of apples and strawberries. Cultivars disappearance of “veggie” aroma (discussed differ greatly in the type and amount of volatile later). Moreover, there is substantial fruit-to- constituents they produce, and although most fruit variation within a cultivar due to, among volatiles have no odor and many esters simply other variables, differences in fruit location smell fruity, these differences largely account (e.g., sun exposure), growth temperature and for the characteristic varietal aroma and flavor light, nutrition, harvest date (i.e., “ripeness”), (i.e., the bouquet) that can be directly attributed and postharvest handling and storage. For 208 6. DEVELOPMENTAL PHYSIOLOGY instance, the continued metabolism of har- et al., 2009). Because of its volatile nature, the vested grapes, especially machine-harvested concentration of methyl anthranilate begins to grapes that are damaged in the process, can decline in mature grapes, as does that of other lead to substantial aroma and flavor loss if the nonglycosidically bound aroma compounds, fruit is picked, transported, or left standing in such as the “veggie,” “herbaceous,” “grassy,” the heat for prolonged periods. On the other “bell pepper,” or “asparagus”-like methoxy- hand, the continuation of berry metabolism, pyrazines typical of Sauvignon blanc, Se´millon, much of it altered in response to dehydration- and Carmene`re, but also of (unripe) Cabernet induced osmotic stress, together with the con- Sauvignon, Cabernet franc, and Merlot berries. centration effect from water loss in harvested However, unlike methyl anthranilate, the cyclic, grapes can also be exploited during a deliberate nitrogen-containing methoxypyrazines, which and prolonged postharvest drying phase to are probably produced from the amino acids produce distinct wine styles such as the dry glycine and isoleucine, are not released into (e.g., Amarone) or sweet (e.g., Recioto, Vin Santo, the air but are accumulated as free volatiles and Vins de Paille) straw wines (Costantini et al., that are released only when the berry cells are 2006; Zamboni et al., 2008). disrupted by frugivores, which they help dis- In contrast to the glycosylated components courage from eating unripe berries. Therefore, discussed previously, the distinctive “foxy” methoxypyrazines accumulate early during V. labrusca aroma components methyl anthrani- berry development to a maximum before verai- late, amino acetophenone, and a furanone are son and are then degraded during ripening to produced in the free volatile form during rip- less than 10% of their maximum concentration ening and are emitted into the air, contributing (Lacey et al., 1991). This decrease appears to to the characteristic smell of ripening Concord be closely associated with that of malate: Most vineyards (Shure and Acree, 1994). Methyl of the decline occurs during the initial ripening anthranilate is also an important aroma compo- phase, with only a slow further decrease above nent of orange flowers and Chinese jasmine approximately 20 Brix (Lacey et al., 1991; green tea, as well as of old strawberry varieties Roujou de Boube´e et al., 2000). In other words, (before their aroma was sacrificed in the pro- grapes high in malate also tend to be high cess of breeding transport-resistant varieties). in methoxypyrazines. Methoxypyrazines are Birds dislike the aroma and thus generally located almost exclusively in the skin of grape avoid Concord grapes; therefore, synthetic berries and in rachis tissues but are extracted forms of methyl anthranilate are used in bird extremely rapidly during juice processing, repellents in a variety of crops. Methyl anthra- before fermentation even starts, and are stable nilate seems to be assembled in the outer meso- in wine (Hashizume and Samuta, 1997). Hexa- carp from a breakdown product of tryptophan nals and hexenals (both are aldehydes and are and methanol (Wang and De Luca, 2005). The characteristic aroma components of strawber- latter may be either directly released from the ries) derived from catabolism of fatty acids cell walls or produced from methane (CH4) (linolenic acid) also contribute to the “fresh released from cell wall pectins when the berries green,” “grassy,” and “herbaceous” aroma of soften during and after veraison (Keppler et al., grapes and are also present in the rachis, but 2008; Lee et al., 1979). The former is thought to their extraction during fermentation might be enable this reaction by conferring on pectin very limited (Hashizume and Samuta, 1997; et al the ability to emit CH4 upon UV irradiation Schwab ., 2008). A close relative of methyl via the production of reactive oxygen species, anthranilate, 2-amino acetophenone (acetophe- thus acting as a photosensitizer (Messenger none is a simple isoprenoid), is present in 6.3. SOURCES OF VARIATION IN FRUIT COMPOSITION 209

Concord and other cultivars with V. labrusca enhance the fruity character, especially of black parentage in both free and glycosidically bound currant and “strawberry/raspberry,” and add form and is reminiscent of “acacia flower,” “truffle” (of which it is a major aroma component) “naphthalene,” or “floor polish.” Although it and “olive” notes (Escudero et al., 2007; Segurel is a regular aroma component of beer and corn et al., 2004). At higher concentrations, however, chips (and of the pheromone of honeybee DMS has an unpleasant “cabbage” odor, which is queens), its formation during wine storage considered an off-flavor. is thought to contribute to the unpleasant Other aroma compounds enter wine not “atypical aging” off-flavor of V. vinifera wines. from the grapes at all but, rather, have their ori- Certain sulfur-containing compounds, vola- gin in storage or packaging materials. One tile thiols, termed mercaptans also contribute example is the infamous TCA (2,4,6-trichloroa- to the aroma complex in wine but probably nisole), which imparts a musty or moldy odor not in grapes. Although they are partially at extremely low concentrations (parts per bound by saliva proteins (Starkenmann et al., trillion) and is responsible for the majority of 2008), they usually have a very unpleasant cork taints in wine. Most of the TCA is pro- smell of rotten eggs. At very low concentra- duced by microorganisms in corks that have tions, however, such thiols are reminiscent of been bleached with chlorinated compounds or “black currant” (mercaptobutanol and mercap- in oak wood that has been treated with topentanol)—characteristic of ripe Sauvignon chlorine-containing insecticides or fungicides. blanc and Cabernet Sauvignon berries—or “passion fruit” and “grapefruit” (mercaptohex- 6.3. SOURCES OF VARIATION IN anol), especially in Sauvignon blanc and FRUIT COMPOSITION Gewu¨ rztraminer but also in Merlot, Muscat, Riesling, Se´millon, and other cultivars (Guth, 1997a; Tominaga et al., 1998). These compounds Uniform fruit composition is often described are bound to a nonvolatile thiol termed gluta- as a critical factor for premium winemaking thione and its breakdown product, cysteine, in and is equally desirable for the production of the grape skin and, to a lesser extent, the meso- grape juice, table grapes, and raisins. It could carp (Peyrot des Gachons et al., 2002a,b). be argued, however, that less uniform fruit Although this binding makes them odorless in may sometimes result in more complex wine, grapes, the odor-active thiols are released by and this might be one of the rationales behind enzymes of bacteria in the human mouth (Star- blending wines from different vineyard sources. kenmann et al., 2008) and by yeast enzymes In many European wine regions, such blending during fermentation (Dubourdieu et al., 2006). was traditionally done in the field, where a The concentration of volatile thiols in wine is range of clones or even cultivars were inter- directly related to the concentration of their planted, sometimes systematically and some- precursors in the grapes, even though only a times randomly. Whereas traits related to minor portion (<5%) of the latter is actually con- color, astringency, acidity, and flavor are key verted to aroma compounds. Yeast also seems to quality attributes of wine grapes, table grape be able to produce dimethyl sulfide ((CH3)2S, often quality depends more on sugar:acid ratios, tex- abbreviated as DMS), perhaps from the S-contain- ture for crunchiness or crispyness, and visual ing amino acids cysteine and methionine or from appearance including color (Mullins et al., the cysteine-containing peptides glutathione and 1992), although Muscat flavor is often also cystine (de Mora et al., 1986). The DMS concentra- sought after. Seedlessness is important in some tion further increases during wine aging and may table grape markets and is usually desirable 210 6. DEVELOPMENTAL PHYSIOLOGY for raisin grapes, for which high sugar content services. The majority of the fluctuations in is much more important than it is for table fruit composition are caused by climate varia- grapes. bility. In fact, weather differences among years, Fruit composition changes over time during in addition to vineyard location, are by far the the berry ripening period as part of the grape- strongest determinants of fruit composition vine’s developmental program and is therefore (Downey et al., 2006). Such climatic differences under genetic control. In addition, like phenol- often trump seasonal differences in soil mois- ogy and yield formation, the extent of these ture (except at the far low and high ends of changes may also be modulated by environ- the moisture spectrum) in both dry-farmed mental factors and by the interaction of these and irrigated vineyards (Keller et al., 2008; factors with the genotype of the vine. It is often Pereira et al., 2006b; van Leeuwen et al., 2004). difficult to separate the influence of one of For instance, seasonal variation accounts for these factors from that of another. For example, an almost 2-fold range in sugar concentration, solar radiation affects both incident light and a 2- or 3-fold variation in acid and anthocyanin tissue temperature. An increase in water sup- content, and at least a 10-fold range in aroma ply leads to an increase in shoot growth, which components (Cacho et al., 1992; Castellarin can decrease the proportion of exposed leaves, et al., 2007b; Downey et al., 2004; Viala and which in turn alters both light quantity and Vermorel, 1909; Wood et al., 2008). The differ- light quality and may also lead to changes in ences in anthocyanin profiles—that is, the rela- canopy microclimate (see Chapter 5.2). Further- tive proportion of individual pigments for a more, the sugar concentration is higher and given cultivar—varies more among years than the malate concentration lower in berries that from veraison to harvest in the same season have fewer seeds, apparently because veraison (Pomar et al., 2005). These differences hold true occurs earlier in such berries (Currle et al., even at the genetic level: Differences in gene 1983; Rapp and Klenert, 1974). Thus, even expression in grape berries between growing within a grape cluster, there is often a natural seasons can be greater than those between variation of sugar concentration in the range pre- and postveraison berries within a season of 5–7 Brix that results from asynchronous (Pilati et al., 2007). Cultural practices can at best development of individual berries (Coombe, be used to fine-tune what nature imposes in 1992). Due to such asynchronous development, any given season. The same applies to clones neither time nor thermal time after bloom of a cultivar and the rootstock used as grafting adequately reflects the developmental stage partner: Season and site still vastly outweigh and maturity of individual berries in a sample the influence of these on fruit composition, from the entire population of berries on a vine, although rootstocks may lead to considerable let alone in a vineyard. differences in yield (Schumann, 1974). Even Any factor that influences vine growth and grapes harvested at identical concentrations of metabolism directly or indirectly impacts fruit soluble solids in different years can have very composition, and this leads to large variation different amounts of anthocyanins and aroma- among growing seasons in terms of fruit qual- active compounds. For a specific genotype, var- ity. Wine grape production is especially sensi- iation in grape composition occurs within one tive to climate variability. This sensitivity is vine, between vines, between vineyards, and the source of considerable variation in vintage between growing seasons due to, among others quality, which in turn forms one of the bases (e.g., some effects of pathogen infection are dis- for the existence of an entire “industry” of wine cussed in Chapter 7.5), the factors discussed in judges, consumer magazines, and related the following sections (see also Jackson, 2008). 6.3. SOURCES OF VARIATION IN FRUIT COMPOSITION 211

6.3.1. Fruit Maturity of hydroxycinnamic acids, others (e.g., euge- nol) seem to remain constant or even decrease Contrary to widespread misunderstanding, (Fang and Qian, 2006). The precursors of some especially among winemakers, grapes are aroma-active esters also appear to decrease physiologically mature when the seeds attain over time (resulting in wines with less fruity their ability to germinate (i.e., immediately aroma), but the majority show no consistent after veraison; see Chapter 2.3), not at some trend. It is not clear which changes late in the arbitrarily defined point thereafter. Neverthe- growing season are indeed due to the con- less, subsequent alterations in fruit composition tinued production or modification of these may be beneficial for the seeds and hence for quality-relevant compounds in the grape the species because they help attract seed dis- berries or simply brought about by the slow persers. As argued in Chapter 2.3, the accumu- dehydration of the berries (i.e., concentration lation of color and aroma volatiles serves as effect, weather permitting). Moreover, physical “advertisement” to signal to potential seed vec- and chemical changes in fruit composition also tors the availability of food material of high lead to changes in the number and composition nutritional and health value. In fact, the major- of microorganisms that call the fruit home. ity of pigments and volatiles are manufactured As the sugar concentration and the pH from nutritionally valuable components such increase, both the number and the diversity of as sugars, amino and fatty acids, and carote- this microflora increase. Yet the consequences noids and thus serve as positive nutritional of these population dynamics for grape juice signals (Goff and Klee, 2006). Many of the asso- and wine composition are not well understood ciated changes during grape ripening were and little appreciated. discussed previously for individual com- Clearly, some of the alterations in fruit com- pounds or specific groups of compounds. In position with increasing grape maturity are addition to the well-known increase in the con- desirable (e.g., more sugar, less acid and bitter centration of sugars, amino acids, and phenolic tannin, and more “good” and less “bad” flavors), compounds (especially anthocyanins in red whereas others are not (e.g., even more sugar, grapes) and the decrease in acidity (with a high pH, and less “good” and more “bad” corresponding rise in pH), flavor and aroma flavors). Moreover, the desired level of fruit components also undergo changes as grapes maturity depends on the intended use of the ripen. For instance, methoxypyrazines decline grapes. Taking soluble solids as a simple measure most rapidly during the early ripening phase of maturity, table and juice grapes may be har- (Lacey et al., 1991; Roujou de Boube´e et al., vested when they have accumulated just 2000), whereas norisoprenoids and most other 16–18 Brix, which is immediately after they have terpenes, or their glycosylated aroma precur- fully turned color. Grapes destined for sparkling sors, appear to continue to increase even at wine production are considered optimally very advanced stages of maturity (>30Brix) ripe at approximately 18–20 Brix. And while the (Fang and Qian, 2006; Hardie et al., 1996a). typical harvest “window” for many white wine Such increases during extended ripening or grapes ranges from 20 to 24Brix, red wine grapes “hang time” of the grapes form the basis for are often left on the vines well beyond the delaying harvest to maximize terpenoid-based physiological sugar maximum of 24 or 25Brix— aroma compounds (Wilson et al., 1984). weather permitting. The factors described in the Whereas the concentration of some volatile following sections exert much of their influence phenols (e.g., guaiacol) increases strongly on fruit composition by accelerating or retarding during maturation, perhaps due to conversion grape ripening and hence maturity. 212 6. DEVELOPMENTAL PHYSIOLOGY

6.3.2. Light leaves are exposed to sunlight (Crippen and Morrison, 1986). Tartaric acid seems to be As the source of energy for photosynthesis, relatively insensitive to low light intensity; sunlight is the most important climatic factor if anything, its production decreases in the affecting grape composition. The effect of light shade, perhaps because the synthesis of its pre- is often expressed in terms of the effects of cursor ascorbate is light sensitive (DeBolt et al., shade, especially as it refers to the within- 2007). In contrast, shaded fruit often has higher canopy microclimate (Jackson and Lombard, concentrations of malic acid, which is probably 1993; Smart, 1985). Apart from weather condi- an effect of the lower temperature that tions, this microclimate, and hence the extent accompanies shade (Pereira et al., 2006a; Viala of sun exposure of the fruit clusters, is a func- and Vermorel, 1909). Regarding amino acids, tion of trellis design, canopy density, shoot although shaded berries have less total nitro- length, and shoot architecture (e.g., plentiful, gen, they may contain more arginine and less overhanging lateral shoots) and can be modi- proline than sun-exposed berries (Pereira fied by canopy management practices such as et al., 2006a). Because light (partly via phyto- shoot thinning and positioning, hedging, or leaf chromes) stimulates CHS and many other removal. It is important to remember, however, enzymes of the pathways involved in the pro- that shade decreases both light intensity and duction of phenolics, shading reduces the temperature during the day, and it is very diffi- production of phenolic components. However, cult to disentangle the separate effects of each different classes of phenolics react differently of these variables. to specific portions of the light spectrum. Shade on the leaves depresses the rate of Anthocyanins and hydroxycinnamic acids are photosynthesis, thus limiting the rate of sugar stimulated primarily by visible light, whereas export to grape berries, which may curtail flavonols react predominantly to UVB. berry growth (Rojas-Lara and Morrison, 1989). Although light is necessary for anthocyanin Berry growth can also be slower in shaded clus- production, direct sun exposure of the fruit is ters than in sun-exposed clusters, especially if not nearly as important for anthocyanin accu- shade occurs early during berry development mulation as for flavonol production; indeed, (Dokoozlian and Kliewer, 1996). Moreover, flavonols may be useful markers of light exposing shaded clusters to sunlight at verai- exposure (Downey et al., 2004; Haselgrove son or later does not lead to compensatory et al., 2000; Keller and Hrazdina, 1998; Macheix berry growth. However, where fruit exposure et al., 1990; Pereira et al., 2006a; Price et al., 1995; is associated with high temperatures, sun- Spayd et al., 2002). However, high amounts exposed berries may remain smaller than of flavonols may lead to noticeable astringency shaded berries (Bergqvist et al., 2001; Crippen or even bitterness in grapes and wines (Gawel, and Morrison, 1986; Reynolds et al., 1986). 1998). When shaded leaves die, their nutrients are Because the synthesis of all acids and pheno- recycled to important sinks. If the fruit happens lics is dependent on imported sucrose, episodes to be important while leaves are senescing (e.g., of cloudy or overcast conditions slow anthocya- around veraison), they may receive a dose of nin accumulation and may result in fruit nitrogen, potassium, and other nutrients, which with lower anthocyanin content (Keller and may eventually lead to an increase in juice pH Hrazdina, 1998). Clouds may also induce a shift (Morrison and Noble, 1990; Smart et al., 1985a, in the relative proportion of individual antho- þ b). Accordingly, such increases in K and pH cyanins. In grapes grown under overcast do not occur in shaded fruit as long as the conditions, malvidin-based anthocyanins may 6.3. SOURCES OF VARIATION IN FRUIT COMPOSITION 213 come to dominate the anthocyanin profile tannin accumulation in the skin, but usually because their production seems to be less not in the seeds, of sun-exposed berries up to responsive to low solar radiation than that of veraison and increases the average length other anthocyanins (Keller and Hrazdina, of tannin polymers (Cortell and Kennedy, 1998). Shading of clusters by leaves, on the other 2006; Downey et al., 2006; Koyama and Goto- hand, only interferes with pigmentation if it is Yamamoto, 2008). This light effect seems to be severe and apparently suppresses the genes that more prominent in cultivars that produce low code for anthocyanin biosynthesis (Jeong et al., amounts of tannins than in cultivars with high 2004; Koyama and Goto-Yamamoto, 2008). It tannin content (e.g., Nebbiolo). Contrary to its appears that anthocyanin formation in the skin influence on anthocyanins, sun exposure reaches a plateau at a photon flux at approxi- appears to decrease the extractability of tannins mately 100 mmol m 2s 1 in the fruit zone; above during ripening, which tends to cancel out at this light intensity (under clear-sky conditions least part of the prior gain in tannin content there can be >2000 mmol m 2s 1), temperature (Downey et al., 2006). The nature of this change becomes the dominant factor in berry coloration in tannin extractability is unknown, but consid- (Bergqvist et al., 2001; Downey et al., 2006; Spayd ering that rising temperature and UV radiation et al., 2002; Tarara et al., 2008). Too much radia- enhance methane release from cell wall pectins, tion (of UVB) can even inhibit anthocyanin pro- possibly via the generation of reactive oxygen duction or induce degradation, perhaps due to species (Keppler et al., 2008; McLeod et al., the formation of hydrogen peroxide (H2O2)asa 2008), it is conceivable that the resulting pectate result of oxidative stress. The H2O2 in com- may bind some of the tannin. Nevertheless, bination with the enzyme peroxidase might grapes grown on less vigorous vines, which degrade anthocyanins (and other phenolics) in normally have higher fruit exposure, and the the vacuoles. Nonetheless, all else being equal wine produced from these grapes tend to have (including latitude), grapes produced at higher greater amounts of skin tannins and anthocya- altitudes tend to have higher concentrations of nins and, therefore, ultimately higher concen- total phenolics and anthocyanins than their trations of stable polymeric wine pigments, lower elevation counterparts, although it is not than those from more vigorous vines (Cortell clear if this is due to the increase in UVB with et al., 2005, 2007a,b). However, anthocyanin increasing altitude (Berli et al., 2008). accumulation can be impaired in vines with Cultivars differ in their fruit’s susceptibility very low vigor due to insufficient leaf area to shade; compared with sunlit fruit, the antho- and excessive fruit exposure. cyanin content is often considerably lower in Because carotenoids act as photoprotectants, shaded Pinot noir, Cabernet Sauvignon, or their preveraison synthesis is induced by light Malbec grapes than in Merlot, whereas colora- exposure, whereas episodes of low light (e.g., tion in varieties such as Syrah, Petit Verdot, or due to overcast or rainy conditions) are asso- Nebbiolo appears to be almost unaffected by ciated with a temporary decrease of xantho- shade. Taken together, it appears that sun phyll carotenoids (Du¨ ring and Davtyan, 2002). exposure of grapes may enhance the color of Given that xanthophyll production apparently (young) red wines mainly by increasing the does not respond to temperature, one might amount of cofactors (flavonols) and less so that therefore conclude that grape berries are most of anthocyanins. Nevertheless, sun exposure vulnerable to sunburn (see Section 6.3.3, Tem- may also improve the extractability of antho- perature) if bright, hot days suddenly follow a cyanins during fermentation (Cortell and period of low light conditions or if shaded Kennedy, 2006). Visible light may also enhance berries are suddenly exposed to the sun. 214 6. DEVELOPMENTAL PHYSIOLOGY

Sudden exposure occurs, for example, follow- average light intensity during the ripening ing defoliation of the cluster zone too late in period (Marais et al., 2001). the growing season in an attempt to improve In addition to the direct effects discussed the microclimate in the fruit zone. Another rea- previously, light exposure of grape clusters son for this vulnerability is that the shaded could also have indirect effects on wine quality. berries have not produced sufficient amounts It appears that yeasts are among the fungi of UV-protective flavonols on their skin (Kolb that are most susceptible to UVB radiation et al., 2003). Moreover, if grapes behave similar (Newsham et al., 1997). Although this premise to apples, then only cultivars resistant to sun- has yet to be tested, it is possible that light burn should respond to high light exposure exposure alters the yeast microflora on the with elevated carotenoid production, whereas surface of grapes. This could have implications carotenoids should decrease in exposed fruit for wine flavor because even in musts ino- of susceptible cultivars (Merzlyak et al., 2002). culated with single-strain yeast cultures, the Sun exposure not only strongly enhances the native yeasts may be important during the preveraison production of carotenoids but also initial stages of fermentation. accelerates their postveraison degradation, and this is associated with increased conversion to 6.3.3. Temperature aroma-active norisoprenoids (Baumes et al., 2002; Razungles et al., 1998; Schultz, 2000). Temperature is another aspect of sunlight Although this results in high norisoprenoid that heats plant tissues. The dependence of concentrations in exposed fruit, removing leaves cellular integrity and enzymatic reactions on to increase light exposure can sometimes—for conditions that are neither too cold nor too unknown reasons—diminish the amount of hot makes temperature a very important factor norisoprenoids (Lee et al., 2007). Light might also influencing grape composition. In cool regions, promote methyl anthranilate formation in sunlit low temperatures often limit photosynthesis Concord berries because higher UV radiation and sugar production in the leaves, although favors the release of its precursor methane from growth and sink activity of the fruit decrease cell wall pectins, possibly as a result of oxidative more than photosynthesis under low temp- stress (McLeod et al., 2008; Messenger et al., erature (Klenert et al., 1978; Ko¨rner, 2003; 2009). Other aroma compounds are also affected Wardlaw, 1990). Conversely, in hot regions, by light; the concentration of glycosylated temperatures often exceed the photosynthetic monoterpenoids is reduced in shaded fruit optimum during a large part of the day; thus, (Reynolds and Wardle, 1989), whereas methoxy- the temperature for a considerable proportion pyrazine concentrations are substantially of the growing season can be higher than the increased, even in young berries. Although it is optimum range for photosynthesis. In addition, not clear whether this is due to a direct effect of high nighttime temperatures in hot regions light on aroma synthesis or degradation or an increase the proportion of assimilated carbon indirect effect of lower berry temperature in the that is lost through respiration, which reduces shade, even relatively brief periods of low light, the total amount of sugar available to the clus- such as overcast weather, during ripening can ters. Global climate change is expected to be be detrimental to the flavor quality of the fruit. associated with higher temperature increases Accordingly, the sensory perception in wine of at night than during the day (Intergovernmen- “fruitiness” was positively correlated and that tal Panel on Climate Change, 2007). Indeed, of “vegetative/asparagus/green pepper”’ was on a global scale the average growing season found to be negatively correlated with the temperature for individual wine regions has 6.3. SOURCES OF VARIATION IN FRUIT COMPOSITION 215 been found to correlate positively with average 2001; Smart and Sinclair, 1976; Spayd et al., wine vintage ratings, with a clear temperature 2002; Tarara et al., 2008). Berries facing the sun optimum that varies by region and wine style on a sun-exposed cluster are also heated much (Jones et al., 2005). more than the nonexposed berries on the same The long-distance transport of assimilates in cluster (Kliewer and Lider, 1968). In addition to the phloem is relatively insensitive to tempera- shade, increasing wind speed also reduces the ture but is inhibited by prolonged periods difference between skin and ambient tempera- above 40C, probably due to temporary block- ture (i.e., wind has a cooling effect, at least age of sieve plate pores by callose. Heat sum- during the day) due to the influence of wind mation (usually expressed as growing degree on convective heat loss from the berries. More- days; see Chapter 2.2) has been found to be a over, berries on loose clusters are heated less very good predictor for fruit maturity, espe- than those on tight or compact clusters because cially if a lower temperature threshold of 18C berries that touch one another conduct more is applied (rather than the 10C threshold gen- heat and lose less to convection (Smart and Sin- erally used in grapevine growth models). Tem- clair, 1976). A viticultural strategy employed to perature, in addition to seasonal rainfall, is diminish cluster compactness is the practice thought to be the main factor driving fluctua- of cutting through inflorescences at bloom, tions in grape quality between years; sugars which results in compensatory stretching of may accumulate most rapidly in the tempera- the remaining portion of the cluster but also ture range 22–28 C, provided soil moisture diminishes yield. Another strategy, used and other factors are not limiting (Hofa¨cker mainly for some table grape cultivars (e.g., et al., 1976). Nevertheless, the link between tem- Thompson Seedless and Flame Seedless), is perature and fruit quality is not straightfor- the application of gibberellin sprays at bloom, ward, in part because the temperature of which leads to decreased fruit set (“berry individual berries is also important. In fact, thinning”) and elongation of the rachis and photosynthesis is less restricted by low tem- also increased berry size (Currle et al., 1983; peratures than are most other plant processes, Williams, 1996). The quality of table grapes so lack of sugar supply is not the primary rea- may be negatively impacted by excessive son for the slow ripening during unseasonably temperature due to discoloration and fruit cool periods or the slower sugar accumulation shriveling (Mullins et al., 1992). in cool compared with warm regions; sink Vineyard row direction has a major influence activity and its direct control by temperature on heating of exposed fruit (Figure 6.7). In east– seem to be much more important. However, west-oriented rows, the berries on the south grape sugars appear to accumulate most rap- side of the canopy (north side in the Southern idly in the ambient temperature range from Hemisphere) can be at higher than optimal approximately 20 to 30 C, provided soil mois- temperature during much of the day (Bergqvist ture and other factors are not limiting et al., 2001). In north–south-oriented rows, (Hofa¨cker et al., 1976; Kliewer, 1973). berries on the west side can be considerably Berries in different positions in a canopy warmer than those on the east side because experience different temperatures. The surface ambient temperatures are generally highest after of green berries exposed to full sunlight can solar noon (Reynolds et al., 1986; Spayd et al., be 12 C warmer than the surrounding air, and 2002). Strong solar heating of berries may be that of dark-colored berries can be as much as advantageous in cool climates but may hinder 17 C, whereas shaded berries are usually close fruit ripening in warm climates, so the practice to air (ambient) temperature (Bergqvist et al., of removing leaves in the fruit zone may have 216 6. DEVELOPMENTAL PHYSIOLOGY

berries than in exposed berries. Nevertheless, due to the pronounced temperature difference between shaded and sun-exposed berries, the latter tend to have much lower amounts of malate, albeit not tartrate, and higher pH at maturity (Kliewer and Lider, 1968; Reynolds et al., 1986). The temperature optimum for pre- veraison malate accumulation in immature berries is 20–25C, and there is a sharp drop in accumulation above approximately 40C (Kliewer, 1964; Lakso and Kliewer, 1975, 1978). As a consequence, grapes grown in warm climates or warm seasons may have higher acidity at the beginning of ripening compared with grapes grown in cooler climates or cool seasons, unless excessive sunlight exposure heats the berries above the optimum in the warm climate (Klenert et al., 1978). How- ever, acidity declines rapidly after veraison in warm-climate regions or warm growing sea- sons, whereas the decrease is much more grad- ual in cool-climate regions or cool growing FIGURE 6.7 seasons. Malate degradation also seems to start Sun-exposed grape berries may heat up et al considerably above the ambient temperature. Photo by earlier in warm conditions (Buttrose ., 1971; M. Keller. Ruffner, 1982b). Therefore, the concentration of organic acids at harvest is usually higher and the pH lower during cool growing seasons than to be applied judiciously depending on vine- during warm seasons, mainly because increas- yard location. Whereas fruit sugar content is ing temperature stimulates malate respiration relatively insensitive to temperature, the berry (Currle et al., 1983; Hofa¨cker et al. 1976; Jackson temperature affects metabolic processes that and Lombard, 1993; Klenert et al., 1978). convert sugars to the various acids and second- The French saying, “C’est l’aouˆt qui fait le ary substrates important for fruit quality and gouˆt” (“August makes the taste”), along with a that degrade (malic) acid. neat linguistic detail (French aouˆt ¼ August, Very high fruit temperature limits sugar aouˆte´ ¼ mature), points to the importance of accumulation in sun-exposed berries but may temperature conditions during veraison for have little effect on sugar concentration in high harvest fruit quality. Temperature is one shaded berries, which also seem to have less of the main climatic variables affected by the variation in sugar than their exposed counter- human-induced rise in the amount of atmo- parts (Kliewer and Lider, 1968). Conversely, spheric CO2. The forward shift of phenological although acidity in mature grape berries is also development resulting from current and future lower if the grapes ripened at higher tempera- climate change (see Chapter 2.3) may be of par- ture because of the marked impact of tempera- ticular importance because earlier veraison ture on malate respiration (Kliewer, 1973), this implies that the critical ripening period shifts effect is much more pronounced in shaded toward the hotter part of the growing season. 6.3. SOURCES OF VARIATION IN FRUIT COMPOSITION 217

That this has already occurred during the sec- temperature, and minimum coloration in ond half of the 20th century has been docu- shaded fruit at high temperature, but (daytime) mented in France, where the period between temperature, rather than light, appears to be budbreak and harvest has become shorter and the main driver of anthocyanin accumulation ripening is occurring under increasingly warm (Spayd et al., 2002; Tarara et al., 2008). Inciden- conditions (Ducheˆne and Schneider, 2005; Jones tally, cool autumn temperatures also lead to and Davis, 2000). Although this trend has been more intense coloration of the leaves, especially correlated with greater fruit sugar and lower in combination with high light that results in acid concentrations, and generally better wine energy overload in the leaves. This effect is par- quality, even warmer does not necessarily ticularly intense in grapevines infected with mean even better. In V. vinifera cultivars, antho- leafroll viruses. Unfortunately, the virus inter- cyanin production increases up to an optimum feres with grape ripening and leads to poor berry temperature of approximately 30 C but is fruit color (see Chapter 7.5). The formation of thought to be inhibited above 35 C (Kliewer, tannins in grape berries appears to increase 1977b; Kliewer and Torres, 1972; Spayd et al., with increasing temperature, whereas flavonol 2002), whereas the optimum seems to be some- production may be relatively unaffected by what lower in V. labrusca and its hybrids with temperature but instead is more responsive to V. vinifera (Yamane et al., 2006). Excessive tem- light (especially UV light). Although tempera- peratures, similar to excessive light, may also tures below approximately 20 C are thought induce oxidative stress, which could even lead to stimulate flavonol accumulation in plant to anthocyanin degradation (Mori et al., 2007). tissues, an effect that is apparently enhanced Consequently, if the air temperature is 25C by low nitrogen status (Olsen et al., 2009), this and berries are at 40C, color development will response has yet to be tested in grapes. be drastically impaired. This sensitivity to day- The concentration of amino acids, especially time temperature (night temperatures seem to proline and g-aminobutyric acid, rises with be much less important) at least partially increasing temperature, but arginine is less explains the poor skin color often observed responsive to temperature (Buttrose et al., in warm climates or in warm growing 1971; Kliewer, 1973). Thus, warm growing sea- seasons, especially when the grapes are directly sons may be associated not only with lower exposed to the sun (Haselgrove et al., 2000; acidity (especially malate) but also with higher Ortega-Regules et al., 2006a). Cultivars with amino acid contents (Pereira et al., 2006b). Sim- þ V. labrusca parentage appear to be especially ilarly, the K content also rises with tempera- sensitive to heat; pigmentation stalls at high ture, which in combination with reduced temperatures, which is why Concord produc- malate has the follow-on effect of higher juice tion for grape juice is mostly concentrated in pH. Sun-exposed berries are also subject to cool-climate areas. High temperature also leads sunburn or sunscald (dead, brown patches on to shifts in the relative amounts of individual the skin) and subsequent shriveling. Such dam- anthocyanins. Because the formation of malvi- age may be due to overheating (above 42C) din-based anthocyanins seems to be far less and excess UV and/or visible light leading to responsive to temperature than that of other oxidative stress (collectively termed photooxi- anthocyanins, the former become dominant in dative damage) to the epidermis and hypoder- grapes grown under hot conditions (Mori mis. The impact of temperature on aroma and et al., 2005; Ortega-Regules et al., 2006a; Tarara flavor compounds is not well understood and et al., 2008). Maximum skin coloration often probably varies depending on the chemical occurs in exposed fruit at cool ambient nature of the various volatile compounds and 218 6. DEVELOPMENTAL PHYSIOLOGY their precursors. For instance, methoxypyra- the response of individual grapevines to cli- zine concentrations are highest under very cool matic factors and has a pronounced impact on ripening conditions and, thus, in more north- vine-to-vine variation within a vineyard. In erly latitudes and/or higher altitudes, espe- fact, the variation in vigor and yield among cially in cool growing seasons. Even if grapes vines in the same vineyard is often closely from a warm and a cool region (or site) were related to the variation in plant-available water, harvested at the same concentration of soluble both in hot and in relatively cool climates solids (i.e., sugar), the “cool” grapes would still (Cortell et al., 2005; Hall et al., 2002; Lamb typically contain severalfold more methoxypyr- et al., 2004). However, the spatial variation in azines than their “warm” counterparts (Lacey fruit quality is not necessarily the same as the et al., 1991). The temperature prior to veraison spatial variation in yield. Water availability may be more important for methoxypyrazine influences shoot vigor and thus canopy micro- production (less at higher temperature) than is climate. Increasing soil moisture stimulates the effect of temperature on the degradation vigor, which can lead to a denser canopy and (more rapid at higher temperature) of these shaded fruit. A larger total leaf area also has a compounds after veraison. The accumulation greater water demand due to transpiration, of terpenoids, on the other hand, shows a which in turn increases the vine’s susceptibility relatively broad temperature optimum from to drought. approximately 10 to 20 C. Nonetheless, fruit Abundant water supply ordinarily delays monoterpene concentrations may be negatively veraison and slows the rate of fruit ripening. correlated with the average daily maximum Large, dense canopies that result from abun- temperature over the ripening period (Marais dant water and nutrient availability are asso- et al., 2001). Norisoprenoids such as b-damasce- ciated with reduced fruit sugar, high acidity, none or b-ionone appear to be relatively insen- and poor color (Dry and Loveys, 1998; Jackson sitive to temperature, although they may be and Lombard, 1993). Water deficit, in contrast, masked by elevated amounts of methoxypyra- typically reduces yield (see Chapter 7.2) and zines under cool conditions. can increase or decrease berry sugar content, acidity, pH, and color, depending on the extent 6.3.4. Water Status and timing of the deficit. Some degree of water deficit is normally regarded as beneficial for Water supply from both the soil and the fruit composition and wine quality. Grapes, atmosphere is very important for fruit compo- especially wine grapes, have been grown suc- sition due to its influence on vine growth, yield cessfully on rather marginal sites with infertile, formation, and fruit ripening. Most of the dif- shallow soils of low water-holding capacity ferences in grape quality caused by soil-related for many centuries, and controlled application differences in “terroir” may be attributable to of stress is the premise of modern deficit differences in soil moisture rather than, for irrigation strategies (Dry and Loveys, 1998; example, parent rock or soil type and composi- Dry et al., 2001; Keller, 2005; Kriedemann and tion (van Leeuwen et al., 2004). At a given vine- Goodwin, 2003; also see Chapter 7.2). yard site, soil moisture depends on the annual Mild to moderate stress generally seems to amount and temporal distribution of rainfall be most effective if applied during the first and on temperature-mediated evaporation. phase of rapid berry expansion by limiting Site-specific and local variation in soil moisture shoot and berry growth and canopy density due to differences in effective rootzone, water- (Dry et al., 2001). Considerable improvements holding capacity, and drainage can modulate in fruit composition may be attributable to 6.3. SOURCES OF VARIATION IN FRUIT COMPOSITION 219 indirect effects of smaller, more open canopies European regulations, can delay berry develop- and, sometimes, reduced crop load, but water ment and ripening because of a reduction in deficit may also have more direct effects on photosynthesis or, in extreme cases, leaf berry size and composition. When water deficit drop (Williams and Matthews, 1990; Williams is associated with lower canopy density, the et al., 1994). improved light penetration into the fruit zone Small berry size is less important for most may also raise berry temperature (Santos et al., white wine grapes than for red wine grapes. 2005). Whereas any plant water deficit almost Skin components are not usually extracted always limits berry size (see Chapter 7.2), its during white winemaking and are not nearly influence on sugar accumulation is generally as critical for the quality of non-wine grapes. less pronounced (Hofa¨cker et al., 1976; On the contrary, large rather than small berries Matthews and Anderson, 1988; Santesteban are typically desired for juice, table, and raisin and Royo, 2006; Stevens et al., 1995; Williams grapes. and Matthews, 1990). This apparent difference Soil moisture has little effect on tartrate might arise from a decrease in the ability of content per berry, although water deficit early berries growing under limited sugar supply to during berry development has been found to accumulate water so that the sugar concentra- limit tartrate accumulation (Eibach and tion, albeit not the amount per berry, remains Alleweldt, 1985). Malate tends to decline with constant. The same principle may apply to the a decrease in soil moisture so that the concen- þ accumulation of mineral nutrients such as K tration of titratable acidity is often lower at har- þ and Ca2 in the berries. Although the amount vest (Keller et al., 2008; Stevens et al., 1995). of these nutrients may be reduced under water Because ABA was found to inhibit enzymes deficit, the concentration in the juice may involved in malate respiration and gluconeo- remain unaffected due to the concomitant genesis (Palejwala et al., 1985), it is possible that decrease in berry size (Etchebarne et al., 2009; ABA mediates the response to water deficit in Keller et al., 2008). In addition, the decrease in ripening berries. Indeed, the amount of ABA berry size may be mostly due to a smaller in grape berries correlates positively with the mesocarp, whereas the weight of the skin and concentration of ABA in the xylem sap at all seeds seems to be less affected by water deficit stages of berry development (Antolı´n et al., (Roby and Matthews, 2004). Moreover, the 2003). Nevertheless, the reduction in malate decrease in photosynthesis and sugar export content is more pronounced when water deficit from the leaves under more severe stress can occurs before veraison than after veraison curtail berry sugar accumulation, especially if (Matthews and Anderson, 1988; Williams and the deficit occurs during ripening (Currle Matthews, 1990). The concentration of arginine et al., 1983; Dry et al., 2001; Hardie and is lower in berries of water-stressed vines, Considine, 1976; Matthews and Anderson, whereas proline may increase in some cases 1988; Peyrot des Gachons et al., 2005; Quick under water deficit (Coombe and Monk, 1979; et al., 1992; Rogiers et al., 2004b; Santesteban Matthews and Anderson, 1988). and Royo, 2006). Thus, although mild water The improved color often observed with deficit may increase sugar accumulation, limit mild to moderate water deficit, especially pre- berry size, and improve fruit composition veraison water deficit (Kennedy et al., 2002), is by restricting shoot growth or by reducing can- to some extent simply due to smaller berry size opy density (Kennedy et al., 2002; Ojeda et al., (see Chapter 7.2), which increases the skin:pulp 2002; van Leeuwen et al., 2004), more severe ratio. In addition, there is also a more direct stress, contrary to winemaking lore and influence of water deficit enhancing the 220 6. DEVELOPMENTAL PHYSIOLOGY production of anthocyanins (Dry et al., 2001), (Peyrot des Gachons et al., 2005). More severe probably via the stimulating effect of root- stress, however, seems to curtail this aroma derived ABA on the genes stipulating flavo- potential. This finding may be particularly rele- noid (especially anthocyanin) biosynthesis vant for white wine grapes, for which a high and, hence, on the activity of the corresponding aroma potential is much more desirable than a enzymes (Castellarin et al., 2007a,b; Jeong et al., high amount of potentially bitter phenolics. 2004). Accordingly, table grape growers some- Clearly, wine grape quality benefits from care- times apply ABA as a spray at or just before fully controlled irrigation in dry climates or veraison to boost red coloration of the fruit. dry growing seasons. Application of the synthetic compound ethe- phon also improves color formation (Szyjewicz 6.3.5. Nutrient Status et al., 1984), perhaps because its breakdown product ethylene also stimulates ABA produc- Of all mineral nutrients, nitrogen is the most tion. Water deficit may also shift the relative potent in its ability to influence vine growth, proportion of individual anthocyanins, favor- morphology, fruit production, and tissue com- ing the production of pigments with higher position (Bell and Henschke, 2005; Jackson degrees of hydroxylation and methoxylation, and Lombard, 1993). Increasing soil nitrogen notably malvidin- and petunidin-based antho- availability enhances photosynthesis, which cyanins (Castellarin et al., 2007a,b). On the means that more sugar is available for growth other hand, irrigation using partial rootzone and fruit ripening. Yet it is commonly accepted drying (see Chapter 7.2) may leave malvidin- that, just as with water, there can be too much based anthocyanins unchanged while enhanc- of a good thing. High nitrogen supply favors ing accumulation of the glucosides of the other vegetative growth, including growth of lateral four anthocyanins (Bindon et al., 2008). shoots (Keller and Koblet, 1995a; Keller et al., Severe water stress, especially before verai- 1998, 1999, 2001b), which can result in dense son, can lead to uneven ripening with some canopies associated with reduced fruit sugar, berries remaining green or coloring poorly. high acidity, and poor color. Growing shoot The accumulation of tannins, flavonols, and tips may also compete with the clusters for sup- cinnamic acids, on the other hand, appears to ply of assimilates (Sartorius, 1973), and fruit be quite insensitive to changes in plant water ripening is delayed (Spayd et al., 1994). In addi- status, both before and after veraison (Downey tion to this indirect influence on fruit composi- et al., 2006), unless water deficit enhances fruit tion, the effects of nitrogen can also be more exposure to sunlight. Carotenoid concentration direct because nitrate uptake leads to a repro- is often lower in grapes grown at low soil mois- gramming of the expression of many genes ture; much of this effect can probably be attrib- involved in metabolism (Scheible et al., 2004). uted to higher sun exposure due to the reduced Thus, high nitrogen status stimulates the pro- canopy density, which accelerates carotenoid duction but not the respiration of organic acids degradation. However, it is unclear whether including malate, which is used as a countera- this is associated with an increase in conversion nion that substitutes for nitrate to prevent of carotenoids to aroma compounds such as tissue alkalinization (Scheible et al., 2004; Stitt norisoprenoids. Nevertheless, mild water defi- et al., 2002). Nevertheless, although this is cit is thought to enhance the grapes’ aroma sometimes associated with higher malate con- potential, for instance, by increasing the centrations in grape berries, juice from berries amount of volatile thiol precursors, irrespective grown on a high nitrogen “diet” still tends to of the influence of water status on berry size have a higher pH, possibly because high rates 6.3. SOURCES OF VARIATION IN FRUIT COMPOSITION 221

of nitrate (NO3 ) uptake by the roots are often shoot growth during the growing season may þ associated with high K uptake (Keller et al., only make matters worse because it wastes 1999; Ruhl et al., 1992; Spayd et al., 1994). More- the vine’s resources and eliminates young, over, carbon skeletons are necessary to assimi- photosynthetically active leaves while leaving late nitrate into amino acids. Increased old, inefficient leaves behind (Coombe, 1959; formation of organic acids and amino acids in Keller et al., 1999). Moreover, the generation turn diverts carbon away from sugar produc- of multiple new shoot apical meristems by tion and inhibits the formation of phenolic (repeated) stimulation of lateral shoot growth components, such as anthocyanins, tannins, fla- due to the elimination of apical dominance vonols, and hydroxycinnamic acids. Even more may draw resources away from the clusters directly, whereas nitrogen deficiency induces and the storage pool in the permanent organs enzymes of the shikimate pathway (Weaver of the vine. and Herrmann, 1997), nitrate supply sup- High soil nitrogen also inhibits root growth, presses the expression of genes involved in which could make vines more vulnerable to phenolics production (Fritz et al., 2006; Olsen water stress in subsequent seasons—a consid- et al., 2009; Scheible et al., 2004). In other words, eration that is especially important during abundant nitrate keeps these genes “switched establishment of young vines. Nitrogen defi- off” or, if they were “on” before nitrate supply, ciency, on the other hand, generally reduces it switches them off, which does not allow them fruit set and bud fruitfulness, leading to loss to design and build the necessary enzymes. of yield potential. Despite the low yield, how- In contrast, carotenoid accumulation and ever, fruit sugar concentration may be inade- terpenoid release are increased by high nitro- quate because there is insufficient nitrogen gen status, but it is not known whether this available for efficient photosynthesis (Keller enhances or suppresses isoprenoid aroma et al., 1998). Whereas soil nitrogen status appar- production and accumulation. Nevertheless, ently has little effect on berry tartrate, malate moderate nitrogen availability is thought to concentration tends to be lower at low nitrogen maximize the grapes’ flavor and aroma poten- availability, but this response may be modified tial, whereas both nitrogen deficiency and by other environmental variables (Keller et al., nitrogen excess ostensibly diminish the aroma 1998; Spayd et al., 1994). potential (Peyrot des Gachons et al., 2005). A typical reaction of plants to low nitrogen The shade problem created by excessive status is the accumulation of phenolic com- nitrogen supply cannot be overcome by the pounds. It appears that the stimulation of phe- popular canopy management technique of leaf nolics production is a response to low nitrate removal in the cluster zone in an attempt to content rather than low amino acid status. improve fruit exposure to sunlight because the Therefore, pigmentation of red grapes during high nitrogen and low flavonol and carotenoid veraison is maximized at low to moderate (e.g., xanthophylls) contents in the berries make nitrogen availability, and it is minimized when them more susceptible to sunburn (Du¨ ring high vine nitrogen status, such as due to earlier and Davtyan, 2002; Kolb et al., 2003). Another fertilizer applications, coincides with overcast common “Band-Aid” action is to tip or hedge conditions (Hilbert et al., 2003; Keller and (i.e., cut off) the excess shoot tips, which can Hrazdina, 1998; Kliewer and Torres, 1972). This temporarily improve assimilate supply to the nitrogen–light interaction affects not only total fruit clusters and eliminate shade from over- grape color but also the distribution of individ- hanging shoots. However, repeated hedging ual anthocyanins. It seems that conditions on fertile sites or in areas that permit continued favoring color accumulation also lead to the 222 6. DEVELOPMENTAL PHYSIOLOGY most balanced distribution of pigments (Keller other amino acids may be somewhat less and Hrazdina, 1998). Because the formation of responsive to nitrogen status (Bell and malvidin-based anthocyanins is less susceptible Henschke, 2005; Kliewer and Torres, 1972; to stress conditions than that of other antho- Spayd et al., 1994). Thus, yeast-assimilable cyanins, the former can become dominant in nitrogen increases strongly as soil nitrogen grapes grown with excessive nitrogen, particu- status increases; nitrogen supply during ripen- larly in combination with poor light or exces- ing is more effective at raising fruit nitrogen sive heat. Under nonlimiting light conditions, status than is nitrogen supply earlier in the and in contrast to the situation with water defi- season (Rodriguez-Lovelle and Gaudille`re, cit, low nitrogen status favors the production of 2002). This has implications for winemaking: pigments with lesser degrees of hydroxylation Because yeast cells cannot metabolize proline, and methoxylation (i.e., cyanidin- and peoni- nitrogen-deficient grapes often lead to sluggish din-based anthocyanins). Vine nitrogen status or stuck fermentations (Bell and Henschke, therefore has a direct influence on the produc- 2005; Spayd et al., 1995). Such fermentations tion of individual pigments in the grape skin can also be associated with malate production in addition to the indirect effect brought by yeast and often result in the formation of about by its influence on vigor and fruit set. H2S (which is perceived as a “reduced” smell Moreover, low nitrogen status also favors the at low concentration but worsens to a smell of accumulation of flavonols in preveraison rotten eggs at high concentration) from SO2 berries (Keller and Hrazdina, 1998). and S-containing amino acids (cysteine and Nitrogen supply influences the transport of methionine) and of acetaldehyde and acetic nitrate and amino acids to the berries and acid (i.e., higher volatile acidity, which also amino acid production and accumulation increases at very high nitrogen concentration). inside the berries (Bell and Henschke, 2005). It Moreover, low plant nitrogen status, especially has been estimated that approximately two- in combination with water stress, is suspected thirds of the berries’ total nitrogen is derived to be one reason for premature aging of white from the leaves after veraison, and that the wines, although increasing nitrogen fertilizer crop’s demand for nitrogen is mainly a func- application has sometimes exacerbated the tion of the number of berries per vine (Treeby problem (Linsenmeier et al., 2007). This phe- and Wheatley, 2006). Because nitrate cannot nomenon is termed atypical aging and is asso- be transported in the phloem, all of the leaf- ciated with the development of an off-odor derived nitrogen would have to be in the form reminiscent of “acacia flower,” “naphthalene,” of amino acids (see Chapter 5.3). Considering or “floor polish” at the expense of varietal that fruit nitrogen at harvest correlates posi- aroma. A threshold of approximately 150 mg tively with leaf nitrogen at veraison NL1 of yeast-assimilable nitrogen (which (Holzapfel and Treeby, 2007), increasing the includes a number of other amino acids and nitrogen status of the leaves by veraison may ammonia in addition to arginine) seems to be be a possible strategy to enhance the nitrogen required for grapes to readily complete fermen- content of the berries by harvest. tation (i.e., to <2gL 1 of residual sugar) (Bell Arginine accumulation in grapevines is and Henschke, 2005). Higher concentrations of restricted at low soil nitrogen availability but amino acids in the berries often result in higher reacts strongly to nitrogen supply (Kliewer concentrations of fruity and floral esters (e.g., and Cook, 1971). Thus, arginine increasingly ethyl acetate, ethyl butyrate, and other similar accumulates in the berries as nitrogen supply compounds) and thiols and in lower concen- rises, whereas the production of proline and trations of higher alcohols (so-called fusel 6.3. SOURCES OF VARIATION IN FRUIT COMPOSITION 223 alcohols, such as propanol, butanol, hexanol, other factor that inflicts physical injury to cell and related compounds) in wine, which typi- membranes (e.g., insect feeding or fungal cally enhances the wine’s sensory properties. infections) or due to senescence if grapes are On the other hand, high concentrations of left on the vine too long. In other words, nitrogen in grape juice often result in the yeast increasing K supply may increase berry K con- producing more SO2 during fermentation. centration and tends to increase the juice pH, Because SO2 inhibits bacterial growth, malolac- and consequently the wine pH (Jackson and tic fermentation by lactic acid bacteria (O. oeni) Lombard, 1993; Mpelasoka et al., 2003), rather in such wines can be difficult to achieve. When than the grape pH. Incorporation into the soil grapes from vines with high nitrogen status are of excessive rates of K fertilizer (up to 900 kg dried during raisin production, they tend to Kha1 for 5 years) may sometimes (Morris turn dark brown due to the involvement of et al., 1980) but not always (Morris et al., arginine in Maillard reactions producing 1987) lead to a higher juice pH. One reason pigmented melanoidins from sugars. Grape for this inconsistency might be that higher K proteins (e.g., PR proteins) also increase at high status is often associated with higher berry plant nitrogen status, which can increase the malate concentrations (Hale, 1977; Hepner requirement for bentonite fining in the result- and Bravdo, 1985; Ruhl et al., 1992). Nonethe- ing wines (Spayd et al., 1994). less, the presence of a high amount of K in Indirect effects of high plant nitrogen status grape juice or wine may lead to precipitation include heightened susceptibility to fungal dis- of K tartrate during storage (Morris et al., eases, such as Botrytis bunch rot and powdery 1980). In contrast, high soil Mg or Ca may mildew (both partly related to vigor and can- decrease K uptake by the roots and sometimes opy density), which may be detrimental to lead to lower juice and wine pH. The effect of grape and wine quality (Bell and Henschke, high Ca and Mg availability on juice pH 2005; Christensen et al., 1994; Valde´s-Go´mez appears to be enhanced by a concomitant et al., 2008; see also Chapter 7.5). The optimum increase in grape tartrate and malate content, availability of soil nitrate to supply the vine’s although the reasons for this increase are demands and satisfy fruit quality requirements unclear. Therefore, contrary to intuition, high depends on cultivar, intended use of the grapes, soil pH, which is associated with high soil Ca and climatic conditions. In a warm, dry, sunny status, may be coupled with low rather than climate or growing season, the optimum nitro- high juice and wine pH. gen supply may be higher than that under cool, Little is known of the influence of nutrients humid, and cloudy conditions. Nitrogen avail- other than N on the accumulation of phenolic ability also depends on soil moisture because components and flavor volatiles in grape the roots can take up only nitrogen ions berries. The stimulation of anthocyanin pro- dissolved in the soil water (see Chapter 5.3). duction in leaves of K- and P-deficient plants þ Although it is often stated that abundant K (see Chapter 7.3) suggests that low nutrient sta- availability in the soil can increase the pH of tus in general might also enhance the produc- grape berries (due to enhanced K uptake and tion of phenolics in the fruit. transport to the berries), it is more likely that the pH only increases after cellular compart- 6.3.6. Crop Load mentation breaks down (due to disruption of cell membranes) during crushing or pressing. The classical view of the relationship Of course, compartmentation can also break between grape yield and quality is that of a down due to freezing temperatures or any linear decrease in quality with increasing yield 224 6. DEVELOPMENTAL PHYSIOLOGY per vine (Currle et al., 1983). However, this is As a general rule, a leaf area of 10–15 cm2 is an oversimplification, and there are many required to fully ripen 1 g of fruit, and this nor- instances in which the quantity and the quality mally results in a yield:pruning weight ratio of of the crop are not related (Hofa¨cker et al. 1976; approximately 5–10 (Kliewer and Dokoozlian, Keller et al., 2005, 2008) or are increased simul- 2005). If the crop load is lower than this (i.e., taneously (Chapman et al., 2004b). In a study undercropping or sink limitation), then the vine conducted over 4 years with 80 individual will invest comparatively more resources in veg- Riesling vines, the between-vine variation in etative growth, which can, in extreme cases, yield was approximately fivefold higher than reduce fruit quality via the follow-on effects of that in either juice soluble solids or titratable a dense canopy. Moreover, berry size may acidity (Geisler and Staab, 1958). A meta- increase to compensate for the low number of analysis of Riesling clonal trials conducted at berries relative to leaf area (Keller et al., 2008; 16 locations over 37 years found that yield Santesteban and Royo, 2006). Conversely, if and soluble solids both increased over time, there is insufficient leaf area to ripen the fruit whereas titratable acidity decreased (Laidig (i.e., high crop load, overcropping, or source et al., 2009). Much of the variation in yield limitation), then the rate of ripening will decline. was attributed to site effects, whereas the This is why vines of medium vigor often pro- change in fruit composition was clearly linked duce both higher yields and better-quality fruit to the rise in average temperature during the than vines at either end of the vigor spectrum. same period. Although yields and mean tem- Such vines are said to be balanced—that is, their peratures continued to vary considerably from crop size matches their vegetative growth and year to year, the variation in fruit composition leaf area development. Furthermore, the leaf declined over time, indicating that composi- area required to ripen 1 g of fruit also depends tion was not greatly affected by yield and that on the trellis and training system; it appears to improved vineyard management contributed be considerably higher in a typical vertically to the change in fruit composition. shoot-positioned canopy than in vines that are It is not so much the crop size or yield per sprawl trained or trained to divided canopies. se that is important but, rather, the crop load, Overcropping ordinarily delays fruit matu- which is a reflection of a vine’s sink:source ration and therefore decreases grape sugar ratio (Bravdo et al., 1984; Jackson and and color if harvest cannot be delayed (Bravdo Lombard, 1993; Santesteban and Royo, 2006; et al., 1984; Weaver et al., 1957; Williams, 1996; see also Chapter 6.1). For instance, an increase Winkler, 1954). However, the effect of crop in vine spacing is typically associated with a load on berry composition depends on how a higher yield per vine, but vine size, and thus difference in crop load is achieved. An out- the leaf area per vine, also increases, so break of pests or diseases, or a hail storm, fruit composition and wine quality may be can reduce photosynthetically active leaf area completely unaffected (Winkler, 1969). Con- after the yield potential has been established, versely, when a higher planting density, which results in reduced sugar accumulation. which is associated with lower yields per vine, If an increase in yield is accompanied by dete- leads to competition among shoots for rioration in the canopy microclimate, then the sunlight, the fruit may have higher titratable changes in fruit and wine composition are acidity while at the same time the juice pH negative (Reynolds et al., 1994b). In other is also higher (Falcetti and Scienza, 1989), words, so-called overcropping effects are often possibly due to recirculation of potassium actually shade effects caused by poor pruning from shaded, aging leaves to the clusters. practices or other vineyard management 6.3. SOURCES OF VARIATION IN FRUIT COMPOSITION 225 errors. For example, if pruning is too light (i.e., and enhance ripening. This can be especially toomanybudsleft),thentheremaybetoo beneficial in cultivars that are prone to over- many shoots, which leads to dense canopies. cropping due to their large clusters, such as Alternative, if pruning is too severe (i.e., too Syrah, Mourve`dre, Grenache, or Zinfandel. fewbudsleft),thenthefewremainingshoots One form of cluster thinning is cutting away may grow too vigorously and produce too the distal one-third or half of flower clusters many laterals, which leads to shade in the during bloom, which not only decreases yield fruiting zone. In contrast, where an increase but also often leads to less compact clusters in yield is accompanied by an improvement due to the compensatory stretching of the in the canopy microclimate, berry composition rachis. It is possible that the advanced maturity may also be improved. For example, the con- observed following such early thinning may be centration of undesirable methoxypyrazines attributed less to the smaller crop than to the in grapes grown on high-yielding minimally tendency for berries in the proximal portion of pruned vines can be dramatically reduced a cluster to ripen more rapidly than those compared with lower yielding spur-pruned in the distal portion (Weaver and Ibrahim, vines. Indeed, when yields are increased by 1968). In table grapes, where large berries are decreasing the pruning level (i.e., leaving desirable, cluster thinning is sometimes more buds), the resulting wines are often fruit- supplemented by berry thinning. ier and less vegetative, perhaps due to the The size of individual berries is often more reduced shoot vigor (Chapman et al., 2004a,b; important than the crop level or even crop load see also Chapter 6.1). If, on the other hand, in determining fruit composition. For instance, yields are stimulated by abundant water sup- large increases in berry size after veraison ply, the result is usually the opposite. may be coupled with concomitant increases Therefore, balancing shoot growth and fruit in berry sugar and K due to phloem import production is an important viticultural goal. (Rogiers et al., 2006). The concentration of K in For an individual vine, the crop load/fruit the berries decreases as the crop load increases quality relationship generally follows an opti- (Hepner and Bravdo, 1985). However, because mum curve with increasing quality as crop tartrate synthesis inside the berry ceases load is increased from a very low level, fol- at veraison, an increase in berry size can lead lowed by a plateau, and finally a reduction in to a substantial decrease in tartrate due to a quality when crop load is further increased. “dilution effect.” Of course, more K and Under changing external conditions (including less tartrate in the berries will result in a cultural practices), this curve can be shifted corresponding increase in juice and wine pH. upward or downward. Rather than setting a In addition, larger berries have a relatively specific, inflexible target yield, economically smaller skin:pulp ratio, which has implications minded vineyard managers aim to achieve the for red wine composition and quality due to highest possible yield without compromising the importance of the extraction of skin-derived quality. Moreover, where weather conditions compounds (mainly anthocyanins, tannins, and permit, delayed ripening can sometimes be flavonols) during fermentation. In contrast to compensated by delayed harvest. grapes used for (red) winemaking, large size On overcropped vines, cluster thinning is and crispness are important quality traits of typically employed to reduce the crop load table grapes. CHAPTER 7

Environmental Constraints and Stress Physiology

OUTLINE

7.1. Responses to Stress 227 7.4.2. Cold Acclimation and Freeze Damage 276 7.2. Water: Too Much or Too Little 231 7.4.3. Heat Acclimation and Damage 286 7.3. Nutrients: Deficiency and Excess 243 7.5. Living with Other Organisms: 7.3.1. Macronutrients 249 Defense and Damage 289 7.3.2. Transition Metals and 7.5.1. Bunch Rot 298 Micronutrients 262 7.5.2. Powdery Mildew 302 7.3.3. Salinity 270 7.5.3. Downy Mildew 304 7.4. Temperature: Too Cold or 7.5.4. Bacteria 306 Too Warm 274 7.5.5. Viruses 308 7.4.1. Chilling Stress 274

7.1. RESPONSES TO STRESS water surplus or deficit, and nutrient deficiency or toxicity) or biotic (e.g., pest or disease attack). Grapevines, like other plants, require just A stress limits either the availability of one or three categories of resources to grow and pro- several resources to the plant or the plant’s duce fruit: carbon, water, and nutrients (Bloom ability to put these resources to use. Although et al., 1985). Nonetheless, they are often exposed there is a general perception that “stressing” to suboptimal growing conditions called envi- grapevines in the vineyard will improve fruit ronmental stress. Such stresses can be abiotic quality, stresses adversely affect plant growth, (e.g., overcast or too bright sky, heat or cold, development, or productivity. However, the

The Science of Grapevines 227 Copyright # 2010 Markus Keller. Published by Elsevier Inc. All rights reserved. 228 7. ENVIRONMENTAL CONSTRAINTS AND STRESS PHYSIOLOGY environment per se does not constitute abiotic because they determine which sink is preferen- stress; external conditions are neutral. It is the tially supplied with the remaining resources ability of plant mechanisms to function in a par- and which ones are abandoned in order to guar- ticular set of environmental conditions that antee the survival of the plant (Geiger and Ser- determines whether these conditions are “stress- vaites, 1991). Thus, many plant reactions to ful.” Depending on the severity and duration of stress involve morphogenic responses that are a stress event and on the rate at which it starts characterized less by an overall cessation of and ends, stress can trigger acclimation pro- growth and more by a redirection of growth that cesses in the entire vine. Shoot and root apical entails inhibition of cell expansion, local stimula- meristems give vines the flexibility to integrate tion of cell division, and changes in cell differen- developmental decisions with continuous tiation (Potters et al., 2007, 2009). Plant hormones changes in the environment. Their high degree are important mediators of these responses, and of developmental and morphogenic plasticity the various organs of a grapevine respond differ- enables grapevines to maximize their photo- ently to hormones. synthetic efficiency, long-term survival, and The ratio of auxin to cytokinin, rather than reproductive potential. Such plasticity is charac- their absolute concentrations, may determine teristic of plants that evolved in resource-rich which organs are to be favored during a partic- environments (Bloom et al., 1985). ular stress, and tissue auxin and cytokinin con- The optimum resource allocation hypothesis centrations are usually inversely correlated. For holds that plants respond to insufficient example, a low auxin:cytokinin ratio stimulates resource availability by investing biomass in shoot growth over root growth, whereas a high those organs and processes that enhance the auxin:cytokinin ratio favors (lateral) root acquisition of the resource that most strongly growth and results in shorter shoot internodes limits growth, often at the expense of investment and smaller leaves—but probably stronger api- in plant parts that have a large requirement of cal dominance. In addition, auxin also appears that resource (Bloom et al., 1985; Poorter and to suppress cytokinin biosynthesis and pro- Nagel, 2000). In general, plants exposed to car- mote gibberellin and ethylene biosynthesis, bon limitation (e.g., due to cloudy weather) but ethylene interferes with auxin transport often increase partitioning to the shoots (espe- (Woodward and Bartel, 2005). Whereas auxin cially the leaves), whereas plants exposed to produced by the expanding leaves near the nutrient deficiency typically increase root shoot tip and transported basipetally from cell growth or at least limit shoot growth more than to cell and in the phloem inhibits lateral shoot root growth (Chapin, 1991; Keller and Koblet, growth, a local increase in shoot-derived auxin 1995a). Consequently, if a nutrient deficiency is in the roots prompts pericycle cells to resume relieved by fertilizer applications, the vine may cell division and produce lateral and adventi- respond by shifting its carbon investment to tious roots while at the same time inhibiting favor shoot growth (to expand the leaf area in root elongation. Conversely, cytokinins pro- order to enhance the now more limiting carbon duced in the root tips and transported acrope- acquisition) at the expense of the roots. The same tally in the xylem inhibit root growth but response would be expected when irrigation activate dormant lateral buds and stimulate lat- water is applied to relieve drought stress eral shoot growth, thus counteracting apical (provided nutrients are not limiting). Moreover, dominance. Cytokinins apparently can also be the number and size of sinks competing for car- produced in very young, expanding leaves, bon during stress periods and their develop- probably by the dividing cells (Nordstro¨m mental stage or relative priority are important et al., 2004), and in general increase sink 7.1. RESPONSES TO STRESS 229 strength by promoting cell division and differ- consequence of the effects of primary stresses, entiation (except in roots, where they inhibit namely osmotic and ionic stresses. Such oxida- cell division) and delay senescence. The ability tive stress can result from an inability to use of auxin to promote root formation and that of photosynthetic energy because light capture cytokinin to induce shoot formation are used proceeds while carbon fixation declines so that to regenerate plants from undifferentiated cal- stresses that curtail photosynthesis typically lus in tissue culture. In addition, the stress hor- increase the leaves’ susceptibility to bright mone abscisic acid (ABA) normally inhibits light. The excess energy then interacts with growth by inhibiting production of the cell oxygen to form so-called reactive (or active) elongation hormone gibberellin and by stiffen- oxygen species. Whereas molecular (atmo- ing the cell walls, although at low concentra- spheric) oxygen (O2) is rather inactive (if this tion ABA may instead activate growth (del were not so, we would all burst into flames), Pozo et al., 2005; Hartweck, 2008). most of the aptly named reactive oxygen spe- Various stresses (e.g., drought, nutrient sur- cies are highly reactive. They are formed as plus, salinity, and chilling) cause cell dehydra- intermediates and by-products of the reduction tion and hence osmotic stress. One universal of O2 to water (H2O), in which electrons (e ) mechanism by which plants cope with this are added in a stepwise manner. This can be challenge is the accumulation of so-called com- written in simplified form as follows [modified patible solutes inside their cells (Bohnert et al., from Lane (2002) and Apel and Hirt (2004)]: 1995; Bray, 1997; Zhu, 2002). Compatible þ ! þ þ þ ! þ solutes are relatively small (i.e., low molecular O2 e O2 e 2H H2O2 e þ þ þ ! weight), hydrophilic (i.e., highly soluble), stable ! OH e H H2O organic compounds that cannot be easily meta- bolized and do not disrupt cell functions. These The reactive agents of decay include free • compounds include sugars (mainly sucrose radicals (e.g., superoxide, O2 , hydroxyl radi- • 1 and fructose), sugar alcohols (e.g., mannitol or cal, OH, singlet oxygen, and O2) and some- glycerol), and amino acids (e.g., proline), and what less unstable nonradicals (e.g., hydrogen they probably serve to reduce the cells’ osmotic peroxide; H2O2). The production in the photo- C potential ( p). This osmotic adjustment sup- synthetic chloroplasts of O2 and its associated ports continued water uptake or prevents dangers are discussed in Chapter 4.1. In the excessive water loss and helps plant tissues to mitochondria, the reduction of O2 to H2Ois maintain a higher water potential (C). Compat- carried out by the enzyme cytochrome oxidase ible solutes also act as osmoprotectants, pro- as part of ATP generation (see Chapter 4.4) tecting enzymes and membranes from osmotic without releasing any reactive oxygen species, stress (and thereby stabilizing them), and also but other respiratory reactions are not so seem to play a role in protecting the tissues “electron-tight.” Thus, reactive oxygen species against oxidative stress. are normal by-products of photosynthesis and, When grapevines are exposed to drought, to a small extent, respiration (Halliwell, 2006; cold or heat, intense light, nutrient deficiency, Mller et al., 2007; Noctor and Foyer, 1998). • salinity, and other environmental stresses (i.e., Plants also use H2O2 and O2 as key compo- abiotic stress), as well as attack by pathogens nents of the cascade of signals involved in the (i.e., biotic stress), they generally suffer from adaptation to changing environments (Foyer oxygen toxicity, termed oxidative stress and Noctor, 2005; Yang and Poovaiah, 2002). (Apel and Hirt, 2004). Oxidative stress nor- However, the formation of reactive oxygen mally is a secondary stress that develops as a species increases under stress, which can lead 230 7. ENVIRONMENTAL CONSTRAINTS AND STRESS PHYSIOLOGY to oxygen toxicity (Apel and Hirt, 2004; Mittler, acid (commonly known as vitamin C) and then 2002). In contrast to the comparatively “lazy” recovering (reducing) its oxidized form, partly O2, these highly reactive molecules crave elec- with the help of the peptide glutathione. Chem- trons because their oxygen atom is just one ically, oxidation typically means loss of an short of filling its outer shell. Consequently, electron from a compound, whereas reduction they damage and, at excessive concentrations, means gain of an electron so that the com- even kill cells by rapidly oxidizing (i.e., taking pound’s charge is reduced; reactions involving away electrons from) the cells’ components reduction and oxidation are termed redox reac- such as membrane lipids, proteins, cell wall tions. The so-called redox state of a cell or tis- polysaccharides, and DNA (Lane, 2002; Mller sue is often estimated by the proportion of et al., 2007). Oxidative damage to DNA is a reduced ascorbate and/or glutathione relative major cause of mutations, whereas damaged to their total amount (Potters et al., 2009). Fur- membranes become leaky and damaged pro- thermore, the production of carotenoids and teins (e.g., enzymes and transporters) are inac- flavonoids (e.g., anthocyanins) in addition to tivated. In leaves, it appears that regardless of compatible solutes and stress proteins also the initial source and type of reactive oxygen contributes to the detoxification process by species, they all ultimately result in over- scavenging active oxygen species or preventing 1 production of O2 by excess light energy, which them from damaging the cells’ structures. oxidizes membrane lipids and triggers the pro- Flavonoids may be oxidized and hence cesses culminating in cell death (Triantaphy- degraded by peroxidase, thereby consuming et al et al et al lide`s ., 2008). Therefore, failure to quickly H2O2 (Pe´rez ., 2002; Ros Barcelo´ ., dispose of (scavenge) the reactive culprits leads 2003). Oxidative stress, therefore, results from to inhibition of photosynthesis and develop- an imbalance between production and degra- ment of chlorotic and necrotic leaves. Note that dation of reactive oxygen species. • the herbicide paraquat acts by inducing O2 Although many stresses reduce the leaves’ and H2O2 production (Halliwell, 2006). photosynthetic rate or the total photosynthetic To cope with oxygen toxicity, plants have leaf area, the causes of these decreases and the evolved a suite of antioxidant defense systems impact of a stress factor vary with the type of designed to prevent oxidative damage by stress. For instance, whereas low nutrient avail- detoxifying active oxygen species (Apel and ability decreases photosynthetic rates in the Hirt, 2004; Halliwell, 2006; Lane, 2002; Mittler, leaves, this decrease is not as great as and starts 2002; Noctor and Foyer, 1998). These defense later than the reduction in growth (Chapin, systems employ antioxidants, which are 1991). This is because nutrients are needed to defined as compounds that “consume” or produce building blocks (e.g., proteins) for the quench active oxygen species without them- addition of new biomass by the vine’s sinks, selves being converted to damaging radicals whereas the photosynthetic apparatus in the in the process. Several of these systems use source leaves is already established. Therefore, the amino acid cysteine, which is a component although vines grown under conditions of of proteins and peptides and is easily and nutrient deficiency show diminished growth, reversibly oxidized (Mller et al., 2007). One they experience a relative excess of photosyn- system employs the enzymes superoxide dis- thate rather than a shortage, and they accumu- mutase and catalase to convert superoxide late sugar and starch in their leaves. Low water into water. Another, the so-called ascorbate– availability also decreases photosynthesis, but glutathione cycle, turns H2O2 into H2Oby this does not result in starch accumulation in allowing it to oxidize the antioxidant ascorbic the leaves because the decrease in growth due 7.2. WATER: TOO MUCH OR TOO LITTLE 231 to water deficit approximately matches the of seed development (but not berries with decrease in photosynthesis (Poorter and Nagel, mature seeds), are usually protected from shed- 2000). ding by their high auxin levels. However, the Eventually, a stress may induce a depletion of original trigger (i.e., initiating signal) and par- assimilates available for export. Carbon deple- tial executioner of the senescence program tion in turn stimulates processes involved in again appears to be oxidative stress, namely 1 et al photosynthesis and reserve mobilization (“fam- in the form of O2 (Triantaphylide`s ., 2008). ine” response), whereas abundant availability Different tissues within the same organ do of carbon favors carbon utilization, export, and not die at the same time. In senescing leaves, storage (“feast” response) (Hellmann et al., the mesophyll cells are the first to be disas- 2000). When they are starved for sugar, plant sembled, followed by the epidermis and, cells initially consume their available starch finally, the vascular bundles. The earliest and reserves and then sacrifice their cell membrane most dramatic change in the cellular structures phospholipids (mainly linoleic and linolenic during senescence is the breakdown of the acids) to recycle fatty acids and other metabo- chloroplasts, which contain the cell’s photosyn- lites (Aubert et al., 1996). Under prolonged and thetic machinery and the majority of the leaf severe stress that occurs as a consequence of a protein and are the site of major biosynthetic deficit or surplus of water or nutrients, extremes processes. This means that photosynthesis of temperature, or pest and pathogen attack, declines due to rubisco and chlorophyll degra- senescence of cells and, eventually, whole dation. However, the chloroplasts in the guard organs sets in. Senescence is defined as the cells remain green and functional, probably to ordered (organized or programmed) degrada- keep the stomata closed to prevent desiccation tion of cell constituents that ends in death and before nutrient recycling is complete (Tallman, is generally followed by shedding (abscission) 2004). The carbon, nitrogen, and other nutrients of the dead organs by breakdown of the cell stored in the leaf (and inflorescence) proteins, walls in the preformed abscission zones. The nucleic acids, polysaccharides, and lipids are moment of death of a cell occurs when the vacu- rapidly remobilized during senescence and olar membrane (tonoplast) ruptures. For a can be used to sustain the growth and metabo- grapevine to sacrifice an organ or part of its lism of important sink organs such as young structure constitutes an adaptive strategy to leaves, clusters, or roots. Of course, the parti- survive a stress period. tioning of resorbed nutrients from leaves that Several plant hormones interact to bring undergo senescence depends on the relative about and coordinate the (premature) abscis- strength or importance of the various sinks of sion of leaves, flowers, or fruit. Abscisic acid the vine. Moreover, the presence of strong sinks stimulates the senescence process, and ethylene (e.g., high crop load) may increase this nutrient operates as an accelerator of abscission, recycling, whereas weak sinks will decrease it. whereas auxin acts as a brake (Roberts et al., 2002). Therefore, ABA and ethylene can trigger senescence and abscission only in organs 7.2. WATER: TOO MUCH OR TOO whose auxin concentration is low. For instance, LITTLE although a variety of soil-related stresses result in ABA production in the roots and transport to the shoots, the developing leaves and flowers Water availability influences canopy devel- (but not fully developed leaves and flowers), opment, vine microclimate, yield, and fruit as well as the grape berries during the period composition, and lack of water constitutes the 232 7. ENVIRONMENTAL CONSTRAINTS AND STRESS PHYSIOLOGY overwhelming limitation to plant growth and in order to compensate for the lower soil water yield formation (Kramer and Boyer, 1995). extraction potential. Similarly, a coarse soil Under natural conditions, water is supplied favors greater rooting depth than a fine- by snow and rainfall and temporarily stored textured soil because less water is available C in the soil for extraction by plant roots. for extraction at low soil in the surface layers However, the amount of rainfall varies greatly of a coarse soil. Variation in soil moisture due from region to region and from year to year, to differences in water-holding capacity and which may drastically impair vine performance effective rootzone strongly influences vine per- and the economics of viticulture in some formance both between and within vineyards regions and some years. Water availability (Hall et al., 2002; Lamb et al., 2004). depends not only on how much rainfall a vine- Waterlogging occurs when the soil moisture yard receives but also on when the rain falls is well above field capacity, which is also called and how rapidly it evaporates. In addition, soil drained upper limit, whereby the soil pores water-holding capacity and hence the amount with a diameter greater than 30 mm retain water of plant-available water varies with soil depth, against gravity after all the water in the larger C texture (i.e., the relative proportions of clay, pores has drained away and the soil M silt, and sand particles), and organic matter 0.01 MPa. Grapevines can be grown hydro- content. For example, a fine loam soil has up ponically, which shows that they do not suffer to six times more available water than coarse from an excess of water during waterlogging. sand, although a larger fraction of the water is However, at high soil moisture, such as during held too tightly by the loam to be accessible to flooding due to heavy rainfall or excessive irri- the roots. Although roots can extract water gation, water drives gases out of the soil pores more easily from water-saturated sand than so that there is insufficient oxygen (O2) for from loam, a coarse soil dries much more rap- proper root function. This condition is termed idly so that water extraction becomes much hypoxia (i.e., O2 deficient) or, in extreme cases, more difficult in the sand as soil water poten- anoxia. This is because gases (including O2) dif- C tial ( soil) decreases. This is because the fuse four orders of magnitude less rapidly in hydraulic resistance to water movement in the water than in air. Due to the dependence of res- r soil ( soil) is approximately 10 times greater in piration on O2 supply, waterlogged roots can a water-saturated sand (due to its large pore become so O2 deficient that they cannot respire size) than in a loam but increases by the same and switch to anaerobic fermentation of pyru- mechanism that causes cavitation in the xylem vate to recycle NADH to NAD for continued (see Chapter 3.3): The pores of a drying soil fill ATP production via glycolysis, which is far less with air, which results in failure of capillary efficient than respiration and uses up storage forces. Just as larger xylem vessels are more carbohydrates while producing lactate and, vulnerable to cavitation, so too are larger soil later, volatile ethanol (Bailey-Serres and pores (i.e., coarser soil) more susceptible to cap- Voesenek, 2008; Zabalza et al., 2009). Inhibition illary failure. The coarser the soil type, the of respiration is accompanied by an increase in more limiting the soil is to plant water use, reactive oxygen species and a decline in pH whereas in a fine-textured soil the vulnerability of the root cells’ cytosol, and the excess protons of the xylem to cavitation is more limiting in turn block the aquaporins in the cell (Sperry et al., 1998). Grapevines growing in membranes so that the roots become less water sandy soils therefore often invest more permeable (Tournaire-Roux et al., 2003). Conse- resources in root growth than vines growing quently, but somewhat paradoxically, flooding- in loamy soils to increase the root:shoot ratio induced anoxia rapidly increases root hydraulic 7.2. WATER: TOO MUCH OR TOO LITTLE 233 resistance and decreases water uptake, which is increasing root growth in the nonaffected por- associated with a decline in stomatal conduc- tion (Stevens et al., 1999). Anoxia also increases tance and transpiration rate (Flore and Lakso, the xylem sap pH, which is a response similar 1989; Keller and Koblet, 1994). Thus, similar to to that in vines experiencing water deficit. This water deficit, an excess of water can lead to par- leads to reduced shoot growth, marginal leaf tial closure of the stomata, which over time burn, leaf yellowing, and, in severe cases, even diminishes photosynthesis and may damage death of the vine. Inflorescence differentiation the photosynthetic apparatus (Else et al., 2009; as well as pollen germination and pollen tube Stevens and Prior, 1994). In addition, energy- growth decrease with decreasing soil O2 con- intensive processes such as cell division come centration, so flooding can be detrimental to to a halt. bud fruitfulness and fruit set; the latter may Vitis species vary in their susceptibility to be reduced to zero if the soil O2 concentration waterlogging (Mancuso and Marras, 2006); for decreases below 5% (Kobayashi et al., 1964; instance, Vitis riparia can tolerate root flooding Stevens et al., 1999). for several days by rapidly suspending root Flooding during the dormant winter season metabolism, especially nutrient uptake and ostensibly has little effect on grapevines assimilation, in order to conserve energy (i.e., (Williams et al., 1994). Moreover, many wild to reduce ATP demand). On the other hand, grapes that have evolved alongside or near susceptible species such as V. rupestris are riverbanks frequently experience flooding unable to adapt their root metabolism and liter- during or soon after budbreak, when the melt- ally run out of energy. In addition, the inability ing snow in the mountains upstream leads to of susceptible species to suppress nutrient an annual peak in river runoff (Mastin, 2008). þ uptake can lead to ion (especially K ) poison- Beyond periodic spring floods, overly abun- ing, membrane leakage, and death. Excessive dant water supply during the growing season soil water, similar to the effect of soil compac- is a predicament that mainly concerns coastal tion, also seems to increase ethylene production regions and areas on the windward side of by the roots and prevents the diffusion of this mountain ranges, as well as some subtropical gas away from the roots (Feldman, 1984; Sharp and tropical regions that experience frequent and LeNoble, 2002). Small doses of ethylene summer rainfall. Most Chinese grape growing produced by the roots from the amino acid areas, for example, suffer from the fact that the methionine normally promote root growth, rainy season coincides with the growing season. perhaps by increasing tissue responsiveness to Because rainfall requires a cloud cover, such auxin and gibberellin (Kende and Zeevaart, regions also often experience low light inten- 1997; Potters et al., 2009). Together with auxin, sities that curtail photosynthesis (see Chapter ethylene also induces the formation of lateral 5.2). Furthermore, grapevines grown in pots are roots and root hairs, as long as the ethylene quite often at risk from hypoxia. The soil water gas can easily diffuse away from the roots. If content after drainage of irrigation water from C diffusion is inhibited, however, ethylene inter- pots is usually much higher ( P 0.001 to feres with cell division in the root meristem 0.003 MPa near the surface) than that in a vine- and delays the inactivation by gibberellin of yard at field capacity and depends on the height the growth-suppressing DELLA proteins so of the pot, which is why (frequently irrigated) that root growth slows (Dugardeyn and Van potted plants are more likely to suffer from water- Der Straeten, 2008; Feldman, 1984). If only a logging than from drought (Passioura, 2006). portion of the root system experiences water- The majority of the world’s grapes, however, logging, then vines may compensate by are grown in more or less Mediterranean 234 7. ENVIRONMENTAL CONSTRAINTS AND STRESS PHYSIOLOGY climates, which experience cool, moist winters occurs with daily and seasonal changes (due and warm, dry summers with high evaporative to radiation and humidity) in vapor pressure demand of the atmosphere. Thus, natural water deficit of the air (Williams et al., 1994). The supply and carbon supply for growth and fruit relationship between plant (leaf) water potential C production are often inversely related (Bloom ( leaf) and these variables can be expressed as et al., 1985). In Mediterranean regions and areas follows: on the leeward (rainshadow) side of mountain C ¼ C Er leaf soil ranges, winter rains often, but by no means r always (Davenport et al., 2008b), saturate the where h is the total resistance to water flow in soil profile to field capacity. Such refill of soil the vine (see Chapter 3.3). water stores can be enhanced by cover crops When transpiration exceeds water absorp- that reduce runoff and improve infiltration, even tion by a vine’s root system, cell turgor (P), rel- though the cover crop may later compete with ative water content (RWC; percentage of tissue grapevines for water (Battany and Grismer, water relative to the water content of the same 2000; Celette et al., 2008; Klik et al., 1998). The tissue at full turgor), and cell volume decrease, soil then progressively dries down as tem- whereas the concentration of solutes in the cell peratures rise and water evaporates, and the increases, reducing the cell’s osmotic potential growing season is often characterized by low soil (Cp) and C. Consequently, the response of the moisture and high vapor pressure deficit cells to water deficit involves a response to (i.e., warm, hot, dry air). The resulting increase osmotic stress. Mild plant water stress has C in air spaces in the soil and water retention by been defined as a decrease of leaf by several successively smaller pores leads to an increase bars or of relative water content by 8–10% in surface tension, which in turn is responsible below corresponding values in well-watered for the development of increasingly negative plants under mild evaporative demand (Hsiao, C C pressure ( P). In arid soils, P can decline 1973). Moderate water stress corresponds to a C C to 3 MPa, compared with P 0.01 to decrease of leaf by more than several bars 0.03 MPa in a soil at field capacity. Water in but less than 1.2–1.5 MPa or of RWC by pores less than 0.2 mm cannot be extracted by 10–20%, and severe water stress corresponds C the roots (extraction from these pores would to a lowering of leaf by more than 1.5 MPa or require a suction of 1.5 MPa to overcome the of RWC by more than 20%. Initially, the decline C P soil M in these pores) and thus constitutes the in and RWC reduces shoot growth and lower limit (termed permanent wilting point) stomatal conductance for water vapor (Hsiao, of plant-available water (Watt et al., 2006). The 1973). It appears that shoot extension and leaf growing season in regions with a Mediterra- expansion in grapevines decline linearly nean-type climate is often characterized by low with decreasing C and stop completely at C ¼ soil moisture and high vapor pressure deficit 1.0 to 1.2 MPa, even though leaf expansion due to the warm, dry air. The water balance of is relatively independent of P (Schultz and grapevines and their cells is determined by Matthews, 1988b; Shackel et al., 1987). the amount of water lost in evaporation to the The primary function of stomata is to avoid atmosphere (transpiration; see Chapter 3.2) and damaging water deficits that would cause the amount of water absorbed from the soil. xylem cavitation (Brodribb and Holbrook, A vine can become water stressed as a result 2003; Jones, 1998; see also Chapter 3.3). Grape- C of both decreased soil water potential ( soil), vines seem to be quite vulnerable to cavitation which generally occurs progressively over time, and are usually regarded as near-isohydric spe- r and fluctuating transpiration rate (E), which cies whose sensitive stomata rapidly increase s 7.2. WATER: TOO MUCH OR TOO LITTLE 235 and decrease transpiration in response to low increasingly blurred as more information C soil, which enables them to maintain almost becomes available. C constant leaf throughout the day and regard- Isohydric species may have evolved in wet C et al less of soil (Du¨ ring, 1987; Galme´s ., 2007). habitats; they are more susceptible to xylem This effect can override the influence of high cavitation and can be regarded as “pessimists”’ light intensity on stomatal opening so that soil modifying their behavior to conserve resources. water deficit in a vineyard often leads to a mid- Although this conservative strategy keeps water r et al day increase in s (Correia ., 1990, 1995; demand by the transpiring leaves well below Loveys and Du¨ ring, 1984). Nonetheless, pro- levels that would challenge the xylem’s supply C nounced diurnal changes in leaf have also capacity (Sperry, 2004), it comes at the cost of a been reported; with dry soil, the difference decline in photosynthesis and hence potential C between predawn and midday leaf may some- losses in CO2 uptake. Anisohydric plants, on times exceed 1 MPa (Williams and Matthews, the other hand, probably originate mostly from 1990). Because grapevine species and cultivars more arid regions, are less susceptible to xylem differ in their vulnerability to cavitation, they cavitation, and can be viewed as “optimists” also vary in their stomatal sensitivity to using available resources in expectation of more drought (Currle et al., 1983; Escalona et al., arriving. Due to the narrow safety margin from 1999; Liu et al., 1978; Schultz, 2003; Soar et al., hydraulic failure, where xylem cavitation 2006b; Winkel and Rambal, 1993). So-called iso- becomes irreversible, the anisohydric approach hydric species or cultivars have more sensitive is riskier, and such vines may pay by shedding stomata than anisohydric species or cultivars. leaves when a drought becomes too severe. r The latter maintain lower s and higher trans- However, it probably enables the plant to more C piration rates, and they markedly decrease leaf efficiently match water supply with demand so during the day and in response to soil that it can maximize CO2 uptake by pushing water deficit or atmospheric moisture stress the xylem to its carrying capacity, at least as (high vapor pressure deficit). These differences long as it can retain its leaf area (Sack and among cultivars are also reflected in the radial Holbrook, 2006; Sperry, 2004). Indeed, Syrah r hydraulic resistance ( h) and activity of aqua- maintains vigorous shoot growth under water- porins in the roots, which are regulated deficit conditions that would completely inhibit to adjust water flow to transpiration rate growth of Cabernet Sauvignon shoots. More- (Vandeleur et al., 2009). Note, however, that the over, if left to its own devices, rather than being distinction is mostly a matter of degree and constrained by a trellis system and foliage wires, not nearly as clear-cut as the terminology sounds. the droopy growth habit of Syrah shoots also C Because leaf never remains absolutely constant, differs from that of Cabernet Sauvignon or it may be more appropriate to “divide” grape- Grenache, whose shoots are much more erect. vines into near-isohydric or pseudo-isohydric Anisohydric plants also would be expected to and near-anisohydric or pseudo-anisohydric have a larger root:shoot ratio than isohydric species and cultivars. The former include V. ber- plants, which may be a drought-avoidance landieri, V. rupestris,andtheV. vinifera cultivars strategy. In addition to V. vinifera cultivars, root- Cabernet Sauvignon, Grenache, Tempranillo, stocks derived from American Vitis species also and Carignan, whereas the latter include Syrah, differ in their susceptibility and response to C r Sangiovese, Chardonnay, Se´millon, Thompson drought and may alter leaf, s, and photosyn- Seedless, and Concord. Only a few species thesis of the scion leaves via the production and cultivars have been studied to date, and and export in the xylem of ABA (Bauerle et al., it is likely that the boundaries will become 2008b; Soar et al., 2006a). 236 7. ENVIRONMENTAL CONSTRAINTS AND STRESS PHYSIOLOGY

In addition to its obvious consequences for the leaf cells. A decrease in turgor may be C, dehydration of plant cells also stimulates the sufficient to trigger ABA synthesis in the synthesis of ABA from the carotenoid zeaxan- leaves; thus, cell turgor is a key signal of water thin in the parenchyma cells, especially near stress. the cambium, of the root, shoot, and leaf vascu- Whereas ABA may be involved in inhibiting lar system (Endo et al., 2008; Nambara and shoot growth of water-stressed plants, it Marion-Poll, 2005). The main function of ABA appears to have the opposite function in roots. is to regulate plant water balance and osmotic Moreover, the pH of xylem and other apoplas- stress tolerance. The role of ABA in water bal- tic (extracellular) sap generally increases in ance is mainly through the regulation of stoma- response to soil drying (Hartung and Slovik, tal conductance (see Chapter 3.2), whereas its 1991; Wilkinson and Davies, 2002). The pH of role in osmotic stress tolerance is through the xylem sap from well-watered plants is approx- production of dehydration-tolerance proteins imately 6.0 (see Chapter 5.1), whereas that of in most cells. Another function of ABA is to act sap from water-stressed plants is approxi- as an antagonist of auxin, which induces cell mately 7.0. This increase in xylem sap pH alone wall loosening necessary for cell expansion could constitute a root-sourced signal to the during growth (see Chapter 3.1). It is possible leaf, inducing stomatal closure and reducing that ABA prevents auxin from exerting its influ- the rate of cell expansion (and hence growth). ence on wall loosening. ABA also directly inter- ABA synthesized in the roots can be leached feres with the cell cycle machinery, thus out into the soil solution, especially in alkaline inhibiting cell division. In addition to the ABA soils (Wilkinson and Davies, 2002). Conse- produced in drying leaves, the amount of root- quently, a high soil pH could weaken the sourced ABA in the xylem sap can increase root-sourced ABA signal. substantially as a function of reduced soil water Roots must absorb more water than what is availability (Loveys, 1984). In contrast to ABA, lost in transpiration to enable plant growth the production of cytokinins in the roots and because growth is mainly caused by cell expan- their transport in the xylem sap decrease in sion due to water import (see Chapter 3.3). response to soil drying (Davies et al., 2002; Yang Water uptake by cells is driven by accumula- et al., 2002). Both ABA and cytokinins can act as tion of solutes (e.g., sucrose) inside the cell stress signals that mediate conditions in the and thus depends on an osmotically generated rootzone via the xylem to the shoots, leaves, DC between cell interior and exterior (see and clusters. The production of ABA in the roots Chapter 3.1). Because water stress reduces C is stimulated rapidly in response to water stress, xylem, a major cause for growth inhibition whereas cytokinin production decreases much under water deficit may simply be the smaller more gradually. Nevertheless, when water DC, which reduces cell water uptake and con- stress occurs suddenly, production and trans- sequently hampers the generation of the neces- mission of the root ABA signal may be too slow sary turgor pressure (Hsiao and Xu, 2000). to enable the stomata to close in time to avoid In addition, cell expansion involves cell wall leaf dehydration. Under these conditions, ABA loosening, which requires acidification by pro- þ is produced directly in the shoot and the leaves tons (H ) pumped from the cell interior to the þ in response to a hydraulic signal from the roots cell wall in exchange for K maintaining the (Christmann et al., 2007; Soar et al., 2004). cell’s electrical charge balance (Stiles and Van Changes in xylem pressure in the roots are Volkenburgh, 2004). It is possible that the transmitted virtually instantaneously to the increase in apoplast pH due to water deficit shoot, where they alter the turgor pressure of also directly interferes with cell expansion 7.2. WATER: TOO MUCH OR TOO LITTLE 237

(Bacon et al., 1998). Therefore, growth rates Rootzone conditions are generally heteroge- C change rapidly with fluctuating xylem and neous, and soil moisture is no exception to this pHxylem, and a decline in shoot growth and leaf rule. When roots experience both wet and dry appearance is the first visible sign of water def- soil, the reaction of shoot growth depends on icit; shoot growth is even more sensitive to whether the difference in soil moisture is per- water stress than is photosynthesis (Hsiao and ceived by the same or different roots. When Xu, 2000; Stevens et al., 1995; Williams et al., the surface soil is dry while the subsoil is still 1994). Moreover, tendrils are also a sensitive wet, there is no reduction in shoot growth as indicator of vine water status: On nonstressed, long as the roots have access to the subsoil vigorously growing shoots, the uppermost ten- water (Phillips and Riha, 1994). However, drils extend beyond the shoot tip. As water when separate roots of the same vine experi- stress sets in and growth begins to slow, new ence dry and wet soil columns, shoot growth tendrils remain small so that the shoot tip is suppressed (Dry and Loveys, 1999; Dry catches up with them. With more severe stress, et al., 2001; Lovisolo et al., 2002a). This can be growth stops and the youngest leaf expands exploited in another irrigation strategy termed beyond the shoot tip. Before they become ligni- partial rootzone drying (PRD), in which water fied, tendrils are highly sensitive to water stress is supplied alternately to only one side of a vine and start wilting before the leaves do. while the other side is allowed to dry down Deliberate application of water deficit is an (Dry and Loveys, 1998). This strategy attempts important management strategy in premium to separate the physiological responses to water r wine grape production. The time between fruit stress (e.g., ABA production and increased s) set and veraison is the period when shoot from the physical effects of water stress, such C et al et al growth and berry size can be most effectively as low leaf (Davies ., 2002; Dry ., controlled by water deficit. This principle is 2001; Kriedemann and Goodwin, 2003). The exploited by regulated deficit irrigation (RDI), ABA produced by the drying roots induces whereby water deficit is applied for a brief partial stomatal closure and reduced shoot period as soon as possible after fruit set growth while the fully hydrated roots maintain (Dry et al., 2001; Keller, 2005; Kriedemann and a favorable plant water status (i.e., no decrease C Goodwin, 2003). In general, deficit irrigation in leaf). Water delivery to the shoots by the simply means that less water is applied than wet roots may increase during PRD (Kang is required by evapotranspiration. During the et al., 2003), and the wet roots also sustain the RDI period, the soil profile is allowed to dry drying roots by supplying water to them (Stoll down until control of shoot growth has been et al., 2000). Eventually, however, water flow achieved. Obviously, this is possible only in from the dry roots becomes so slow that ABA areas with sufficiently low seasonal rainfall or delivery from these roots ceases and the sto- high evaporative demand and on soils with mata begin to reopen (Dodd et al., 2008; Dry limited water storage capacity. In addition to et al., 2000a). To prevent such adaptation by limiting berry size, RDI also tends to be asso- the vines, irrigation is alternated every week ciated with improved sun exposure of the or two between the two sides of the root sys- grapes. Once shoot growth stops and especially tem. In addition to increased ABA production, after veraison, vines are stressed only suffi- the drying roots produce lower amounts of ciently to discourage new shoot growth. At cytokinins, which inhibits lateral shoot growth the end of the growing season, the rootzone is (Stoll et al., 2000). Most of the reduction in refilled to field capacity to avoid cold injury to canopy size of vines subjected to PRD may be roots during the winter (see Chapter 7.4). due to inhibition of lateral shoot growth 238 7. ENVIRONMENTAL CONSTRAINTS AND STRESS PHYSIOLOGY

(dos Santos et al., 2003; Dry et al., 2001). xylem conduits (De Boer and Volkov, 2003; C þ Provided leaf is maintained under PRD, berry Roberts and Snowman, 2000). This traps K þ size and yield are also maintained, whereas the taken up from the soil (ABA does not affect K lower canopy density often results in improved uptake) and delivered from the shoots in the þ fruit composition. The fundamental difference phloem, and it leads to K accumulation in the þ C between RDI and PRD is that RDI imposes a roots. The osmotic activity of K lowers root, soil water deficit over time, whereas PRD which may enhance phloem water import (see imposes a deficit over space (Keller, 2005). If Chapters 3.2 and 5.1). This helps the vine to þ rainfall during the growing season is low maintain root growth while avoiding K trans- enough, RDI always results in a plant water port to the leaves, where it would only worsen C deficit, whereas PRD usually does not if man- the water deficit by lowering leaf. However, at aged properly (de Souza et al., 2003; Dry et al., higher concentrations (i.e., more severe stress) 2001; Kriedemann and Goodwin, 2003). The ABA inhibits root growth. In addition, the roots, PRD effect may be lost if the switching interval especially the growing root tips, also osmotically is too long, the wet soil volume is too small for adjust, or osmoregulate, by accumulating sugars C a given vine size, two discrete root systems and amino acids in order to lower root to favor cannot be established (e.g., following conversion water uptake, continued growth, and possibly of furrow- or sprinkler-irrigated vineyards), reduce the risk of xylem cavitation (Du¨ ring and where vines have access to a relatively high Dry, 1995; Schultz and Matthews, 1988a). At water table, or in poorly structured clay soils. more severe water stress, however, the vessels An additional response to PRD seems to be that embolize, which interrupts water flow in the grapevines shift root growth to deeper soil affected xylem conduits altogether. In fact, layers (Dry et al., 2000b, 2001), whereas under embolisms may be quite common in grapevines standard drip irrigation roots are often concen- and may be important in inhibiting shoot trated in the surface soil. Although this adaptive growth at moderate water deficits (Lovisolo behavior makes vines more drought resistant, it and Schubert, 1998; Lovisolo et al., 2008; Schultz also interferes with the application of the PRD and Matthews, 1988a). Of course, soil drying principle on soils with a deep rootzone and also reduces the rate of nutrient transport to the ready access of the roots to subsurface water. roots so that overall nutrient availability Root growth of water-stressed vines declines, decreases, especially if the water deficit is suffi- but less so than shoot growth, because roots cient to slow root growth (Hsiao, 1973; see also can grow at lower C than shoots, partly because Chapter 7.3). Thus, decreasing soil moisture higher activity of expansin proteins may makes nutrient uptake increasingly difficult, increase cell wall extensibility in the root tips and a greater proportion of the nutrient demand (Erlenwein, 1965a; Sharp et al., 2004; Wu and by the developing canopy and especially the rip- Cosgrove, 2000). In addition, the effect of water ening fruit has to be supplied from stored stress is more pronounced on the roots’ diameter reserves in the woody parts of the vine. A than on their length: Stressed roots become thin- decline in soil moisture is also associated with a ner (Mapfumo et al., 1994a). These adaptations decrease in root respiration and, below approxi- increase the root:shoot ratio and maintain water mately 5% volumetric soil moisture, leads to a and nutrient supply to the shoots (Hsiao and Xu, concomitant loss of membrane integrity so that 2000). By inducing a blockage of ion channels in roots begin to die back under severe drought the stele (see Chapter 3.3), ABA reduces the conditions (Huang et al., 2005b). þ r release of K (and possibly other nutrient ions, The stomatal closure (high s), discussed pre- such as H2PO4 ) from the root cortex into the viously, diminishes water loss by transpiration 7.2. WATER: TOO MUCH OR TOO LITTLE 239 ) ) − 1 − 1 s s − 2 − 2 m 2 O m 2 Transpiration (mmol H Transpiration Photosynthesis ( µ mol CO

−2 −1 −2 −1 Stomatal conductance (mol H2O m s ) Stomatal conductance (mol H2O m s ) FIGURE 7.1 Relationship between stomatal conductance and transpiration (left) and photosynthesis (right) of mature leaves of two grapevine cultivars during soil drying (M. Keller, unpublished data). Note the steeper decline of photosynthe- sis compared to transpiration at low stomatal conductance. but also limits photosynthesis (Figure 7.1) photosynthate available for export. Nonethe- because CO2 diffusion into grape leaves is less, as long as the water deficit is mild enough much more dependent on open stomata than to restrict shoot growth more than photosyn- is H2O vapor diffusion out of the leaves (Boyer thesis, there might be relatively more sugar et al., 1997; Escalona et al., 1999; Flexas et al., available for other sinks (Lawlor and Cornic, 1998, 2002; see also Chapter 4.2). This is called 2002), which could benefit fruit ripening. stomatal regulation or stomatal limitation of Whereas leaf sugar concentrations may photosynthesis and is accompanied by a remain high for some time, starch becomes decrease in ATP and NADPH consumption depleted, and assimilate export decreases pro- because less energy is needed for CO2 fixation. gressively with increasing severity of water Not surprisingly, the effect on photosynthesis stress (Du¨ ring, 1984; Quick et al., 1992). In addi- and assimilate export of short episodes of high tion, when faced with water stress, expanding vapor pressure deficit is similar to the effect of leaves and growing root tips commonly accu- longer periods of soil water deficit (Shirke and mulate solutes, especially inorganic ions such þ þ Pathre, 2004). However, even a stress that is as K and Ca2 , but also sugars such glucose mild enough not to diminish photosynthesis and fructose and, to a lesser extent, amino acids can reduce shoot growth (i.e., cell expansion), such as proline and organic acids such as malate and hence canopy development, because the (Cramer et al., 2007; Patakas et al., 2002; Sharp reduction in growth usually occurs before the et al., 2004). This osmotic adjustment, or stomata begin to close (Hsiao, 1973). The com- osmoregulation, maintains turgor and allows bination of smaller total leaf area and decreased continued, albeit slower, growth (Du¨ ring, 1984; photosynthesis will result in reduced daily Morgan, 1984). When expansion is complete assimilate production of the grapevine canopy (e.g., in fully grown leaves), tissues gradually (Perez Pen˜a and Tarara, 2004). Because the net lose this ability to osmoregulate (Patakas et al., assimilation rate of the canopy determines the 1997), but leaves developing later in the season export rate from the canopy, a reduction in can- can achieve a greater capacity for osmotic opy photosynthesis will reduce the amount of adjustment than leaves that are formed earlier. 240 7. ENVIRONMENTAL CONSTRAINTS AND STRESS PHYSIOLOGY

C Under severe stress—that is, when leaf organic acids from the citric acid cycle, rather decreases below approximately 1.5 MPa than assimilates directly from the chloroplast (depending on cultivar), RWC decreases below (as under nonstress conditions), so that there approximately 75%, and stomatal conductance is a decrease in organic acids (Lawlor and g ( s) declines to less than 20% ( 50 mmol H2O Cornic, 2002). 2 1 et al g m s , according to Flexas ., 2002) of s in When the leaves lose turgor due to severe well-watered vines—growth slows strongly or water stress, they will wilt; the leaf surface area stops. Moreover, photosynthetic metabolism is decreases, and leaves hang down and become progressively impaired, initially by a reduction parallel to the solar rays (Lawlor and Cornic, of electron transport and ATP synthesis that 2002; Smart, 1974). Because water stress inhibits the Calvin cycle and, at very low RWC, reduces photosynthesis so that fewer electrons by inhibition of light harvesting and photosys- are needed, wilting is an effective mechanism tem [especially photosystem II (PSII)] function for reducing light absorption (i.e., the number (Escalona et al., 1999; Hsiao, 1973; Tezara et al., of photons captured by the leaf; see Chapter 1999; see also Chapter 4.1). Although the amount 4.1). This can substantially reduce a potential of rubisco protein in the leaves also declines “energy overload” that would be damaging to under severe drought stress (Bota et al., 2004), the photosynthetic system (Flexas et al., 1999b; this might be a consequence rather than a cause Lawlor and Cornic, 2002). Nevertheless, water of reduced photosynthesis. This depression of stress can be especially damaging at high light photosynthesis is called metabolic regulation or intensity with large photon fluxes that are asso- metabolic limitation, and in contrast to stomatal ciated with a relative energy surplus in the limitation, it is irreversible (Escalona et al., 1999; chloroplasts (Lawlor and Tezara, 2009). More- Lawlor and Cornic, 2002). The decrease in pho- over, because the cuticle allows small amounts tosynthesis is accompanied by only a slight of water vapor to pass through by diffusion, decrease in respiration (see Chapter 4.4) and, leaves cannot avoid water loss completely, no possibly, an increase in the proportion of photo- matter how tightly the stomata are closed respiration relative to photosynthesis, which (Boyer et al., 1997; see Figure 7.1). Such cuticu- may serve to counter an energy overload by lar transpiration increases exponentially with “consuming” excess electrons (Cramer et al., rising temperature because both the water per- 2007; Du¨ ring, 1988; Lawlor and Cornic, 2002; meability and the vapor pressure deficit that Lawlor and Tezara, 2009; see also Chapter 4.3). provides the driving force for water loss Thus, as water stress intensifies, an increasing increase as the temperature rises (Riederer proportion of fixed CO2 is lost from the leaf. and Schreiber, 2001). The problem is particu- r The more severe the water stress, the more of larly acute when drying wind reduces b and this CO2 is derived from stored carbohydrates stimulates transpirational water loss. Although (Lawlor and Cornic, 2002). As reserve carbohy- young leaves may be more sun-exposed and drates are consumed, amino acids, particularly thus experience higher evaporative demand proline and glutamate, accumulate because their than older leaves, the old leaves wilt at higher C production (supply) exceeds consumption leaf than the young leaves because the latter (demand). This in turn seems to lead to nitrate are better able to osmoregulate (Patakas et al., accumulation in leaves, possibly due to feedback 1997). The wilting older leaves produce plenty inhibition of nitrate reductase (Patakas et al., of ABA, which is exported to the young leaves 2002; see also Chapter 5.3). The carbon skeletons to reduce water loss. for the production of amino acids in water- ABA can also result in oxidative stress via stressed leaves are provided by glycolysis and increased production of active oxygen species 7.2. WATER: TOO MUCH OR TOO LITTLE 241 that,whenproducedinexcess,damageand and wine pH (see Chapter 6.2). This effect is eventually kill the plant cells due to uncon- probably most pronounced if severe water trolled oxidation (see Section 7.1). This stimu- stress occurs during a period in which the lates the vine’s antioxidant defense system to clusters’ sink strength is at its maximum (i.e., prevent oxidative damage (Cramer et al., during and immediately after veraison). Even 2007), leading to increased foliar concentra- under drought conditions leading to complete tionsofantioxidantssuchascarotenoids defoliation, it is rare for the perennial parts of (especially xanthophylls), glutathione, ascor- grapevines to dieback. Restoring water supply bate (vitamin C), and a-tocopherol (vitamin to defoliated plants usually results in bud- E). If the water stress is not relieved by rainfall break and regrowth, presumably aided by the or irrigation, the increased ABA and reduced generation of root pressure (see Chapter 3.3). cytokinin contents accelerate leaf aging and Nevertheless, partial or complete dieback, lead to senescence of older leaves (Yang et al., beginning in the youngest (and smallest) por- 2002). Senescence, which is initiated when tion of the shoots and roots, can occur due to leaf cytokinins fall below a threshold level, complete cavitation of the xylem (i.e., hydrau- involves the disassembly of the chloroplasts lic failure) for prolonged periods. with their photosynthetic machinery and is It is often stated that the reduction in vegeta- accompanied by a decline in chlorophyll and tive growth due to water stress is more severe remobilization of carbon, proteins (e.g., degra- than that of fruit growth (Williams, 1996; dation of rubisco), and nutrients from these Williams et al., 1994). Water deficit, similar to leaves. It culminates in leaf abscission, which nutrient deficiency, typically reduces yield, can also be induced by excessive xylem cavita- particularly if the deficit occurs early in the tion, whereas the meristems inside the buds growing season (Williams and Matthews, remain alive. This drought-induced deciduous 1990). The sensitivity of reproductive growth, behavior also promotes early inception of bud and hence fruit production and yield forma- dormancy and enables vines to survive severe tion, depends on the developmental stage of drought by drastically reducing evaporation the vine. Soon after budbreak, the developing and conserving resources. Once initiated, the flower clusters begin competing successfully rate of senescence accelerates with increasing with the growing shoots for limited water and temperature so that a heat wave following assimilates (Hale and Weaver, 1962). Once drought stress can lead to severe defoliation inflorescences contribute assimilates via their of vines. The cytosolic glutamine synthetase own gas exchange (Leyhe and Blanke, 1989), (GS1) in the phloem may be involved in the they may survive even relatively severe production of glutamine used for nitrogen drought sufficient to stunt shoot growth. Closer export and of proline used for storage (see to and during bloom, however, the flowers’ Chapter 5.3) during water stress or senescence. metabolic requirements far exceed their own Some of the sugars, amino acids, and phloem- contribution—after all, the majority of the sto- mobile mineral nutrients (see Chapter 7.3) mata are located in the cap that is shed at can be recycled to the fruit, which may par- anthesis (Blanke, 1990b; Blanke and Leyhe, tially sustain ripening. However, because 1988, 1989a; Vivin et al., 2003)—but the flower nutrients are “swept” into the fruit by the clusters continue to be weak sinks until fruit sugar-driven mass flow in the phloem (see set. Whereas the female floral organs are Chapter 5.1), this nutrient recycling may thought to be relatively insensitive to water þ lead to an undesirable increase in fruit K stress (although smaller ovaries are more sus- content that may subsequently increase juice ceptible to stress than are larger ovaries within 242 7. ENVIRONMENTAL CONSTRAINTS AND STRESS PHYSIOLOGY the same fruit cluster), water deficit during following growing season. Water deficit nor- meiosis in the anthers (which occurs 1–12 mally decreases cluster initiation, which can weeks before anthesis) may cause pollen steril- reduce bud fruitfulness and thus result in low ity (Dorion et al., 1996; Saini, 1997). Even mod- numbers of clusters per shoot (Alleweldt and erate water stress for a brief period during Hofa¨cker, 1975; Buttrose, 1974b). In contrast, male meiosis can hamper pollen development if the water deficit is just mild enough to because water stress may inhibit sugar trans- decrease canopy density, this may actually port to and starch accumulation in the pollen enhance cluster initiation due to the improved grains (Dorion et al., 1996; Saini, 1997); how- canopy microclimate (Smart and Coombe, ever, this has yet to be investigated in grapes. 1983; Williams and Matthews, 1990; Williams Starch is required to fuel respiration for pollen et al., 1994). development and germination and for pollen Once fruit set has passed and the vine has tube growth, so their low “fuel” status prevents made a significant investment in seed produc- the pollen tubes from reaching the ovary in tion, vines experiencing water stress generally time for fertilization, if they are able to germi- maintain fruit development at the expense of nate at all (see Chapter 2.3). Reduced pollina- shoot and root growth and replenishment of tion and fertilization result in poor fruit set or, storage reserves in the roots (Eibach and Alle- in more severe cases, abscission of inflores- weldt, 1985; Mullins et al., 1992; Williams cences (Callis, 1995; Smart and Coombe, 1983). et al., 1994). Of course, this can have long-term In other words, reproductive development is implications because the vine is heavily depen- most sensitive to water stress from meiosis to dent on stored reserves for budbreak the fol- fruit set. The effects of water deficit are likely lowing spring. Moreover, a weak root system C mediated by a combination of low inflorescence can predispose vines for drought stress when and changes in xylem sap pH, ABA, and cyto- there is insufficient water available. A small kinins in addition to supply of photosynthate root system supplying water to a large canopy via the phloem. For instance, although ABA would result in a very steep water potential DC ¼ C C stimulates vacuolar invertase in the leaves in gradient ( soil leaf; see Chapter 3.3), order to maintain turgor to sustain photosyn- which could render vines vulnerable to xylem thetic activity and assimilate export, it sup- cavitation and, consequently, canopy desicca- presses the same enzyme in flowers and tion (Sperry et al., 2002). To balance water loss young fruits, which are subsequently aborted by transpiration and supply by root uptake, C (Roitsch and Gonza´lez, 2004). An involvement such vines have to either lower leaf (e.g., by of ABA and cytokinin is consistent with the osmotic adjustment) or close their stomata, observation that PRD, which maintains high especially around midday when evaporative C leaf, may be associated with poor fruit set demand is at its maximum. (Rogiers et al., 2004b). Moreover, it is possible Under dry conditions, moderate nitrogen that when embolisms develop in the inflores- (N) supply via fertilizer application may con- cence xylem, they may not be as easy to repair serve soil water in the short term. However, a as in leaves. Note that it is unknown whether decrease in the root:shoot ratio due to fertilizer deliberate prebloom water stress could be used (especially N) addition can further increase a in breeding programs to sterilize the female vine’s susceptibility to drought, particularly in breeding partner. irrigated vineyards should irrigation be with- In addition to reducing berry number, water held (Erlenwein, 1965a; Sperry et al., 2002). stress during bloom could also have implica- The increase in nitrate concentration in the tions for a vine’s yield potential in the xylem sap that accompanies abundant N 7.3. NUTRIENTS: DEFICIENCY AND EXCESS 243 supply (see Chapter 5.3) seems to be associated plant water status (Creasy and Lombard, with a decrease in xylem sap pH, which in turn 1993; Du¨ring et al., 1987; Findlay et al., 1987; reduces the sensitivity of the stomata to ABA Greenspan et al., 1994, 1996; Ollat et al., 2002; (Jia and Davies, 2007; Wilkinson and Davies, Rogiers et al., 2001). Nevertheless, due to the 2002). This means that the stomata close later reversal of xylem flow at veraison (see Chapter and more slowly when a soil with high N sta- 6.2), postveraison water deficit, especially pro- tus dries compared with a relatively N-poor longed deficit, can lead to berry shrinkage by soil. In nonirrigated plants, however, fertilizer dehydration when xylem efflux plus berry tran- application is thought to improve the capacity spiration exceed phloem influx (Keller et al., for osmotic adjustment and the resistance to 2006). Thus, the extent of preharvest shrinkage xylem cavitation, which tends to counter the is inversely related to the water status of the vine. lower root:shoot ratio (Bucci et al., 2006; Sperry The response of berry growth to water defi- et al., 2002), although excessive N supply can cit also depends on the crop load. Berry size reduce water uptake due to the buildup of may be curtailed more on plants bearing a solutes in the soil water, similar to the effect heavy crop (i.e., many berries per vine) than of salinity discussed in Chapter 7.3 (Keller on vines with a light crop load because lower and Koblet, 1994). sugar import by each berry also limits water Although fruit growth may be relatively uptake by those berries (Fishman and Ge´nard, insensitive to water stress compared with shoot 1998; Santesteban and Royo, 2006). If a stress growth, limited water supply during the period occurs early enough to reduce fruit set, the size of berry cell division and cell expansion of the remaining berries may increase and par- restricts berry enlargement, which limits berry tially compensate for the loss in yield potential, size (Roby and Matthews, 2004; Williams and which would offset prospective benefits of Matthews, 1990). In keeping with the effect on smaller berry size for wine quality (see Chapter C shoot growth, reduced xylem and pHxylem 6.2). Water stress, however, may prevent com- could also be partly responsible for the smaller pensatory berry growth on vines that start the berry size of water-stressed vines, particularly growing season with a small crop, whereas it if the stress occurs before veraison. Yield reduc- does not seem to have an additional effect on tions due to water stress can still be severe at heavily cropped vines, where berry size is this stage, whereas after veraison berries seem already limited (Keller et al., 2008). Although to become increasingly insensitive to water def- plant water stress symptoms become apparent icit. Thus, the same extent of water deficit when more than 50% of plant-available water occurring during the postbloom cell division has been extracted from the soil, grape berries phase of berry growth normally reduces berry (both before and after veraison) do not begin size more than if it occurs after that phase to shrink until 80% of the available water has (Currle et al., 1983; Hardie and Considine, been transpired by the vine (Keller et al., 2006). 1976; Hofa¨cker et al., 1976; Matthews and Anderson, 1989; McCarthy, 1997; Williams and Matthews, 1990). Applying more water 7.3. NUTRIENTS: DEFICIENCY later in the season cannot compensate for the AND EXCESS decrease in berry size due to early season defi- cit, possibly because increased phloem flow and greatly reduced or halted xylem flow into In a growing grapevine, each new cell has a the berry after veraison strongly decrease the relatively specific requirement for a well- sensitivity of berry water status to soil and defined set of inorganic mineral nutrients 244 7. ENVIRONMENTAL CONSTRAINTS AND STRESS PHYSIOLOGY

(often abbreviated “minerals”) in addition to water and carbon. Some of these nutrients, especially during budbreak and during leaf fall, are supplied from stored reserves in the permanent structure of the vine or from senes- cing leaves. However, the majority of nutrients throughout the growing season must be acquired by the roots in the form of ions dis- solved in the soil water or “soil solution.” Roots can either grow toward nutrient-rich areas or patches in the soil in a process called root inter- ception or wait for the nutrients to arrive with the soil water. In the latter case, the nutrients must move through the soil either by diffusion, which is usually the dominant process, or by mass flow, which is possible when plants tran- spire rapidly and thus remove copious amounts of soil water. In fact, one adaptive advantage of transpiration may be its capacity to enhance mass flow of nutrients to the root surface, which may be important in soils with relatively low nutrient availability—provided water is not limiting (Cramer et al., 2008). Accordingly, grapevines seem to transpire more rapidly when the nutrient concentration FIGURE 7.2 Nutrient availability in the soil is depen- in the soil solution is low than when abundant dent on soil pH. Illustration by M. Keller, after Lucas and nutrients are present, but the leaves of the high- Davis (1961). nutrient vines wilt more readily when they run out of water (Keller and Koblet, 1994; Scienza storage capacity in both clayey and sandy soils and Du¨ ring, 1980). due to its beneficial effect on soil structure Soils vary not only in their capacity to store (aggregate formation). Although the nutrient water but also in the amount and composition concentration in a dry soil is usually higher of mineral nutrients they contain and in the than that in a wet soil, these nutrients are extent to which these nutrients are available less available in the dry soil because the lack for uptake by the roots. Nutrient storage capac- of water slows diffusion and mass flow ity and accessibility are influenced by soil tex- (Marschner, 1995); the diffusion rate decreases ture, rooting depth, and organic matter roughly with the square of the soil water con- (humus) content, but availability is modified tent. Moreover, mineralization of organic þ ! by soil moisture and pH (Figure 7.2). For matter and nitrification (NH4 NO3 ) also instance, just as a loamy soil can store consider- slow down in drying soil. The concentration ably more water than a sandy soil (see Chapter of exchangeable cations (e.g., calcium) rises at 7.2), it also adsorbs more nutrient ions than the expense of anions (e.g., phosphate), and sandy soils where the nutrients are leached roots may lose contact with soil particles due away by percolating water. Organic matter, on to shrinkage so that nutrient availability to the the other hand, increases water and nutrient roots declines steeply as the soil dries out 7.3. NUTRIENTS: DEFICIENCY AND EXCESS 245

(Bloom et al., 1985; Jackson et al., 2008). plants together. This increases the roots’ absorb- Although this diminishes nutrient uptake and ing capacity well beyond the depletion zones delivery of nutrients to the shoots (Davies caused by the growing root tips. The fungi et al., 2002; Mpelasoka et al., 2003), the tissue transfer most of the absorbed nutrients to the nutrient concentration may nevertheless plant roots in exchange for sugar, especially increase because water stress curtails carbon glucose, which is the energy source for mycelium assimilation and shoot growth more than nutri- growth, spore production, and active nutrient ent uptake (Bloom et al., 1985). Nonetheless, uptake. The symbiotic microorganisms are both the concentration and the total amount of mainly known for their ability to enhance 2þ nitrogen in the leaves and shoots tend to be H2PO4 and zinc (Zn ) uptake, but they also þ lower in grapevines whose growth is slowed transfer sulfur (S) and copper (Cu2 ) (Marschner, by competition with cover crops for access to 1995; Smith et al., 2001). They may also facilitate both water and nutrients (Celette et al., 2009; acquisition of N, K, Ca, Mg, and iron (Fe) under Tesic et al., 2007). certain conditions. Some mycorrhizae can Nitrogen availability fluctuates widely apparently tap directly into plant-unavailable throughout the growing season, whereas the organic material, such as decaying plant or ani- availability of the relatively mobile calcium mal remains, where N predominantly occurs as (Ca) and magnesium (Mg), and of the nearly proteins. The fungi promote their decomposi- immobile phosphorus (P) and potassium (K), tion and mineralization, and then they capture is much more constant (Nord and Lynch, and transfer to the roots the resulting inorganic et al 2009). Potassium and phosphate (H2PO4 ) can N in addition to P (Hodge, 2006; Hodge ., be taken up by mature root sections, whereas 2001; Leigh et al., 2009). Furthermore, these þ þ absorption of Ca2 and Mg2 seems to be fungi grow readily toward organic, and hence restricted to the unlignified fine roots. Iron nutrient-rich, patches in the soil, where their þ (Fe2 ) uptake is confined to the growing root mycelium proliferates for the benefit of their 2 tips, whereas sulfate (SO4 ) uptake is concen- host plant. However, their carbon requirement trated in the elongation zone immediately makes the mycorrhizae sinks for photosyn- behind the meristematic region. In addition to thetic assimilates (Hall and Williams, 2000; the fibrous fine-root system most effective in Smith et al., 2001). The supply of sugar to the nutrient uptake, the older regions of grapevine fungi represents the price paid by the vine for roots usually form symbiotic (i.e., mutually enhanced nutrient uptake. Once established, beneficial) units with mycorrhizal fungi the fungi ostensibly suppress lateral root (Gebbing et al., 1977; Possingham and Groot growth, making the plant more dependent on Obbink, 1971; Stahl, 1900). These microbes nutrients supplied by the mycorrhiza (Osmont infect the roots and provide an additional link et al., 2007). Because of the carbon cost to the between the vine and the soil, living partly plant of establishing and maintaining the sym- within the root cortex, whereby the fungal biosis, young plants initially tend to grow more and plant cytoplasms remain separated by the slowly in mycorrhiza-“contaminated” soil cell membranes of the two partners, and partly than in fumigated soil, especially if the soil is within the surrounding soil (Bucher, 2007). The adequately supplied with nutrients (Clarkson, fungi can absorb water and nutrients at con- 1985). In soils with abundant nutrient avail- siderable distance from the root and thereby ability, root colonization by mycorrhizae greatly extend the soil volume exploited by the is usually much slower because in an environ- roots by forming an extensive and interconnected ment with plentiful resources there is no need hyphal network that can even link different for a plant to “waste” carbon on the symbionts. 246 7. ENVIRONMENTAL CONSTRAINTS AND STRESS PHYSIOLOGY

Nutrient ion uptake is mainly controlled by abundantly available in the soil solution—for demand of the vine and varies according to example, after fertilizer application or incor- growth requirements, even as soil nutrients poration and decomposition of cover crops. differ in concentrations over several orders of Although growth and crop yield may not magnitude. Thus, growth is the “pacemaker” increase further, the extra supply leads to accu- for nutrient uptake, and the rates of uptake and mulation inside the vine (Delas and Pouget, root growth reflect the demand created by plant 1984; Keller et al., 1995; Pouget, 1984). Because growth (Clarkson, 1985; Clarkson and Hanson, grapevines cannot uproot themselves and relo- 1980; Gastal and Lemaire, 2002; Tester and cate to search for new resources, this inappro- Leigh, 2001). Correlations between plant growth priately named “luxury consumption” can be and nutrient supply may simply indicate that useful for temporary storage of nutrients, such nutrient availability limits grapevine growth. as nitrate or phosphate, that are subject to wide Therefore, both nutrient uptake (Keller et al., temporal and spatial fluctuations in the soil 1995) and shoot growth (Keller and Koblet, (Clarkson, 1985; see also Chapter 5.3). Storage 1995a; Spayd et al., 1993; Williams and within plant cells occurs in the vacuoles, Matthews, 1990) of grapevines show satura- whereas the cytoplasm is well buffered against tion-type responses to increasing soil nitrogen. changes in nutrient concentration (Forde, 2002; Recycling in the phloem of xylem-delivered Tester and Leigh, 2001). As a result, the concen- nutrients or of their assimilated versions from tration of nutrient ions may be three to four the leaves to the roots acts as a feedback signal orders of magnitude lower in the cytoplasm system to regulate nutrient uptake. For example, than in either the vacuole or the apoplast. The nitrate transporters in the roots can be “shut” storage pools serve as an insurance against in response to high concentrations of amino temporary shortages in supply; they can be acids or sugars, and sulfate transporters accessed to sustain growth when nutrient avail- are repressed by cysteine and glutathione ability is low or to permit regrowth in spring or (Amtmann and Blatt, 2009; Forde, 2002; following catastrophic events such as drought, Grossman and Takahashi, 2001). Such feedback fire, or cold damage (Bloom et al., 1985). regulation also fine-tunes potassium uptake However, even if their availability increases in according to shoot demands (Tester and Leigh, unison, all nutrients are not necessarily taken 2001; Ve´ry and Sentenac, 2003). Furthermore, up and accumulated equally; for example, a plant’s carbon status impacts nutrient uptake, N, P, and K may accumulate throughout the at least partly because of the need for energy to vine, including the fruit, at the expense of Ca run ATP pumps. Under conditions of carbon and Mg (Delas and Pouget, 1984). depletion—for instance, when photosynthesis When vacuolar nutrients reach a critical is limited by environmental constraints such as minimum concentration, the cytoplasmic con- low light—vines reduce nutrient uptake and centration cannot be sustained and metabolism transport in the xylem (Keller et al., 1995). A glu- is disturbed. Insufficient supply of a nutrient cose phosphate is thought to be the sugar signal ion slows shoot growth to a rate consistent with that synchronizes nutrient uptake to photosyn- supply (Clarkson, 1985; Gastal and Lemaire, thesis (Lejay et al., 2008). The decrease in vine 2002). Leaf expansion, for example, is particu- growth due to water deficit (see Chapter 7.2) larly sensitive to fluctuations in N supply also decreases the nutrient requirements of (Forde, 2002). Nutrient deficiency triggers a grapevines. range of responses in the vine, some of which Nevertheless, nutrient uptake can exceed are general stress responses, whereas others growth requirements when nutrients are are specific for the nutrient in question 7.3. NUTRIENTS: DEFICIENCY AND EXCESS 247

C (Grossman and Takahashi, 2001). General no effect on leaf), restricted leaf expansion and responses include cessation of cell division senescence of older leaves, and increased root: and cell expansion (i.e., reduced growth), shoot ratio (Clarkson et al., 2000; Wilkinson changes in vine morphology (e.g., increased and Davies, 2002). This response leads to a root:shoot ratio), starch accumulation, decrease decrease in water uptake and transport, which is in photosynthesis, and modification of metabo- probably mediated by closure of aquaporins lism to adapt to the limited nutrient supply. (Maurel et al., 2008). If different roots of the same Accumulation of reactive oxygen species also vine experience differences in nutrient availabil- appears to be a general response that is com- ity, it makes sense for the plant to reduce water mon to at least N, K, P, and S deficiency uptake from the depleted soil and compensate (Schachtman and Shin, 2007). Another frequent by increasing uptake from the nutrient-rich areas. reaction to mineral nutrient deficiency is the Nutrient limitation interferes with cell divi- accumulation of amino acids such as arginine, sion by preventing the normal progression of glutamine, and asparagines and the polyamine the cell cycle so that the time it takes for a cell putrescine (Rabe, 1990). to divide increases (Gastal and Lemaire, 2002; Specific responses to limited supply of a par- Grossman and Takahashi, 2001; Lemaire and ticular nutrient include induction of transport Millard, 1999). Moreover, meristematic cells systems to enhance nutrient uptake and remo- are not directly connected to the vine’s vascular bilization of stored reserves of that nutrient. In tissues, and the very small vacuoles of these addition, because plants can only make opti- small, dividing cells permit little nutrient stor- mum use of a particular nutrient if no other age. Therefore, these cells are more sensitive nutrient is limiting, deficiency in one nutrient to fluctuations in nutrient delivery via the tran- often also impacts the uptake and transport of spiration stream than are mature cells. In devel- other nutrients (Amtmann and Blatt, 2009; oping leaves, both cell division and cell Keller et al., 2001). When a portion of a root sys- expansion decline rapidly in response to N tem is in soil that is depleted of a particular deficiency. This reaction is, at least in part, nutrient, plants often enhance growth and caused by a decrease in the production of cyto- uptake mechanisms in another portion of the kinin from isoprenoids in the root tips (nitrate roots growing is soil where supply of that and phosphate stimulate cytokinin production) nutrient is still adequate. Although such local and its transport in the xylem to the shoots in increases in nutrient acquisition can compen- addition to reduced cytokinin production in sate for differences in nutrient supply arising the young, expanding leaves (Coruzzi and from highly heterogeneous soil conditions Zhou, 2001; Forde, 2002; Kakimoto, 2003; Takei (Nibau et al., 2008; Scheible et al., 1997), a et al., 2002). Lack of cytokinin also seems to decline in shoot growth similar to that in inhibit lateral bud outgrowth (Ferguson and response to PRD (see Chapter 7.2) often occurs Beveridge, 2009) so that low-N vines produce when nutrients such as N or P are available to fewer and shorter lateral shoots (Keller and only a portion of the roots (Baker and Milburn, Koblet, 1995a). Root growth, on the other hand, 1965; Robinson, 1994). Nutrient deficiency, par- may well increase, notably in response to limit- ticularly N and P deficiency, can also trigger other ing supplies of N (Keller and Koblet, 1995a) plant responses that are very similar to effects of and P but probably also for K and S. In contrast soil water deficit, namely increased ABA and to their action in shoot organs, cytokinins reduced cytokinin production, increased pHxylem inhibit root growth (Kakimoto, 2003; Werner which increases guard cell sensitivity to ABA), et al., 2003), perhaps by stimulating the produc- r r increased h and s, decreased transpiration (but tion of the growth-inhibiting gas ethylene 248 7. ENVIRONMENTAL CONSTRAINTS AND STRESS PHYSIOLOGY

(Dugardeyn and Van Der Straeten, 2008). As root:shoot ratio (Clarkson and Hanson, 1980). the amount of cytokinins declines, root growth When grafted grapevines are planted, root- increases. This means that when nutrients limit stocks with efficient nutrient uptake could be overall plant growth, roots become relatively chosen to minimize fertilizer input. On the stronger sinks for assimilates than shoots in other hand, inefficient rootstocks might reduce order to alleviate the deficiency by improving vigor on fertile sites. nutrient uptake from previously untapped soil Among mineral nutrients required for plant regions where higher nutrient concentrations growth, N, P, K, Ca, Mg, and S are usually may be found (Clarkson, 1985). classed as macronutrients, and the remaining Slow growth and the appearance of chlorosis essential nutrients Cl, B, Fe, manganese (Mn), (chlorophyll breakdown leading to leaf yellow- zinc (Zn), copper (Cu), molybdenum (Mo), ing), necrosis (cell death), and senescence of and nickel (Ni) are classed as micronutrients. leaves are typical symptoms of mineral nutrient This division is arbitrary and merely reflects deficiency in grapevines. The shedding of older the fact that different nutrients are required in leaves is often accompanied by remobilization vastly different amounts to satisfy vine growth. of nutrients in those leaves and redistribution The tissue concentrations of both macro- and to developing organs such as young leaves micronutrients vary severalfold across different and grape berries. This is particularly true of grape cultivars (Christensen, 1984). Depending the phloem-mobile ions of N, P, K, S, Mg, Na, on cultivar, rootstock, and seasonal weather and chloride (Cl), whereas phloem-immobile conditions, the amounts of macronutrients nutrients, such as those of Ca and boron (B), can- removed from a vineyard per ton of harvested not be redistributed. Consequently, deficiency grapes are in the range 1–3 kg N, 0.2–0.4 kg P, symptoms of phloem-mobile nutrients are first 1.5–4 kg K, 0.2–1 kg Ca, and 0.05–0.2 kg Mg apparent on older leaves, whereas deficiency (Bettner, 1988; Conradie, 1981a; Currle et al., symptoms of phloem-immobile nutrients are 1983; Mullins et al., 1992; Schreiner et al., usually confined to young, growing organs. 2006), with additional amounts being The amount of minerals accumulated by dif- incorporated into the growing permanent ferent Vitis genotypes may vary two- or three- structure of the vines. The lower end of these fold. Moreover, rootstocks influence the ranges applies only where stalks and pomace concentration of nutrients in scion leaves (Trieb are recycled to the vineyard. Deficiency of and Becker, 1969), which has implications for micronutrients, especially B, Fe, Mn, and Zn, fertilizer recommendations based on tissue is relatively common in vineyards throughout nutrient tests. Species, cultivars, and rootstocks the world (Mullins et al., 1992). Although that are more efficient in nutrient uptake than grapevines naturally acquire nutrients via root others can produce more growth from a given uptake from the soil solution, it is possible to amount of absorbed nutrient ion (Clarkson alleviate nutrient deficiencies in the short term and Hanson, 1980; Scienza et al., 1986). This is by foliar sprays. Nutrient ions that are dis- especially important when nutrient availability solved in water (and only then) can diffuse is limited. Nutrient use efficiency is the recipro- through plant cuticles, probably via aqueous cal of the nutrient cost of growth (Bloom et al., polar pores in the cuticular membrane, and 1985). Therefore, efficient species or cultivars can then be taken up by leaf cells and frequently have lower average tissue concen- distributed via the apoplast (Scho¨nherr, 2000, trations of nutrients than those of inefficient 2006). Diffusion increases in proportion to the species or cultivars. However, the former often amount of nutrient ions applied and is inde- produce larger root systems and hence a larger pendent of temperature but increases with 7.3. NUTRIENTS: DEFICIENCY AND EXCESS 249 increasing air humidity (Scho¨nherr, 2000; budbreak of unfertilized vines (Roubelakis- Schreiber et al., 2001). In addition, nutrients Angelakis and Kliewer, 1979). Even newly may also be taken up through the stomata planted rooted cuttings initially grow almost (Eichert and Goldbach, 2008). exclusively from stored reserves (Groot Obbink et al., 1973). Remobilization from the reserve 7.3.1. Macronutrients pool seems to be independent of soil N avail- ability, and poor N reserve status due to inade- Nitrogen quate refilling in the previous growing season The role of N and its uptake, assimilation, can restrict early shoot growth and canopy and transport in the vine are discussed in development and probably flower develop- Chapter 5.3. Grapevines normally acquire N ment as well (Keller and Koblet, 1995a). Uptake in the form of inorganic nitrate (NO3 ), increases progressively through bloom, fruit although most of the N in a typical vineyard set, and the first phase of berry growth and soil is bound in organic matter, which is nor- may increase further after veraison. In some mally unavailable for direct uptake by the cases, uptake has been found to pause once roots. Such organic material is decomposed shoot growth has ceased (Lo¨hnertz et al., and mineralized, or converted to plant- 1989), but in other cases the uptake rate was available NO3 , by soil microbes. In addition, at its peak during this preveraison period, rain and lightning may deposit some N that is sometimes—especially in warm climates— present in the atmosphere due to fossil fuel followed by another rise after harvest and volcanic emissions. However, the distri- (Conradie, 1980, 1986). It seems likely that such bution of both organic matter and available discrepancies reflect differences in soil mois- inorganic N in the soil is extremely heteroge- ture during summer because both shoot growth neous, both in time and in space. Nitrate con- and nutrient uptake slow in drying soil (Keller, centrations in the soil water can vary over 2005). When water is not limiting, maximum N several orders of magnitude, from a few micro- uptake may occur during the warmest portion moles to approximately 100 mM, even over of the growing season. In any case, uptake until short distances and time spans (Keller et al., the end of bloom constitutes less than 30% 1995, 2001b). Because of the rapid shoot growth of the seasonal demand, although the leaves in spring, a vine’s N demand is greatest may accumulate up to 60% of their seasonal N between budbreak and bloom, even though requirement. Thus, storage reserves reach a most of the N uptake from the soil occurs after minimum around bloom time or even later bloom, provided there is sufficient soil mois- (Schaller et al., 1989; Weyand and Schultz, ture (Currle et al., 1983; Peacock et al., 1989). 2006b), which makes vines vulnerable to defi- During this period, the vine is heavily depen- ciency if insufficient N is available in the soil. dent on the N reserves stored in the permanent However, the strong dependence on reserve N structure because the roots absorb very little N after budbreak also means that fertilizer appli- from the soil before five or six leaves have cation is ineffective at the start of the growing unfolded on the shoots (Lo¨hnertz et al., 1989; season to alleviate an existing deficiency that Treeby and Wheatley, 2006). In fact, nutrient arises from low reserve status, whereas the application has little effect on bleeding sap ability of vines to absorb N after harvest in suf- composition (Andersen and Brodbeck, 1991), ficiently warm climates permits enhanced and the bleeding sap of grapevines fertilized refilling of storage reserves through late-season with up to 672 kg N ha 1 contained similar fertilizer supply (Conradie, 1980, 1986; Holzapfel amounts of NO3 as that exuded during and Treeby, 2007; Treeby and Wheatley, 2006). 250 7. ENVIRONMENTAL CONSTRAINTS AND STRESS PHYSIOLOGY

Whereas overall high N soil status inhibits Takei et al., 2002). Conversely, general N starva- lateral root growth and leads to a reduction in tion results in reduced plant growth and yield, the root:shoot ratio (Erlenwein, 1965a; Keller gradual chlorosis of older leaves (i.e., they turn et al., 1995), localized N supply (e.g., through pale green and later yellow), and early abscis- a drip irrigation system) to a low N soil stimu- sion of those leaves (Scienza and Du¨ ring, lates lateral root growth in the new high N 1980). It seems likely that such premature leaf zone to enhance N uptake there (Lo´pez-Bucio senescence is triggered by a combination of et al., 2003; Nibau et al., 2008; Scheible et al., sugar accumulation and low N status in the 1997). Despite the increase in root growth, leaves (Figure 7.3) (Wingler et al., 2009). Old water flow through the roots generally leaves become an alternative source of N for decreases due to higher hydraulic resistance the roots and other sink organs when N uptake (Chapin, 1991). Like other plants, grapevines by the roots fails to meet sink demand with an optimum supply of N and water make (Hikosaka, 2005). longer use of the growing season by extending Nitrogen starvation increases ABA produc- the effective life of their leaves (Alleweldt et al., tion in the roots and decreases the amount of 1984a; Lawlor, 2002). When vines are grown on cytokinins, whereas N resupply increases cyto- N-rich soil, the chlorophyll degradation that kinins (Chapin, 1991; Wilkinson and Davies, accompanies autumn senescence of the leaves 2002). Both ABA and cytokinins transported to is delayed (Keller et al., 2001b), perhaps at least the shoots in the xylem sap mediate informa- partly because nitrate uptake leads to increased tion on nutrient status in plants. Enhanced production and export from the roots of the cytokinin delivery to the shoots may be one antisenescence hormone cytokinin (Hwang possible reason for the delayed lignification and Sakakibara, 2006; Sakakibara et al., 2006; that is sometimes found in vines with high N ) − 2 Chlorophyll (mg dm Chlorophyll

ha−1

µ −2 −1 Photosynthesis ( mol CO2 m s )

FIGURE 7.3 Association between leaf photosynthesis and chlorophyll content in Mu¨ ller-Thurgau grapevines grown under two long-term N fertilizer regimes (left; from Keller, 2005) and increased lateral shoot growth and delayed leaf senes- cence on vine with high N status (left plant on right) compared with low-N vine (right plant on right; photo by M. Keller). 7.3. NUTRIENTS: DEFICIENCY AND EXCESS 251 status; cytokinins reduce xylem formation and N supply decreases the demand for carbon lignification (Groover and Robischon, 2006). skeletons for N metabolism and generally sup- Because cytokinins stimulate cell division, the presses growth more than photosynthesis, growth-depressing effect of N deficiency could sugar export declines and starch accumulates also be related, at least in part, to the effect of N in the leaves (Chapin, 1991; Chen and Cheng, on cytokinin synthesis: All cytokinin molecules 2003b; Gastal and Lemaire, 2002; Lawlor, 2002). contain five N atoms within the structure At least part of the decrease in photosynthesis formed by the attachment of an isoprene func- of N-deficient leaves may be caused by feedback tional group to ATP or ADP. Through the sup- inhibition as a consequence of carbohydrate pression of cell division, N deficiency can accumulation (Hermans et al., 2006). When this inhibit shoot growth at any time during the is accompanied by oxidative stress arising from growing season (Gastal and Lemaire, 2002). too much light absorption, the leaves—even Whereas cytokinins stimulate cell division in those of white cultivars—also produce photo- the shoot apical meristem and promote lateral protective anthocyanins in the petioles, leaf bud outgrowth (Ferguson and Beveridge, veins, and sometimes in the leaf blade and 2009), they inhibit cell division in the root api- cluster peduncles, which therefore turn red cal meristem. A decrease in cytokinin produc- (Figure 7.4) (Currle et al., 1983; Ga¨rtel, 1993; Lillo tion thus decreases main and lateral shoot et al., 2008 ). Incidentally, although malvidin- growth and enhances root growth. The sensi- based pigments tend to dominate the anthocya- tivity of leaf initiation and expansion to fluctua- nin profile in the berries of many dark-skinned tions in N supply results in marked changes in cultivars (see Chapter 6.2), cyanidin and peoni- the leaf area:plant weight ratio. din derivates are much more abundant in Approximately half of the leaves’ N is the leaves (Wenzel et al., 1987; M. Keller, unpub- invested in photosynthetic hardware, especially lished data). Conversely, the yellowing of rubisco and other enzymes, and photosynthetic deficient and senescent leaves is caused by capacity is strongly correlated with N supply unmasking and partial retention of carotenoids (see Chapter 5.2 and Figure 5.5). By limiting (see Chapter 4.1) that accompanies chlorophyll the amount and activity of these proteins, N breakdown rather than by new production of deficiency also restricts photosynthesis (Chen yellow pigments. and Cheng, 2003b). This enzymatic inhibition Surplus sugar in the leaves may also become r in turn increases s and also results in a poten- available for export to the roots (Lemaire and tial “energy overload.” To avoid excessive light Millard, 1999). In contrast to the response to absorption that would damage the photo- water deficit, the leaf sugar is not needed for synthetic apparatus, leaves disassemble a por- osmotic adjustment. Similar to their reaction tion of their chlorophylls (i.e., they become to water stress, however, leaves decrease the chlorotic) and activate their thermal energy- angle between leaf blade and petiole (see dissipation and antioxidant systems (Chen Figure 7.4), which further curbs light absorp- and Cheng, 2003a; Keller, 2005). In other words, tion to avoid an energy surplus. In addition, the reduced chlorophyll content of N-starved leaves of N-deficient grapevines tend to have vines (Keller et al., 2001b; Spayd et al., 1993) is a a thicker epidermis (Ru¨ hl and Imgraben, 1985). consequence, not a cause, of the decrease in Another similarity with water stress is that photosynthesis (see Figure 7.3). The N contained under prolonged N stress grapevines start in the leaf proteins is recycled to young leaves shedding older leaves following the remobiliza- (see Chapter 5.3) so that older leaves are the tion of nutrients in those leaves and redistribu- first to become chlorotic. Because inadequate tion to developing organs such as young leaves 252 7. ENVIRONMENTAL CONSTRAINTS AND STRESS PHYSIOLOGY

FIGURE 7.4 The angle between the leaf blade and the petiole in a nitrogen-sufficient (left) and a nitrogen-deficient Mu¨ ller-Thurgau grapevine (right). Also note the absence of lateral shoot growth and the red pigmentation of the petiole on the right. Photos by M. Keller. and growing fruit (Currle et al., 1983; Ga¨rtel, also seems to aggravate the vulnerability of 1993; Lawlor, 2002). Taken together, the similar- fruit set to water deficit (Keller et al., 1998). ity of vine responses to water and N deficit Low N status may sometimes (Hilbert et al., suggests that the combined effects of these 2003; Keller et al., 1998; Rogiers et al., 2004b), two stress factors are predominantly additive although not always (Keller et al., 2001a), be rather than interactive. Thus, vines growing associated with berries growing larger. This is under low soil moisture, present during a probably a consequence of diminished sink drought or deficit irrigation, may require a number due to poor fruit set. A restriction in somewhat higher N supply to achieve photosyn- crop load at the beginning of berry develop- thetic rates as high as those of fully irrigated ment tends to result in compensatory growth vines (Alleweldt et al., 1984a; Rogiers et al., of the remaining berries, unless such compen- 2004b). Insufficient N also appears to sensitize sation is prohibited by the effects of water defi- plants to subsequent water stress, possibly cit (Keller et al., 2008). Compensatory berry because the increased ABA production induces growth might partly be possible because of lim- C the stomata to close at higher leaf, and acceler- ited competition from shoot meristems, whose ates nutrient remobilization followed by leaf growth is curtailed by N deficiency. This may senescence and abscission during water stress. leave more photosynthates available for export On the other hand, abundant water and/or N to the grapes, which in turn would also may make vines more susceptible to stress when increase water uptake by the berries. In any one of them suddenly is in short supply. case, grapes with a low N content often result Insufficient N availability during bloom in musts that are prone to sluggish or stuck fer- interferes with flowering and reduces fruit set mentations (Bell and Henschke, 2005; Spayd and cluster initiation in the buds, although too et al., 1995; see also Chapter 6.2). much N also decreases cluster initiation (Ewart Excessive application of N fertilizer can also and Kliewer, 1977; Keller and Koblet, 1995a, pose problems, especially in young vines 2001a; Spayd et al., 1993). Low plant N status whose small root system may temporarily be 7.3. NUTRIENTS: DEFICIENCY AND EXCESS 253 exposed to very high concentrations of soil N Phosphorus following fertilizer application. Very high The anion phosphate (H2PO4 ; often abbre- NO3 concentrations may decrease the osmotic viated P for inorganic phosphate) is a major potential (Cp) of the soil solution, which can i inhibit growth and decrease stomatal conduc- substrate in energy metabolism (ATP contains tance due to insufficient water uptake (Keller three phosphate groups; see Chapters 4.1 and and Koblet, 1994), similar to the impact of salin- 4.4) and synthesis of nucleic acids (as phosphate ity discussed later. In bearing vines, the vigor- groups in DNA and RNA), membranes (as a ous growth and often dense canopies that constituent of phospholipids), carotenoids, and accompany high soil N status can have unde- gibberellins. It also plays an important role in sirable consequences for fruit composition (see photosynthesis (via photophosphorylation), res- Chapter 6.2) and vulnerability to diseases (see piration, and the regulation of a number of Chapter 7.5). In addition, vigorous vines with enzymes. Furthermore, phosphorylation of high N status ostensibly are also more vulnera- aquaporins regulates water transport across ble to physiological disorders such as bunch membranes: Phosphorylated aquaporins are active—that is, open (Maurel et al., 2008). In stem necrosis (Christensen and Boggero, 1985; 4 Currle et al., 1983; Keller et al., 2001a). In some addition, inorganic pyrophosphate (PPi;P2O7 ), cases, however, N deficiency may cause a a by-product of starch production, is essential higher incidence of bunch stem necrosis (Capps for phloem function (Koch, 2004). Because of its and Wolf, 2000). importance, the leaf P content increases roughly Soil N status may also have implications for as the four-thirds power of the leaf N content: P 4/3 the availability of other nutrients. Higher N (Niklas, 2006; Niklas and Cobb, 2006). amounts of available N can enhance the solubil- This relationship also holds approximately true þ þ þ ity of cations such as K ,Ca2 , and Mg2 and across different grape cultivars (calculated from data in Christensen, 1984). Thus, the seasonal reduce it for anions such as H2PO4 (Keller et al., 1995). Higher cation availability may be pattern of P uptake by grapevines from the soil coupled with enhanced uptake by grapevines more or less mirrors the pattern for N uptake (Keller et al., 2001b), but it may also increase the (Conradie, 1980, 1981). potential for rainfall-driven leaching of these Phosphorus is one of the least available of all nutrients (Perret and Roth, 1996). Over time, essential nutrients because it is extremely insol- excessive N application to vineyard soils might uble, and hence immobile, due to its affinity to þ þ þ therefore increase the likelihood for nutrient cations such as Ca2 ,Mg2 , and Al3 , with deficiencies to develop. In contrast, abundant N which it forms insoluble complexes, and its supply sometimes (Spayd et al., 1993), but not conversion into organic forms in the soil always (Hilbert et al., 2003; Keller et al. 1995, (Raghothama, 1999). Under weakly acidic con- 2001b), decreases P uptake, perhaps because an ditions, P occurs mostly in the form of N-induced reduction in root carbohydrate status H2PO4 , whereas under weakly alkaline condi- 2 occasionally limits carbon availability for tions it changes to HPO4 . Its concentration in mycorrhizal fungi. Furthermore, because NO3 the soil solution is generally below that of þ uptake is coupled to influx of protons (H ) and many micronutrients; the typical P concentra- release of OH , the pH in the immediate vicinity tion is less than 10 mM, whereas the concentra- of the roots—the so-called rhizosphere—rises tion in the cell cytosol is 5–10 mM. Especially with increasing N uptake. In extreme cases, such in acid soils (pH 5.5), which are common in alkalinization can lead to iron-deficiency- Bordeaux, France, and in the northeastern induced leaf chlorosis. United States, P occurs as H3PO4 and is the 254 7. ENVIRONMENTAL CONSTRAINTS AND STRESS PHYSIOLOGY major limiting nutrient (Kochian et al., 2004). enhance the P available to the roots (see Fig- This is one reason why such soils are often ure 7.2), as well as increase the availability of þ 2þ ameliorated by the application of lime (CaCO3) potassium (K ) and manganese (Mn ). Grape- during vineyard establishment. Moreover, P vines also rely heavily on mycorrhizal fungi for availability is normally highest in the topsoil P uptake, and P-starved plants ostensibly and decreases sharply with soil depth. As a encourage colonization of their roots by mycor- consequence, fertilizers with high phosphate rhiza via exudation of sesquiterpenes that help concentration are often applied to improve P the fungi to establish root contact (Bais et al., availability; unfortunately, however, a consid- 2006). erable portion of this supplementary P can A decrease in leaf number and size is one of leach from vineyards into nearby rivers and the earliest and most reliable symptoms of P lakes, where it may lead to proliferation of deficiency. Low P supply limits cell division, algae (algal blooms), oxygen depletion, and restricting leaf initiation in the shoot apical death of fish. meristem and expansion of newly developed et al Roots take up P mainly as H2PO4 (Gross- leaves (Chiera ., 2002). Reduced cell divi- man and Takahashi, 2001). Growing roots initi- sion may also be responsible for the inhibition ally absorb P in the root tips and root hairs, but of cluster initiation or differentiation and of lat- this uptake rapidly depletes available P close to eral shoot growth in P-deficient vines (Ga¨rtel, the roots (Bucher, 2007; Gilroy and Jones, 2000). 1993; Grant and Matthews, 1996; Skinner et al., Decreasing P availability leads to a decrease in 1988). Phosphate deficiency can also increase r r the growth of main roots and an increase in the s and h so that the reduction in water supply growth of lateral roots and the formation of to growing organs restricts cell expansion, root hairs (Hermans et al., 2006; Lo´pez-Bucio which in turn strongly limits leaf expansion et al., 2003; Nibau et al., 2008; Osmont et al., (Clarkson et al., 2000). Therefore, although as a 2007; Raghothama, 1999). This shift in root phloem-mobile nutrient P can be recycled from growth pattern may be mediated by auxin that old leaves to support growing organs, P defi- accumulates because its movement is blocked ciency primarily restricts the sink activity of and results in a denser but shallower root sys- shoot meristems, and the resulting lack of tem and an increase in the root:shoot ratio demand for assimilates in growth eventually (Malamy, 2005; Potters et al., 2009). Although leads to sugar and starch accumulation in this adaptive strategy enables vines to better source leaves (Hermans et al., 2006; Kochian explore the rather more P-rich surface soil, it et al., 2004). Phosphorus deficiency reduces also makes them more vulnerable to water source activity by decreasing leaf photosynthe- stress when the soil dries out. This may be sis and assimilate partitioning, partly because why the response is limited to situations in there is insufficient Pi for ATP synthesis and which the entire root system, rather than just partly because the leaves do not fully activate portions of it, experiences P deficiency their rubisco (Paul and Foyer, 2001; Woodrow (Schachtman and Shin, 2007). The roots of some and Berry, 1988). The latter could be due to P-deficient species also exude organic acids feedback inhibition by excess sugar and seems such as citrate and malate that acidify the rhi- to be especially pronounced at temperatures zosphere and solubilize P from rock phosphate below approximately 15C (Hendrickson et al., (Dakora and Phillips, 2002; Hammond and 2004b). Nonetheless, sugar export via the White, 2008; Marschner, 1995). In addition, P phloem to the roots may continue to support deficiency results in proton extrusion to acidify lateral root branching (Hammond and White, the soil, and the lower soil pH can greatly 2008). Deficiency also leads to salvage of Pi 7.3. NUTRIENTS: DEFICIENCY AND EXCESS 255

þ from nucleic acids and phospholipids through- synthase, as well as H -ATPases and other out the plant, and the latter are replaced by transport proteins (Amtmann and Blatt, 2009; galacto- and sulfolipids (Schachtman and Shin, Clarkson and Hanson, 1980). In addition to its þ 2007). Moreover, P-deficient leaves remain role in membrane transport, K also stimulates small and dark grayish-green rather than turn- the loading of sucrose into the phloem for ing chlorotic as in other cases of nutrient export, perhaps because it helps maintain the starvation (Ga¨rtel, 1993; Hammond and White, electrical neutrality that is necessary to gener- þ 2008). Severely P-deficient leaves may turn ate pH gradients by H -ATPases (Lalonde red due to anthocyanin production from accu- et al., 2004; Marten et al., 1999; see also Chapter þ mulated sugars that are not exported from the 5.1). The K concentration in the soil solution leaves (Currle et al., 1983; Grossman and Taka- varies from approximately 0.1 to 6 mM, hashi, 2001; Lillo et al., 2008). This pigmentation whereas its concentration in the cell cytosol is is probably a photoprotective reaction because approximately 100 mM (Ashley et al., 2006). þ r the decline in photosynthesis is associated with Transport of K in the xylem affects h and, surplus energy (i.e., light absorption and elec- thus, sap flow rate (see Chapter 3.3). Whereas tron transfer proceed even as CO2 fixation initial growth of grapevines after budbreak is declines) that results in oxidative stress (Feild dependent on K reserves mobilized from the et al., 2001; Hoch et al., 2003). In addition, in- permanent structure of the vine, uptake from sufficient P supply appears to restrict Mg trans- the soil increases well before bloom and pro- port in the xylem, which can lead to symptoms ceeds more or less linearly throughout the of Mg deficiency (Skinner and Matthews, 1990), growing season until a few weeks before leaf whereas excess P may lead to zinc and iron fall (Conradie, 1981b). þ deficiency (Ga¨rtel, 1993). Inadequate K supply strongly reduces xylem sap flow even under well-watered condi- Potassium tions, which limits shoot and fruit growth and þ Potassium (K ) is the most abundant cation greatly increases the risk of drought stress (a molecule with a positive charge) in plant (Currle et al., 1983). Potassium deficiency also cells. Its concentration in grape leaves is suppresses sugar transport in the phloem and approximately half that of N (Ga¨rtel, 1993). In can result in sucrose (but not starch) accumula- contrast with all other macronutrients except tion in the leaves to substitute for the missing þ calcium, K is not incorporated into organic K as osmoticum (Cakmak et al., 1994; Hermans compounds but remains in its ionic form et al., 2006). This explains not only why root (Robinson, 1994). As such, it is one of the cells’ growth cannot increase (and often stops) in major osmotic solutes and plays a key role in K-deficient plants but also why deficiency leads cell expansion (see Chapter 3.1) and stomatal to feedback inhibition of photosynthesis. With movement (see Chapter 3.2). Thus, rapid plant sucrose being “trapped” in the leaves and photo- þ growth and development require large K synthesis declining, the consequences for fruit fluxes to provide this ion to the growing tis- production and ripening can be severe. At the sues. This means that growth in general is sen- same time, its mobility in the phloem enables þ þ þ sitive to the vine’s K status. Cells also use K K to be recycled from older to younger organs to neutralize the negative charge of anions, and to move preferentially toward growing which helps maintain membrane potential and tissues or organs, including the fruit and the per- counterbalance the movement of other cations manent vine structure (Conradie, 1981b; Currle þ such as protons (H ). The protons are needed et al., 1983; Mpelasoka et al., 2003). Visual for the activity of enzymes such as ATP deficiency symptoms include glossy leaves, 256 7. ENVIRONMENTAL CONSTRAINTS AND STRESS PHYSIOLOGY

which results in poor fruit set. Deficiency symptoms are usually associated with an accu- mulation of polyamines (especially putrescine) in the shoots and leaves (Geny et al., 1997b). It has been proposed that it is this putrescine buildup that induces the visible leaf symptoms (see Figure 7.5); however, putrescine applica- tion may improve fruit set (Geny et al., 1997a, 1998). Polyamines are ultimately breakdown products of the amino acid arginine (via orni- thine or agmatine; Kusano et al., 2008), and as FIGURE 7.5 Leaf symptoms of potassium deficiency in K deficiency grows more severe, putrescine Syrah. Photo by M. Keller. may become the main organic soluble N com- pound. The cause and/or purpose of the accu- especially under high solar irradiance, often fol- mulation of polyamines during K deficiency is lowed by bronzelike discoloration from dying unknown. However, given that polyamines epidermis cells and pale-yellow leaf margins can block nonselective cation channels and þ (Figure 7.5). In more severe cases, the leaf mar- enhance active transporters (H -ATPases) in gins roll upward (“cupping”) and eventually plant cells (Kusano et al., 2008), it is possible become necrotic before the leaves are shed that they serve to prevent compensatory but (Currle et al., 1983; Ga¨rtel, 1993). These symp- detrimental accumulation of Na in the absence toms develop more quickly under high irradi- of K. Despite the concomitant decrease in argi- ance because the excited “surplus” electrons nine, however, grapes from K-deficient vines lead to oxidative stress (Marschner and Cakmak, may not result in sluggish or stuck fermenta- 1989). Leaf symptoms are also more severe on tions because yeast can readily metabolize heavily cropped vines due to the translocation polyamines. of K to the growing berries (Currle et al., 1983; Putrescine also accumulates in leaves, espe- Ga¨rtel, 1993) that, between bloom and harvest, cially of vigorously growing young vines, dur- accumulate far more K than any other nutrient. ing the stop-and-go growth caused by widely Indeed, mature grapes contain almost twice as fluctuating spring temperatures. This can lead much K as N and approximately 10 times as to a syndrome referred to as “spring fever,” much as P and Ca, which are the next most abun- whose symptoms are virtually identical to dant minerals in grape berries. Depending on those of K deficiency, even though the leaves crop level, the harvested fruit contains 50–75% have almost normal K concentrations (but ele- of a vine’s total amount of K (Conradie, 1981b; vated N concentrations). It is possible that the Rogiers et al., 2006). If the deficiency is not accumulation of putrescine is part of a cold relieved, shoot vigor declines gradually over acclimation response that enhances the leaves’ several years (Ga¨rtel, 1993). ability to survive freezing temperatures Deficient vines also become more suscepti- (Cuevas et al., 2008). The link between putres- ble to powdery mildew and to cold injury in cine and low temperature may also be the rea- winter (Currle et al., 1983; Ga¨rtel, 1993). More- son why K deficiency symptoms appear to be þ over, due to the importance of K as an osmo- more severe under cool conditions. In contrast ticum for pollen hydration and germination, to K deficiency, however, spring fever symp- and for pollen tube growth, K deficiency also toms are confined to the oldest leaves, and the interferes with pollination and fertilization, shoots usually outgrow the condition within a 7.3. NUTRIENTS: DEFICIENCY AND EXCESS 257 few weeks. Nevertheless, the berries often proteins in concert with iron-containing proteins remain small, and ripening is delayed. (via the so-called ferredoxin/thioredoxin sys- Potassium deficiency can result from compe- tem) regulate starch production and remobiliza- tition for root uptake by other cations present at tion in response to light and sugar supply. 2 high concentration in the soil solution. This is Sulfate (SO4 ) can be transported in both the possible under saline conditions, where sodium xylem and the phloem and temporarily stored þ (Na ) is the predominant dissolved cation but in the cell vacuoles. Glutathione is the major þ also where NH4 is the predominant nitrogen transport and storage form of reduced S; storage þ form. This is typical of acid soils (NH4 -based occurs mostly in the bark (Cooper and Williams, fertilizers in turn contribute to soil acidifica- 2004; Kopriva, 2006). Nevertheless, plant growth tion), defined as soils with a pH ¼ 5.5 (Kochian seems to depend on sustained uptake, usually et al 2 ., 2004). The roots often respond to inade- as SO4 , from the soil and transport in the þ quate K by growing sideways; that is, they xylem to the leaves, where it is reduced and deviate from their normal gravitropic growth, assimilated into cysteine, methionine, and other which may help them to explore (and exploit) organic compounds or partially reexported in 2 previously untapped soil patches that contain the phloem as SO4 or glutathione (Grossman þ more K (Ashley et al., 2006). Rootstocks differ and Takahashi, 2001; Leustek and Saito, 1999). þ in their ability to take up K and to release Insufficient S supply leads to a depletion of cys- þ K into the xylem; for instance, it appears that teine and glutathione pools, which is associated rootstocks derived from V. berlandieri do not with an increase in reactive oxygen species þ take up K as readily as those derived from (Schachtman and Shin, 2007). This is especially V. champinii. important because the production or release þ þ þ Excess K can decrease leaf Ca2 and Mg2 (possibly from glutathione or cysteine degrada- concentrations and induce symptoms of Mg tion) of elemental S or sulfide may be involved deficiency due to competition for uptake by the in the vine’s defense strategy against certain roots (Morris et al., 1980). This is particularly pro- fungal and bacterial pathogens (Cooper and nounced in young, nonfruiting vines, especially Williams, 2004; see also Chapter 7.5). Sulfur on sandy soils with a low pH (Ga¨rtel, 1993). deficiency could thus result in increased suscep- þ At very high concentrations (100–200 mM), K tibility to pathogen attack because only plants 2 can even inhibit enzymes, thus becoming toxic. growing at high SO4 availability are able to release sufficient S to counter invasion. Defi- Sulfur ciency symptoms include yellowing (chlorosis) Sulfur is an essential constituent of the amino of young leaves and stunted shoot growth. acids cysteine and methionine (and hence of pro- Sulfur-starved plants normally accelerate root teins), lipids, intermediary metabolites (acetyl- elongation, form prolific lateral roots close to CoA) in energy (ATP) generation and electron the root tip to enhance uptake, and decrease transport, and molecules (e.g., the cysteine- shoot growth so that the root:shoot ratio containing peptide glutathione) involved in the increases, perhaps in response to increased protection of tissues against oxidative stress auxin production (Lo´pez-Bucio et al., 2003; (Kopriva, 2006). Sulfur-containing compounds Schachtman and Shin, 2007). Application of (e.g., cysteine and glutathione) are used by cells S-based fungicides (predominantly elemental S; to counteract reactive oxygen species brought e.g., for powdery mildew control) may have on by such environmental stressors as drought, the added benefits of enhancing vine defense cold, heat, high light, and fungal attack (Noctor and at the same time countering S deficiency and Foyer, 1998). In addition, S-containing (Cooper and Williams, 2004). However, foliar S 258 7. ENVIRONMENTAL CONSTRAINTS AND STRESS PHYSIOLOGY application during hot conditions (>32C) can second messenger, whereby an external sti- induce burn symptoms on leaves, shoots, and mulus leads to an increase in reactive oxygen berries. species that results in temporary oscillations þ in the cytosolic Ca2 concentration; the period, Calcium frequency, and amplitude of these oscillations þ Calcium is unique among macronutrients in amount to a characteristic “Ca2 signature” þ that a high proportion of a plant’s Ca2 is that encodes information about the nature and located in the cell walls, where it serves as a strength of the stimulus (Hetherington and reinforcing agent. In fact, cell walls probably Brownlee, 2004; McAinsh and Pittman, 2009). þ owe their rigidity to the cross-linking of pectins For instance, ABA promotes release of Ca2 þ by Ca2 (Ferguson, 1984; Jarvis, 1984), which from the guard cell vacuoles and influx from also holds adjacent cells together (i.e., prevents the apoplast to induce stomatal closure (Allen þ them from sliding). Indeed, the Ca2 concentra- et al., 2001; Amtmann and Blatt, 2009), and tion in the apoplast is 300–1000 times higher auxin and gibberellin coordinate cell expansion þ than that in the cytoplasm, and most of this by inducing a rise in cytosolic Ca2 released þ Ca2 is tightly bound to pectic acids (as Ca- from intra- and extracellular stores. Calcium pectate) in the cell walls (Boyer, 2009). binds to a small protein termed calmodulin to Although, like K, Ca is not actually incorpor- modulate the activity of other proteins ated into organic molecules (Amtmann and (Scrase-Field and Knight, 2003; Snedden and þ þ Blatt, 2009), the ionic bonding of Ca2 contrasts Fromm, 2001). Moreover, Ca2 influx and a þ þ with apoplastic K , which is mostly soluble. cytosolic Ca2 gradient with increasing concen- þ Another important site of Ca2 accumulation tration toward the tip are necessary for the and storage, in the form of Ca-oxalate, is the directional growth of root hairs and pollen cell vacuole. Crystals of Ca-oxalate, especially tubes (Dutta and Robinson, 2004; Holdaway- the needle-shaped raphides, may serve a Clarke and Hepler, 2003; Ve´ry and Davies, defense role by deterring herbivores. Because 2000). þ of its ability to connect lipids and proteins at At high concentration, Ca2 can disrupt þ membrane surfaces, Ca2 is important in main- metabolism by precipitating phosphate as taining membrane integrity; it prevents mem- CaHPO4 (Plieth, 2005); thus, high amounts of brane damage and leakiness to solutes Ca are extremely toxic to cells. Plants utilize (Clarkson and Hanson, 1980; Hirschi, 2004). ATP-driven pumps (often regulated by cal- Because membrane leakage (i.e., loss of semi- modulin) to keep the concentration of cytosolic permeability) results in cell death, Ca delays Ca three or four orders of magnitude lower þ senescence and organ abscission. Ca2 ions than that of apoplastic and vacuolar Ca, allow- þ inhibit the Mg2 -dependent reactions of respi- ing only small Ca oscillations potentially gener- ratory and intermediary metabolism (Clarkson ated as signals by the pumps (Hetherington and Hanson, 1980) and alter the water perme- and Brownlee, 2004; Hirschi, 2004; McAinsh ability of membranes by modulating aquaporin and Pittman, 2009). Because plants have little þ activity (Johansson et al., 2000). Plants also control over Ca2 uptake, which occurs across require Ca to produce electrons, protons, and nonselective cation channels, they sequester Ca oxygen during photosynthesis (see Chapter as Ca-oxalate crystals inside specialized cells 4.1). In addition, Ca is involved in intracellular called idioblasts to prevent toxicity (Franceschi signaling in response to osmotic stress that and Nakata, 2005). The idioblasts are located arises from many environmental impacts in roots, leaves (especially along veins), (Plieth, 2005; Zhu, 2002). Calcium serves as a petioles, and fruit. This biomineralization takes 7.3. NUTRIENTS: DEFICIENCY AND EXCESS 259 the soluble Ca out of “circulation,” and the status can also improve survival after freezing crystals may serve as a storage form that can (see Chapter 7.2). be rapidly mobilized during Ca starvation and A shortage in Ca supply, similar to salinity, during early spring growth. leads to downregulation of aquaporins, which The Ca concentration in grape leaves is sim- in turn increases the hydraulic resistance in ilar to or higher than that of N (Ga¨rtel, 1993). roots and interferes with water flow to the þ Because Ca2 is virtually phloem immobile, it xylem (Luu and Maurel, 2005). In addition, is preferentially supplied to rapidly transpiring Ca deficiency also makes plants more suscepti- organs such as mature leaves and becomes ble to damage by salinity or low soil pH immobile once deposited in a particular organ; (Hirschi, 2004; Plieth, 2005). Natural Ca defi- in addition to the leaves, the bark seems to be a ciency is rare and occurs primarily on soils major sink for Ca in grapevines (Conradie, with very low pH because the highly toxic alu- þ 1981b). Therefore, deficiency symptoms appear minum (Al3 ) becomes very soluble at pH < 5 þ þ mainly in young, expanding leaves. Symptoms and blocks Ca2 uptake. Excessive soil K þ of Ca deficiency develop when Ca supply to (Delas and Pouget, 1984) and/or Mg2 and also cells cannot keep pace with Ca movement salinity can induce Ca deficiency. However, the þ þ to the vacuoles. Deficiency causes cell walls to availability of Ca2 (and Mg2 ) at the root sur- disintegrate and membranes to become leaky, face often greatly exceeds the demand by which causes cell death and can lead to the grapevines, especially in high-pH soils. There- collapse of affected tissues (Hirschi, 2004). fore, vines grown on calcareous soils generally This can lead to marginal leaf necrosis and shri- have considerably higher tissue Ca contents veling of fruit clusters beginning at the tip, (and often higher Mg but lower K contents) which resembles symptoms of bunch stem than vines grown on other soils. On such soils, necrosis (Ga¨rtel, 1993). Indeed, the appearance the amount of Ca in a vine (especially in its per- of bunch stem necrosis has been associated manent structure) can even exceed its content þ þ with insufficient Ca2 (and often Mg2 ) relative of N, which is normally the most abundant þ to K in rachis tissues, especially in the epi- mineral element in grapevines. Moreover, dermal and subepidermal layers (Currle et al., in soils with a pH > 7.5, the soils’ calcium car- et al 1983; Feucht ., 1975). Due to its importance bonate (CaCO3) can lead to precipitation of for pollen tube growth, Ca deficiency is also phosphorus, iron, zinc, copper, and manganese, detrimental to fertilization and fruit set. making these nutrients less plant-available. Although xylem-supplied Ca tends to accumu- Different Vitis species are adapted to a wide þ late in the stomatal regions of grape berries range of Ca2 in different soils and are often (Blanke et al., 1999), local deficiency in the fruit grouped as either calcicole (lime-tolerant or may be promoted by excessive canopy tran- adapted to calcareous or alkaline soils) or calci- spiration and rapid shoot growth diverting fuge (lime-sensitive or adapted to neutral to xylem flow, and thus Ca supply, away from acidic soils) species. For instance, V. labrusca and the fruit. This could be especially pronounced V. riparia are lime-sensitive, whereas V. berlandieri during periods of rapid berry cell expansion is lime-tolerant. þ that requires Ca2 for incorporation in new cell walls and membranes and as an intracellu- Magnesium þ lar signal. Excessive Ca supply, on the other Although Mg2 is a structural component of hand, may retard the rate of fruit ripening, chlorophyll, less than 20% of a vine’s total Mg possibly by preventing cell wall disassembly content is bound in chlorophyll. As a divalent þ (Ferguson, 1984). However, high plant Ca cation, Mg2 is an important cofactor required 260 7. ENVIRONMENTAL CONSTRAINTS AND STRESS PHYSIOLOGY to activate many enzymes (including the sucrose and starch in the leaves, whereas the key photosynthetic protein rubisco, the N- export of sucrose and amino acids declines assimilatory enzyme glutamine synthetase; see (Cakmak and Kirkby, 2008; Cakmak et al., 1994; also Chapters 4.2 and 5.3) and transport pro- Hermans et al., 2006). This explains why the teins (including the ATPase proton pumps roots of Mg-starved plants stop growing, similar responsible for phloem loading; see Chapter to the situation with K and Zn deficiency but in 5.1) (Cakmak and Kirkby, 2008; Clarkson and stark contrast to the response to N and P defi- Hanson, 1980). In contrast with most other ciency. The link between Mg status and sucrose nutrient ions, almost nothing is known about export may also be the reason for the observed þ how roots take up Mg2 and how they regulate negative correlation between petiole Mg content this uptake and subsequent transport. The and berry sugar (van Leeuwen et al., 2004). The þ pattern of Mg2 uptake by grapevine roots buildup of sugar in the leaves, in turn, leads to þ appears to be similar to that of K , starting feedback inhibition of photosynthesis and soon after budbreak and continuing until results in the degradation of chlorophyll, proba- shortly before leaf fall (Conradie, 1981b). Grape bly to mitigate excess light absorption leading leaves contain approximately 10-fold less Mg to oxidative stress (see Chapter 7.1). In fact, than N (Ga¨rtel, 1993). Mg-deficient plants are extremely light sensi- Magnesium deficiency is a quite common tive; high light intensity accelerates the appear- predicament of vines growing in sandy, very ance of the characteristic interveinal chlorosis acidic (pH <4.5) soils, where high concentra- of Mg-deficient leaves, and the deficiency symp- 3þ þ tions of aluminum (Al ), ammonium (NH4 ), toms are more apparent on sun-exposed than on þ and hydrogen ions (H3O ) tend to inhibit Mg shaded leaves (Marschner and Cakmak, 1989). þ uptake (Ga¨rtel, 1993). However, high Ca2 (e.g., The surplus sugar is used to produce antho- þ in calcareous soils with high pH) and/or K cyanins as a photoprotectant in the interveinal availability can also curb Mg uptake and induce areas of the leaves of red but not white cultivars Mg deficiency due to competition among these (Figure 7.6) (Currle et al., 1983). In addition, cations for root uptake (Delas and Pouget, even before the destruction of chlorophyll, the 1984); the same applies to competition with leaves activate their antioxidant system so that þ Na in saline soils (Shaul, 2002). Grapevines ascorbate and glutathione concentrations grafted on American rootstocks may be more increase (Cakmak and Kirkby, 2008). It therefore prone than own-rooted V. vinifera cultivars to appears that vines grown in areas that typically such interference of high soil K with Mg uptake experience high irradiance may require more (Mullins et al., 1992). In addition, although appli- Mg to avoid the risk of enduring oxidative cation of N fertilizers can enhance the (short- stress (Cakmak and Kirkby, 2008). term) availability of Mg in the soil solution and In addition to the detrimental consequences its uptake by the vine, it may also increase the for yield formation and fruit ripening due to (long-term) risk for leaching and depletion of the decrease in phloem export from the leaves, the surface soil. Because Mg is more phloem- insufficient Mg availability (sometimes in con- mobile than Ca, Mg deficiency symptoms first junction with low Ca and high K supply) has become apparent as chlorotic discoloration of also been implicated in the development of a the interveinal areas of old leaves as chloro- physiological disorder termed bunch stem phylls are being dismantled (Currle et al., 1983). necrosis that may develop during ripening However, even before any symptoms become (Cocucci et al., 1988; Keller and Koblet, 1995b). visible, the inhibitory effect of Mg starvation on The rachis of affected fruit clusters develops phloem loading leads to accumulation of reddish-brown to black necrotic lesions, 7.3. NUTRIENTS: DEFICIENCY AND EXCESS 261

FIGURE 7.6 Leaf symptoms of magnesium deficiency in the white cultivar Mu¨ller-Thurgau (left) and the red cultivar Pinot noir (center) and cluster abscising due to severe bunch stem necrosis (right). Photos by M. Keller. beginning with some stomatal guard cells or a Botrytis cinerea. It is conceivable that the role few subepidermal cells following collapse of of Mg in sugar export from the leaves may be their cell walls (Hifny and Alleweldt, 1972; to blame for this disorder, especially because Ja¨hnl, 1967, 1971; Theiler, 1970). The lesions symptom development is often related to probably reflect the oxidation of phenolic com- adverse environmental conditions that limit pounds and rapidly spread into, along, and/or photosynthesis and assimilate supply, and the around the stem and in the process may girdle incidence tends to be higher on cool compared the phloem, which effectively interrupts import with warm vineyard sites (Jackson, 1991; Jack- of assimilates into the berries distal to the son and Coombe, 1988; Keller and Koblet, necrosis, while initially leaving the xylem 1994, 1995b; Pe´rez Harvey and Gaete, 1986). In intact. The girdling effect stops sugar accumu- addition, loss of leaf area during the ripening lation in the berries, whereas the degradation period also seems to favor the appearance of of malate continues, which can lead to poor bunch stem necrosis (Redl, 1984). Finally, dry- fruit quality and berry shrinkage. The rachis ing winds and high vapor pressure deficit fol- eventually dries up, and in severe cases the dis- lowing even brief episodes of rainfall, as well tal portion of affected clusters may be abscised. as high light intensity, seem to favor the sud- Clusters appear to be most susceptible to this den appearance of necrotic lesions on the syndrome before and during bloom, at which rachis. Whether irrigation can substitute for time the disorder is also termed inflorescence rainfall in dry climates is unknown. In Europe, necrosis (see Chapter 6.1), and after the begin- applying Mg sprays, typically in the form of ning of sugar accumulation at veraison (Jack- dissolved Mg-sulfate or Epsom salt et al son and Coombe, 1988; Keller ., 2001a). (MgSO4 7H2O), directly to the fruit zone at ver- Cabernet Sauvignon, Riesling, and Gewu¨ rztra- aison has successfully increased rachis Mg con- miner are particularly sensitive to bunch stem tents and partially alleviated the incidence of necrosis, whereas the Pinots and Chardonnay bunch stem necrosis. þ are relatively insensitive (Currle et al., 1983). Excessive soil Mg2 , defined as greater than Affected rachis tissues are vulnerable to infec- 40% of the soil’s cation exchange capacity, is tions by saprophytic pathogenic fungi such as rare but occurs in serpentine soils that are poor 262 7. ENVIRONMENTAL CONSTRAINTS AND STRESS PHYSIOLOGY in silicates but rich in ferromagnesium miner- consequently inaccurate fertilizer recommenda- als. Such soils occur mainly in the North Amer- tions, resulting from analysis of leaf samples ican Pacific coastal ranges and California’s “contaminated” with dust. Sierra Nevada foothills and are often associated In the presence of oxygen (i.e., in the upper þ þ with high pH; very low K ,Ca2 , and P; and soil layers), iron generally occurs in the oxi- þ high Fe, B, cobalt (Co), and Ni. Although no dized form Fe3 (ferric iron), although the þ symptoms of Mg toxicity are known, it is reduced form Fe2 (ferrous iron) is the soluble thought that very high amounts of Mg in leaves cation and is the form required by plants. þ may impair photosynthesis (Shaul, 2002). However, Fe3 forms soluble complexes Moreover, excessive Mg can lead to poor soil (chelates) with many organic and inorganic structure and may induce K and, sometimes, molecules, including humic and fulvic acids P deficiency, especially toward the end of the (which are important components of soil growing season. Conversely, in high Na soils, organic matter), tannins, and phosphate. Like high Mg contents may ameliorate some of the other plants termed “strategy I” species (Briat adverse effects of Na on soil structure, and Lobre´aux, 1997), Vitis species absorb iron þ provided there is also sufficient Ca in the soil. molecules as Fe3 -chelates, which the enzyme þ ferric chelate reductase (FCR) reduces to Fe2 7.3.2. Transition Metals and on the root plasma membranes (Bavaresco Micronutrients et al., 1991; Schmidt, 2003; Varanini and þ Maggioni, 1982). The Fe2 ions are moved þ Iron across the membranes by Fe2 transporters Iron is the most abundant metal on Earth but but are then transported to the leaves in the is extremely insoluble in oxygen-rich environ- xylem sap mostly as metabolically inactive þ ments, which includes most soils (Schmidt, Fe3 –citrate complexes (Curie and Briat, 2003). 2003). It is generally the most abundant Upon arrival in the leaves, they are reactivated þ micronutrient in grapevines (Ga¨rtel, 1993). to Fe2 by FCR before they can be taken up into Due to its ability to occur in two different ionic the leaf mesophyll. The FCR protein is highly þ þ states (Fe2 and Fe3 ), it is a cofactor or ingredi- sensitive to pH, and enzyme activity declines ent of proteins involved in electron transfer as the apoplast pH increases (Nikolic et al., (e.g., ferredoxin) and many enzymes that cata- 2000). Inside the cells, unused Fe is again che- lyze reduction/oxidation (redox) reactions lated, this time by a nonprotein amino acid (Clarkson and Hanson, 1980; Curie and Briat, called nicotianamine that apparently serves to 2003; Curie et al., 2009). As such, it is involved keep Fe in a soluble form and ensures its cor- in chlorophyll synthesis (although the chloro- rect distribution among the various cell orga- phyll molecule does not contain Fe), photosyn- nelles and in the phloem (Curie and Briat, thesis, and respiration and, via enzyme 2003; Curie et al., 2009; Schmidt, 2003). þ activation, in C, N, and S assimilation, lipid Although Fe3 can be stored in a protein and hormone (e.g., ABA) synthesis and degra- termed ferritin, approximately 80% of the Fe dation, DNA synthesis and repair, as well as in a leaf is located in the chloroplast as part of detoxification of reactive oxygen species. In the photosynthetic machinery. Therefore, Fe addition to leaves, Fe is also abundant in seeds deficiency markedly decreases photosynthesis and pollen and may be necessary for pollen and sugar production for export to the vine’s production (Curie et al., 2009). Its profusion sink organs (Bertamini and Nedunchezhian, in soil particles is the source of many errone- 2005). This in turn strongly limits vine growth ously high apparent Fe concentrations, and and yield (Bavaresco et al., 2003; Gruber and 7.3. NUTRIENTS: DEFICIENCY AND EXCESS 263

Kosegarten, 2002) and results in oxidative vacuoles, where it is chelated by nicotianamine stress from an excess of absorbed light. It is or citrate, or in plastids, where it is bound up possible that such oxidative stress is the reason by ferritin and from where it can also be for the typical decline in leaf chlorophyll. Thus, retrieved on demand to keep cytosolic Fe con- deficiency symptoms include leaf chlorosis centrations stable. However, Fe toxicity is very (from gradual loss of chlorophyll or lack of its rare under natural conditions and is mainly production; Figure 7.7) beginning at the mar- limited to acid soils (Kochian et al., 2004). Tox- gins and progressing to the interveinal areas icity may also occur as a result of incorrect of young leaves, followed by marginal necrosis applications of foliar nutrient sprays (e.g., due and, finally, abscission. Developing lateral to errors in dose calculations). Leaf chlorosis shoots are stunted, often show pink internodes, due to Fe deficiency, on the other hand, fre- and have small, chlorotic leaves that may fail to quently develops when grapes are grown on unfold, although Fe is phloem-mobile and can calcareous soils, which are prevalent, for exam- be remobilized from older and senescing leaves ple, in Burgundy and Champagne in France, (Curie and Briat, 2003). Under severe defi- much of Hungary, and eastern Washington. ciency, the tendrils and inflorescences also Diagnosis of Fe chlorosis (e.g., by tissue analy- become chlorotic, and fruit set is poor (Ga¨rtel, sis) is difficult because the total Fe concentra- 1993), probably because of the Fe requirement tion of chlorotic leaves is often similar or even for pollen development. higher than that of “healthy,” green leaves, Conversely, excess Fe can also cause oxida- partly because the chlorotic leaves are usually tive stress, reduce leaf chlorophyll, and result smaller (Sattelmacher, 2001). However, the Fe in chlorotic or necrotic spots on the leaves content of a leaf is not closely related to its þ (Curie and Briat, 2003). Because Fe2 reacts rap- physiologically active Fe content. In addition, idly with hydrogen peroxide (H2O2) to form Fe-deficient plants also have a tendency to reactive oxygen species in the Fenton reaction, accumulate Zn, Mn, Co, and cadmium (Curie plants tightly control the availability of both and Briat, 2003). 2þ et al Fe and H2O2 (Curie ., 2009; Halliwell, Although Fe can be abundant in calcareous þ 2006; Lane, 2002). Excess Fe can be stored in soils, it is often precipitated as insoluble Fe3

FIGURE 7.7 Leaf symptoms of lime-induced chlorosis in Concord grapevines. Photos by M. Keller. 264 7. ENVIRONMENTAL CONSTRAINTS AND STRESS PHYSIOLOGY oxides and hydroxides, making it unavailable condition is also termed lime-induced chlo- for the roots. A one-unit increase in soil pH rosis, although it should probably more can decrease Fe solubility 1000-fold (see Fig- appropriately be called bicarbonate-induced ure 7.2). The ability of grapevines to cope with chlorosis (Currle et al., 1983; Ga¨rtel, 1993; such Fe deficiency depends on species and cul- Mengel, 1994). Due to their diverse origin, how- tivar. Species that have evolved in calcareous ever, Vitis species and thus rootstocks differ in conditions (e.g., V. vinifera and, to a lesser their tolerance of calcareous soils (see Chapter extent, V. berlandieri or V. champinii) can pump 1.2). Therefore, the chlorosis problem may be þ out protons (H ) and organic acids (malate overcome by grafting susceptible cultivars to and citrate) from the roots, which acidifies the tolerant rootstocks, such as those derived from soil solution and improves Fe solubilization V. berlandieri, V. vinifera (which, however, is and uptake (Brancadoro et al., 1995; Jime´nez susceptible to phylloxera), and, to a lesser et al., 2007; Mengel and Malissiovas, 1982). extent, V. rupestris (Bavaresco et al., 2003; Iron-inefficient species (e.g., V. labrusca, Schumann and Frieß, 1976). V. riparia, and Muscadinia rotundifolia)are Excessive soil moisture, especially waterlog- þ unable or less able to release H , but they also ging, also reduces Fe availability (as well as produce organic acids under Fe deficiency. the availability of N, K, and Mn) in addition þ The H -release strategy works only for species to restricting root growth. In addition, the poor or cultivars that are not sensitive to high soil aeration in waterlogged soils (e.g., due to abun- þ pH. Although release of H may enhance Fe dant rainfall or overirrigation) and in com- uptake and transport to the leaves in sensitive pacted soils also increases the soil HCO3 species as well, and although the apoplast pH content due to reduced diffusion of CO2 from seems to be unaffected by soil pH, uptake of the soil water. These effects may combine to bicarbonate (HCO3 ) from calcareous soils induce chlorosis, especially in calcareous soils et al et al (which contain high concentrations of HCO3 (Currle ., 1983; Williams ., 1994) and that forms from the soil’s CaCO3) leads to soil with a high P content: Pi reacts with soluble changes in the apoplast that inhibit conversion Fe to form insoluble Fe-phosphates. Moreover, þ of the inactive form (Fe3 ) to the active form chlorosis is exacerbated if excessive soil mois- þ of iron (Fe2 ). As a consequence, Fe becomes ture coincides with low soil temperatures “locked up” in the apoplast (Mengel and Bu¨ bl, (Davenport and Stevens, 2006). 1983; Mengel et al., 1984). Therefore, the Fe can- not enter the mesophyll cells and is unavailable Zinc þ for metabolism, which leads to yellowing Zinc (Zn2 ) is the only metal ion that is pres- between the veins of young leaves and eventu- ent in all six classes of enzymes, and much of ally whitening of the whole leaf. Nevertheless, the Zn in plants is strongly bound in proteins because the affected leaves ostensibly signal (Broadley et al., 2007). The majority of Zn- the roots to increase Fe uptake, the total leaf binding proteins are involved in the regulation Fe concentration may be as high as or higher of gene transcription (i.e., copying DNA into than that in nonchlorotic plants; Fe may also RNA) via, among others, effects on DNA and accumulate in the roots (Currle et al., 1983; RNA binding and RNA metabolism. Zinc and Gruber and Kosegarten, 2002). This “iron chlo- copper are integral parts of one form of the rosis paradox” is frequent in own-rooted antioxidant enzyme superoxide dismutase, Concord grapes (whose soil pH optimum is which may also help protect plant tissues 5.5) or scion cultivars grafted to V. riparia against the production of reactive oxygen rootstocks grown on high pH soils. Hence, the species that lead to oxidative stress that 7.3. NUTRIENTS: DEFICIENCY AND EXCESS 265 accompanies many environmental stresses 1983). In addition, insufficient Zn supply can (Apel and Hirt, 2004). The concentration of Zn inhibit pollen formation and, therefore, pollina- in grape leaves is approximately one-third to tion, leading to poor fruit set and a “hens and one-half that of Fe, but their concentrations chickens” appearance of the clusters, mimick- are similar in grape berries, in which the con- ing what occurs during B deficiency. Severe centration, but not the absolute amount, of this Zn deficiency leads to necrotic root tips, which phloem-immobile element declines during rip- is lethal. Zinc toxicity, on the other hand, is rare ening (Ga¨rtel, 1993; Rogiers et al., 2006). Most and occurs predominantly in low pH soils trea- (>90%) of the Zn in a soil is insoluble and ted with sewage sludge or contaminated with hence not plant available; up to half of the sol- other man-made Zn-containing (waste) pro- uble fraction is in the main plant-available form ducts, such as corroding galvanized objects þ Zn2 , although some Zn may be taken up by (Broadley et al., 2007). It decreases uptake of the roots as organic complexes (Broadley et al., P, Mg, and Mn, leads to stunted growth and 2007). Solubility is strongly dependent on pH, reduced yield, and can result in chlorosis and similar to Fe, Zn availability is low in cal- caused by Fe deficiency. careous soils with a pH > 7 (see Figure 7.2) and high bicarbonate content. Prolonged flood- Copper ing, high organic matter content, and high Mg: As a component of copper proteins, such as Ca ratios also seem to decrease Zn availability plastocyanin, cytochrome oxidase, ascorbate (Broadley et al., 2007). Moreover, excessive P oxidase, superoxide dismutase, polyphenol oxi- þ þ supply can immobilize Zn due to the formation dase, and laccase, copper (Cu and Cu2 ) parti- of insoluble Zn phosphate (Zn3(PO4)2) (Currle cipates in photosynthetic and respiratory et al., 1983; Ga¨rtel, 1993). Although insufficient electron transfer, lignification, ethylene percep- Zn availability is the most widespread micro- tion, cell wall metabolism, oxidative stress pro- nutrient deficiency, especially in sandy soils tection (in concert with Zn), and molybdenum with high pH, plants respond readily to foliar cofactor synthesis (Burkhead et al., 2009). Its þ þ Zn application (Broadley et al., 2007). ability to “switch” between Cu2 and Cu not Zinc deficiency results in a decline in protein only makes copper an essential cofactor in and starch production, accumulation of sugar many oxidase enzymes that catalyze redox in the leaves, and stunted shoots (short inter- reactions but also contributes to its inherent nodes) with small, malformed leaves (asym- toxicity (Clarkson and Hanson, 1980; Yruela, metrical with wide petiolar sinus and sharply 2009). Concentrations of Cu in grape leaves toothed margins) that develop a mosaic-like are approximately 15 times lower than those chlorotic pattern between the veins (“mottle of Fe (Ga¨rtel, 1993), although in grape berries leaf”); the veins may become clear with green they are in the same range, and the content of borders (Currle et al., 1983; Ga¨rtel, 1993). High both metals continues to increase as the grapes þ light intensity seems to accelerate symptom ripen (Rogiers et al., 2006). Roots absorb Cu2 development (Marschner and Cakmak, 1989), using high-affinity transporters and probably perhaps as a result of oxidative stress. More transport it in the xylem in the form of Cu-nico- severe deficiency delays shoot maturation tianamine chelate (Curie et al., 2009). High (periderm formation) and stimulates lateral plant N status appears to increase the demand shoot growth, whereas the leaves’ interveinal for Cu (Yruela, 2009). The phloem-mobile Cu areas become reddish-brown or bronze and can be remobilized from old leaves for redistri- then necrotic, leading to rolling of the leaf bution to sinks in need of extra Cu, although blades (Broadley et al., 2007; Currle et al., such recycling does not appear to be very 266 7. ENVIRONMENTAL CONSTRAINTS AND STRESS PHYSIOLOGY efficient so that young leaves are more the surface soil (Toselli et al., 2009). In extreme impacted by Cu deficiency than old leaves cases, young vines may die after replanting of (Burkhead et al., 2009; Marschner, 1995). old vineyard sites or in nurseries. Moreover, Deficiency symptoms include inhibition of the fungicidal activity of Cu may have other root growth; stunted shoots; small, misshapen, undesirable side effects: High Cu content in pale-green or chlorotic leaves, often with curled grapes at harvest can inhibit yeast growth, leaf margins; and decreased fruit set due to leading to sluggish or stuck fermentations poor pollen and embryo viability (Marschner, and, sometimes, poor wine quality (Tromp 1995). However, Cu deficiency is rare and is and De Klerk, 1988). mainly restricted to soils that are very high in organic matter that strongly binds Cu. More- Manganese over, Fe may substitute for Cu because the Like other transition metals, such as Fe and tasks of many Cu proteins can also be carried Cu, Mn exists in various oxidation states, but þ out by equivalent Fe proteins (Yruela, 2009). only Mn2 can be taken up by roots and trans- In contrast, when present in excess, Cu can be ported throughout the plant in both the xylem toxic and, despite its linkage with antioxidant and the phloem (Pittman, 2005). Leaf Mn con- enzymes, may cause oxidative damage due to centrations are similar to those of Zn (Ga¨rtel, the production of reactive oxygen species. Due 1993). In ripening grape berries, the Mn con- to the damage it causes to the photosynthetic centration, but not its content, declines during machinery, Cu can exacerbate the impact of ripening, similar to that of the phloem- high light intensity in leaves. Excess Cu may immobile elements Ca and Zn (Rogiers et al., also inhibit root elongation while stimulating 2006). Like Zn, Mn has antioxidative effects lateral root growth, and it may cause leaf chlo- in plant tissues. It can serve as an antioxidant þ þ rosis or even necrosis (Yruela, 2009). Copper by being oxidized from Mn2 to Mn3 and is availability increases at low soil pH (see Fig- also a structural component of antioxidant ure 7.2), and long-term application of Cu-based enzymes (e.g., superoxide dismutase and cata- fungicides can lead to Cu accumulation in the lase) as well as of the water-splitting (i.e., elec- surface soil (Ga¨rtel, 1993). For instance, downy tron-, proton-, and oxygen-producing) enzyme mildew (see Chapter 7.5) has been controlled in photosynthesis (Pittman, 2005). In addition, since the late 19th century by the copper–sul- Mn is required for the function of enzymes fate–lime mixture (CuSO4 3Cu(OH)2 3CaSO4) such as glucosyltransferases, which attach a called Bordeaux mixture and, more recently, glucose molecule to phenolics and other by copper oxychloride (CuCl2 3Cu(OH)2) and compounds (Marschner, 1995). Therefore, Mn copper hydroxide (Cu(OH)2), which has often deficiency may increase tissue sensitivity to led to more than 10-fold increases in soil Cu. oxidative stress, which can be caused by a vari- Such Cu accumulation may diminish uptake ety of environmental stresses (see Chapter 7.1). of P, Fe, and, in sandy soils, Mg and Ca. It Deficiency symptoms are consequently more can also lead to toxicity (Toselli et al., 2009), severe on sun-exposed leaves (energy “over- especially in vineyards planted on sandy and load”). Chlorotic leaves in response to insuffi- acid soils (frequent application of S-based fun- cient Mn availability occur first in the basal gicides also increases soil acidity). Whereas portion of the shoots, soon after budbreak. Cu toxicity is extremely rare in established Chlorosis appears mosaic-like, and the leaves vineyards because of the older vines’ deeper later acquire a reddish or bronze color, whereas root system, it can result in stunted growth of lateral leaves often remain green, and fruit young vines, whose roots are concentrated in ripening is delayed (Currle et al., 1983; Ga¨rtel, 7.3. NUTRIENTS: DEFICIENCY AND EXCESS 267

1993). The availability of Mn is highly pH involved in the production of both auxin and dependent (see Figure 7.2) and is minimal ABA (Schwarz and Mendel, 2006). A shortage around pH 7, at which deficiency can be a in Mo supply may also result in reduced fruit problem, especially on sandy soils with high set and clusters displaying “hens and chick- amounts of organic matter. However, Mn defi- ens,” especially in susceptible cultivars such ciency is often masked in such soils by the as Merlot (Kaiser et al., 2005). Other deficiency simultaneous development of lime-induced Fe symptoms include short internodes, zig-zag chlorosis (Ga¨rtel, 1993). On the other hand, growth, and pale-green leaves with necrotic plants often seem to take up much more Mn margins. Mo-deficient Merlot may furthermore than they require, and excess Mn can be show flaccid and cupped leaves that wilt easily extremely toxic (Pittman, 2005). Despite its anti- due to excessive transpiration (Kaiser et al., oxidative properties at “normal” concentra- 2005). This is because the hampered ABA pro- tions, excess Mn induces oxidative stress and duction renders Mo-starved vines vulnerable results in symptoms such as stunted growth, to losing stomatal control (see Chapters 3.2 leaf chlorosis, and necrotic lesions (Kochian and 7.2). Merlot appears to be less well able to et al., 2004). Plants therefore sequester and store take up Mo from the soil than other cultivars, þ Mn2 ions and Mn-chelate (i.e., Mn–organic but grafting on rootstocks can overcome this acid complexes) in the vacuoles in addition to problem. Although Mo deficiency is relatively the Mn required in the chloroplasts. However, rare, it can occur on acid soils (Mo availability Mn toxicity is rare and occurs predominantly strongly declines below a soil pH of 5.5) and on acid soils (pH <5.5) with high Mn availabil- soils with high Fe content, especially in cool cli- ity (Kochian et al., 2004). Uptake of Mn may mates. During a cool, wet spring, release of 2 also be increased under conditions of Fe and MoO4 in the soil may be restricted, as is root Zn deficiency, whereas excess Fe inhibits Mn growth. In addition, high sulfate availability uptake (Pittman, 2005). can inhibit molybdate uptake; the two anions compete for root uptake due their similar size. Molybdenum 2 However, MoO4 is highly mobile in plants, so Molybdenum is available to grapevine roots foliar applications can be very effective in distri- 2 et al mostly as molybdate oxyanion (MoO4 ). buting Mo throughout grapevines (Kaiser ., Although it is an essential element, it is 2005). Toxicity due to excess Mo is even more required only in minute amounts by grape- rare than deficiency; symptoms can include vines, mainly as a cofactor that forms the active purple leaves due to anthocyanin accumulation. site of a few proteins called molybdoenzymes (Schwarz and Mendel, 2006). Nitrate reductase Boron is the main molybdoenzyme in plants, and Although boron is an essential element for nitrate induces Mo uptake (Clarkson and plants, its functions are very poorly under- et al Hanson, 1980; Kaiser ., 2005; Reid, 2001). stood. As borate (B(OH)4 ), it binds strongly Therefore, a major effect of Mo deficiency is to pectic polysaccharides and thus is involved the accumulation of nitrate due to reduced in the formation or cross-linking of the pectin nitrate reductase activity and lack of amino network in the primary cell walls, which sup- þ acid synthesis (Currle et al., 1983; see also ports the function of Ca2 in cell walls and is Chapter 5.3). Consequently, the visual symp- essential for normal cell and leaf expansion toms are similar to those of N deficiency: (O’Neill et al., 2004). Therefore, almost all of reduced growth and yield. Another molyb- the B in plants is located in the apoplast, and doenzyme is aldehyde oxidase, which is B must be continually transported to the plant’s 268 7. ENVIRONMENTAL CONSTRAINTS AND STRESS PHYSIOLOGY growing regions to sustain cell wall structure the season and in the oldest leaves. Accumula- and growth. Moreover, the B requirement of tion also continues in grape berries throughout pollen tubes is especially high because they their development and ripening (Rogiers et al., are rich in pectin, particularly in the tip. Boron 2006). Boron toxicity can be induced by undue is available in the soil solution as boric acid B fertilization and is a peril especially of arid (B(OH)3). Thus, it differs from other micronu- regions. Insufficient B availability, on the other trients in that it exists as a neutral molecule at hand, is frequent on acid soils (pH <4.5), espe- physiological pH and appears to be taken up cially under dry conditions, and rapidly results into plants mostly by simple diffusion, espe- in cessation of cell division, whereas cell expan- cially at high external concentration (Clarkson sion is thought to be unaffected (Clarkson and and Hanson, 1980; Reid, 2001). Hence, the roots Hanson, 1980; Currle et al., 1983). However, may not be able to exclude excess B, which the inhibition of meristem activity leads to increases the risk of B toxicity that arises from stunted shoot and root growth, with shoots the ability of B to bind to ATP and NAD(P)H. developing “swollen” internodes and often dis- In addition to passive diffusion, active trans- playing a zig-zag appearance and petioles port into the roots and the xylem probably remaining short and thickened (Currle et al., occurs when the B concentration in the soil 1983; Ga¨rtel, 1993). Symptoms are usually con- water is low (Reid, 2001; Takano et al., 2008). fined to young tissues; although B is phloem- The symptoms of B toxicity resemble those mobile, it apparently cannot be recycled from of B deficiency (Figure 7.8), and the tissue con- old leaves. Bushy, branched shoot growth, centration range between deficiency and toxic- resembling symptoms of fanleaf virus infection, ity is very narrow (Currle et al., 1983; Takano and poor bud fruitfulness may be a carryover et al., 2008). Moreover, B continues to build up effect of insufficient B available for proper pri- in the leaves, and even the petioles, as the mordium formation during the previous grow- growing season progresses (Christensen, ing season (Ga¨rtel, 1993). In addition, reduced 1984). Therefore, in contrast to other nutrients, N uptake in B-deficient vines leads to low leaf the concentration of B is highest at the end of N status and sugar and starch accumulation

FIGURE 7.8 Symptoms of boron deficiency in Chardonnay (left) and boron toxicity in Cabernet Sauvignon (right). Photos by M. Keller. 7.3. NUTRIENTS: DEFICIENCY AND EXCESS 269 in the leaves (Camacho-Cristo´bal and Gonza´lez- Silicon Fontes, 1999). Root growth also ceases, and roots may swell and crack. Because B is impor- Silicon (Si) is the second most abundant ele- tant for pollen germination and pollen tube ment (oxygen is the most abundant) in the soil, growth, B deficiency interferes with fertilization making up approximately 28% of the earth’s (Currle et al., 1983; May, 2004; O’Neill et al., surface. As a component of minerals, it is pres- 2004). This results in poor fruit set and a condi- ent in clay, sand, and rocks in the form of sili- tion termed “hen and chicken” (French milleran- con dioxide (SiO2, the building block of quartz dage), with clusters containing small, seedless and glass), which accounts for 50–70% of the berries among normal berries (Ga¨rtel, 1993). soil mass (Ma and Yamaji, 2006). The lateral In addition, seed development is impaired, roots, but apparently not the root hairs, also and the seeds remain smaller than in nondefi- absorb some Si passively or via transporters as cient vines (Hardie and Aggenbach, 1996). soluble silicic acid (Si(OH)4), which is then Boron deprivation probably also induces oxida- released to and transported in the xylem sap et al tive damage to cells, and high light intensity (Liang ., 2005; Ma and Yamaji, 2006). Thus, consequently exacerbates the symptoms of Si is taken up and distributed with the transpira- B deficiency (Marschner and Cakmak, 1989). tion stream as an undissociated and neu- tral molecule, similar to boric acid (B(OH)3). Nickel Because Si(OH)4 polymerizes at concentrations Compared with the minute amount required greater than 2 mM, it is ultimately precipi- þ < m 2 ) tated throughout the plant as amorphous, by plants ( 0.1 g/g dry weight), nickel (Ni n is relatively abundant in almost all soils. hydrated silica bodies of (SiO2- H2O), which Although many proteins contain Ni, very little are also called silica gel opal or phytoliths is known about the element’s function in (Ma and Yamaji, 2006). Due to its immobility in plants. Several enzymes are activated by Ni; the phloem, xylem-supplied Si accumulates one of them, urease, is involved with nitrogen in older tissues, including the stomatal regions et al metabolism that is required to process urea, of grape berries (Blanke ., 1999). Its existence for example, during arginine mobilization as an undissociated molecule and ability to poly- (Witte et al., 2005). Another such enzyme is merize make Si the only element for which superoxide dismutase, which plays an impor- excessive uptake and accumulation does not tant role in the defense against oxidative stress. result in detrimental effects to plants. Following uptake of Ni by the roots, it is prob- Plants use polymeric silicates to impregnate ably transported in the xylem in the form of Ni- the cell walls of epidermis and vascular tissues, nicotianamine chelate (Curie et al., 2009). Due thereby strengthening the tissues, reducing to its partial mobility in the phloem, Ni can also water loss, and retarding invasion by fungal be remobilized from old leaves and redistribu- pathogens (Clarkson and Hanson, 1980; Ma ted to high-priority sinks. Nonetheless, the Zn and Yamaji, 2006). For example, penetration of concentration in grape berries declines during the powdery mildew fungus Erysiphe necator ripening, although its total amount remains ostensibly leads to localized accumulation of constant after veraison (Rogiers et al., 2006). Si in the cell walls (Blaich and Wind, 1989). In Nickel deficiency results in reduced shoot vigor addition, Si also stimulates the antioxidant sys- following budbreak; dwarfed, thick leaves with tems, which may avert stress-induced oxidative cupped tips (perhaps caused by oxalate accu- damage (see Chapter 7.1). Accordingly, appli- mulation); loss of apical dominance; and brittle cation of Si sprays to the canopy can wood due to poor lignification. reduce powdery mildew infections, perhaps 270 7. ENVIRONMENTAL CONSTRAINTS AND STRESS PHYSIOLOGY by providing a physical barrier and/or by is relatively rich in dissolved salts and because enhancing the production of phenolic com- irrigation tends to raise water tables. Therefore, pounds. Thus, although deemed a nonessential irrigation in arid and semiarid regions over nutrient (i.e., one that is not required to com- prolonged periods can lead to a buildup of salt plete the plant’s life cycle), Si provides many near the soil surface. Most table and raisin benefits, such as improved resistance to pests grapes are grown in rather dry and warm cli- and diseases; tolerance of drought, salinity, matic regions, such as southeastern Asia, heavy metals, and high temperatures (Currie California, Chile, or Australia, and are thus and Perry, 2007; Epstein, 1999; Ma and Yamaji, especially threatened by salinity. 2006); and even improved yield and fruit qual- The dominant soil salts are cations, such as þ þ þ þ ity by enhancing the production of flavonoids. Na ,K ,Ca2 , and Mg2 , and their associated 2- For this reason, Si is integrated in many fertili- anions, such as chloride (Cl ), sulfate (SO4 ), 2- zers. Most of these stress-alleviating effects carbonate (CO3 ), and bicarbonate (HCO3 ). result from the strengthening of cell walls by Small amounts of other ions are also present in Si and by its ability to enhance the binding of the soil solution. The relative amounts of differ- þ þ cations (e.g., Na and Mn2 ) to the cell walls, ent ions vary between water sources and soil which may prevent their buildup to toxic con- types, but the ions most often associated with þ centrations inside the cells (Saqib et al., 2008). the effects of salinity on grapevines are Na Plants high in Si may be able to reduce uptake and Cl . Dissolved ions increase the electrical þ of Na from saline soils and to transport less conductivity of water, and thus salinity of irriga- of it to the leaves. tion water or water extracts of soils is expressed in electrical conductivity units (measured in 7.3.3. Salinity decisiemens per meter or dS m 1). The threshold above which salinity begins to affect V. vinifera The term salinity describes the occurrence of growth and yield formation seems to be approx- high concentrations of soluble salts (i.e., in ionic imately 2 dS m 1, and above 16 dS m 1 vines form) in water and soils. The development of cannot survive (Zhang et al., 2002). Because dis- salinity is termed salinization and occurs in solved ions decrease the osmotic potential (Cp) regions where water evaporation from the soil of water, electrical conductivity is also a measure 1 exceeds precipitation so that salts dissolved in of Cp: 2 dS m corresponds to approximately the soil solution tend to become concentrated 20 mM NaCl generating a Cp 0.1 MPa. at the soil surface. Whereas this process is char- Sodicity is related to salinity and refers to acteristic of arid environments, the opposite the presence of sodium relative to calcium process, called acidification, occurs in regions and magnesium in the soil. Sodicity is where rainfall consistently exceeds evaporation expressed as the sodium adsorption ratio (i.e., especially in the tropics and subtropics) (SAR) because most cations in the soil are þ 2þ and thereby leaches cations such as K ,Ca , attracted to the negative charges of clays. The 2þ and Mg so that the soil pH decreases and sodicity of irrigation water or soil water 3þ 2þ the highly toxic Al , along with Mn and iron extracts is calculated as follows: 2þ Fe , becomes soluble in the soil solution. Rain- þ ½Na water also contains some salt ( 50 mg NaCl SAR ¼ qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 1 þ þ L ), especially in coastal areas (Munns and ½Ca2 þ½Mg2 Tester, 2008). However, irrigated vineyards are at much greater risk from salinization than where [...] denotes the concentration of an ion nonirrigated vineyards because irrigation water in millimoles per liter (mM). 7.3. NUTRIENTS: DEFICIENCY AND EXCESS 271

þ In addition to their elevated Na content, which in turn decreases transpiration and pho- sodic soils are afflicted with a deterioration of tosynthesis and consequently the production of structure due to clay dispersion and a rise in sugar for export to other plant parts (Downton hydraulic resistance. Saline and sodic soils are et al., 1990; Shani and Ben-Gal, 2005). Rising Cl usually classed together as “salt-affected soils.” concentration in the leaves also reduces stoma- Such soils contain a sufficient concentration of tal conductance (Walker et al., 1981), although þ þ þ soluble salts or exchangeable Na to interfere Na tends to counter this by replacing K in with plant growth. The most common cause the guard cells and thereby keeping the sto- þ of salt stress is a high concentration of Na mata partially open. However, the rate of pho- and Cl in the soil solution. Both of these are tosynthesis declines as leaf Cl concentration essential plant nutrients but become toxic at increases well before any visible symptoms of much lower concentrations than other nutri- salt damage become apparent (Downton, 1977). ents. Plant damage due to salt-affected soils is The challenge for grapevines growing in the outcome of a combination of hyperosmotic saline environments is that their roots must stress and hyperionic stress due to a disruption take up nutrient ions while keeping out the of homeostasis (Greek homois ¼ similar, stasis ¼ toxic Na and Cl. “Toxic,” as usual, is a relative stand still, steady) in water status and ion dis- term; it is important to remember that plants tribution (Hasegawa et al., 2000; Zhu, 2001). require some Cl for the water-splitting reac- Initially, buildup of salt ions in the soil tion that produces electrons, protons, and oxy- decreases Cp of the soil solution; the Cp in gen during photosynthesis (see Chapter 4.1). “normal” soils is generally approximately Roots effectively “pick” the nutrient ions from 0.01 MPa but can drop to less than 0.2 MPa the toxic ions in the soil solution or pump the C in saline soils. The resulting decrease in soil toxic ions taken up back out again so that more þ impedes water uptake by the roots (Shani than 95% of Na and Cl is prevented from et al., 1993; see also Chapter 3), increases root entering the xylem (Munns, 2002; Munns and þ hydraulic resistance due to closure of aquapor- Tester, 2008). Nonetheless, although Na is ins (Luu and Maurel, 2005), and results in not an essential nutrient, the ion is taken up C water deficit for the vine and a decline in leaf into cells down the electrochemical gradient, þ (Cramer et al., 2007; Downton and Loveys, competing with K for uptake (Hasegawa 1981; Walker et al., 1981). Thus, the initial et al., 2000). The hypodermal and endodermal effects of a rise in soil salinity are identical to cells of salt-stressed grapevine roots appear to þ þ the effects of drought stress (see Chapter 7.2). selectively accumulate K over Na and Cl The ensuing collapse of the water potential gra- compared with cortical and pericycle cells (Sto- dient necessary for growth curtails shoot rey et al., 2003). In contrast, the cortex and peri- þ growth and leaf expansion and inhibits lateral cycle cells sequester large amounts of Na and shoot development, whereas root growth is Cl in their vacuoles (Storey et al., 2003). How- þ usually less sensitive (Munns and Tester, ever, grapevines take up more Cl than Na 2008). Nonetheless, in some instances, root from saline soils that have equivalent concen- growth of grapevines was found to be more trations of both ions (Walker et al., 1981), and sensitive to salinity than shoot growth (Hawker a small portion of each ion ends up in the and Walker, 1978). Growth may also slow xylem and is transported to the shoot with the through deactivation of gibberellins under salt transpiration stream. Consequently, Cl and, C þ stress (Yamaguchi, 2008). The decrease in leaf to a lesser extent, Na accumulate in the older and increase in root-derived and locally pro- leaves, building up with increasing concentra- duced ABA also induce closure of the stomata, tion in the soil solution, and they continue to 272 7. ENVIRONMENTAL CONSTRAINTS AND STRESS PHYSIOLOGY do so as the growing season progresses (Down- even before such irreversible damage ensues, ton, 1985; Munns, 2002; Shani and Ben-Gal, oxidative stress may increase the vine’s light 2005; Stevens and Walker, 2002). Over time, sensitivity because more photons are being the ions may accumulate to toxic concentra- absorbed than can be used by the declining tions; this ion-specific phase of salinity stress photosynthesis. One way grapevines cope with is associated with premature death of older such an energy overload appears to be an leaves (Munns and Tester, 2008). increase in photorespiration (Cramer et al., et al Excessive Cl uptake interferes with NO3 2007; Downton, 1977; Downton ., 1990; et al nutrition because NO3 uptake seems to Walker ., 1981), which dissipates some of þ respond to the concentration of NO3 Cl the excess energy but comes at the cost of lower rather than to NO3 alone (Clarkson, 1985). photosynthetic efficiency. Another defense This requires application of abundant nitrogen strategy is to boost the antioxidant systems (i.e., þ fertilizer to improve plant N status. High Na glutathione and xanthophyll cycles) that capture concentration, on the other hand, is toxic to and inactivate some of the reactive oxygen þ þ plants because it interferes with K nutrition, species (Cramer et al., 2007). Because Mn2 acts þ which reduces K -stimulated enzyme activities, as an antioxidant in plant tissues, (foliar) appli- metabolism, and photosynthesis. At concentra- cation of Mn-chelates (and possibly Zn-chelates) þ tions exceeding approximately 100 mM, Na might alleviate effects of oxidative stress in and Cl also directly inhibit many enzymes plants grown on saline sites or irrigated with (Munns, 2002; Munns and Tester, 2008; Zhu, saline irrigation water (Aktas et al., 2005). 2001). High salinity eventually overwhelms the The first visible sign of salt stress is an inhibi- leaf vacuoles’ capacity to sequester Cl and tion of shoot growth and leaf expansion (Walker þ Na , leading to toxic concentrations in the cyto- et al., 1981), whereas the roots are more robust so plasm and disturbing the cells’ ionic balance; that their growth is less curtailed (similar to the þ þ Na accumulates at the expense of K and can response to water deficit). However, roots are þ þ even result in a loss of K and Ca2 from the the first and most important organs to experi- þ cells. Sodium can compete with Ca2 and dis- ence salinity, which decreases their ability to place it from the cell wall and thus affect cell wall explore the soil for water and nutrients. Salinity þ þ properties. Because Ca2 in turn can reduce Na impacts growth directly through the effect of þ þ uptake and increase K and Ca2 uptake, addi- ions on the physiology of the plant, whereas þ tion of Ca2 can somewhat alleviate the toxic the influence of sodicity is indirect due to its del- effects of salinity (Hasegawa et al., 2000; Plieth, eterious effects on soil physical properties. Salin- 2005). However, prolonged exposure to high ity and sodicity both impair root growth, þ soil Ca2 may itself be stressful for the plant. respiration, and water uptake, reducing vine Oxidative stress is another characteristic of growth, yield, and fruit quality (Shani and salinity-induced injuries to plant tissues (Munns Ben-Gal, 2005; Shani et al., 1993). In some cases, and Tester, 2008; Zhu, 2001). It is a secondary salinity may be associated with changes that stress that results from the effects of ion imbal- are typical of mild water deficit, such as earlier ance and hyperosmotic stress and from the veraison; higher fruit sugar, proline, potassium, decline in photosynthesis (see Chapter 7.1). and Cl ; and greater decline in acidity during 1 Excess free oxygen radicals (especially O2) and ripening (Downton and Loveys, 1978; Walker et al hydrogen peroxide (H2O2) oxidize membrane ., 2000). As the salinity becomes more severe, lipids and other cellular components, which however, fruit set, berry size, as well as sugar eventually leads to membrane leakage and tis- and anthocyanin accumulation are increasingly sue deterioration (Mller et al., 2007). However, restricted (Hawker and Walker, 1978). 7.3. NUTRIENTS: DEFICIENCY AND EXCESS 273

lesser extent, V. candicans and V. champinii, are more tolerant (Williams et al., 1994). Some root- stocks derived from these species (e.g., Ramsey, 1103 Paulsen, 110 Richter, Ruggeri 140, and 101–14 Mgt) are able to exclude much of the salt from root uptake and root-to-shoot trans- port (Antcliff et al., 1983; Sauer, 1968; Walker et al., 2000). Therefore, these rootstocks and scions grafted to them are only marginally affected by high salt concentrations in the soil (Downton, 1985; Stevens and Walker, 2002; Zhang et al., 2002). However, it appears that at least some of them (Ramsey, 1103 Paulsen, FIGURE 7.9 Leaf symptoms of salt injury on Merlot. Photo by M. Keller. and 101–14 Mgt) progressively lose this salt- exclusion ability (Tregeagle et al., 2006). Under long-term exposure to saline conditions, which The reduction in growth is in part due to the tends to lead to salt buildup in the soil over decrease in photosynthesis and in part due to time, these rootstocks may become less salt inhibition of cell division and cell expansion tolerant. (Zhu, 2001). More severe salinity arrests lateral The impact of salinity on vines seems to be shoot growth and induces necrotic leaf margins more severe on heavier clay loam soils than (“marginal burn” or “salt burn”; Figure 7.9) in on lighter loamy sands. Moreover, irrigation older leaves, followed by progression of the and soil management also affect the extent of necrotic symptoms toward the petiole, whereas physical degradation of salt-affected soils. Irri- the main veins remain green (Williams and gation is a common cause of agricultural land Matthews, 1990; Williams et al., 1994). Such salt degradation because salt dissolved in the irri- þ injury is the result of the accumulation of Na gation water is left in the soil following evapo- and/or Cl in the transpiring leaves (i.e., ion ration. Excessive irrigation, particularly with concentrations gradually increase as the tran- saline water, as well as frequent cultivation spiration stream “deposits” salt ions in the (tillage) and intense trafficking are a good rec- leaves) to the point where the vacuoles can no ipe for rapid loss of soil fertility. However, salts longer contain these ions. Accumulation in the can also build up under highly efficient drip cytoplasm then leads to enzyme inhibition (salt irrigation (Stevens and Walker, 2002), when poisoning), whereas accumulation in the cell ions move down the soil profile and below walls leads to dehydration of the cells; both the emitters and then move laterally and rise outcomes result in cell death (Munns, 2002). again to the soil surface with the evaporating The threshold Cl content for marginal necrosis water. The resulting high-salt zone around the seems to be approximately 2.5% of the leaf dry edges of the wetting zone can restrict root weight (Walker et al., 1981). growth similar to the restriction imposed by a Although they vary somewhat in the extent pot. The “pot” size is smaller in sandy soils þ of Na and Cl uptake and accumulation in than in loam soils. Waterlogging due to the for- the leaves (Groot Obbink and Alexander, mation of impermeable soil layers or as a result 1973), most cultivars of V. vinifera are moder- of excessive irrigation also increases the risk of ately sensitive to salt. American Vitis species, salt damage because waterlogged grapevine þ especially V. riparia, V. berlandieri, and, to a roots lose the ability to exclude Na and Cl 274 7. ENVIRONMENTAL CONSTRAINTS AND STRESS PHYSIOLOGY from uptake. Waterlogging also appears to also inhibits cell division by preventing cell þ increase the amount of Na in the soil solution expansion (see Chapter 3.1). Furthermore, cold þ relative to other ions so that Na uptake is temperatures increase the rigidity of the nor- often favored over Cl uptake (Stevens and mally fluid cell membranes (Chinnusamy Walker, 2002). Even if only a portion of the root et al., 2007). Because low temperatures restrict system is exposed to saline conditions while cell division more than photosynthesis (the other portions continue to have access to fresh- duration of cell division increases exponen- water, the latter ostensibly do not compensate tially with decreasing temperature), sugar and for the decline in water uptake by the former starch tend to accumulate in the leaves during (Shani et al., 1993). Prolonged exposure to a cool episode (Wardlaw, 1990). When the tem- saline soil water ultimately results in vine perature drops too low, it can result in damage death (Shani and Ben-Gal, 2005). On the other to plant tissues. The type and extent of damage hand, where soil salts can be leached out of depend on whether or not the temperature the rootzone by using a fresh source of irriga- drops below the freezing point and on the tion water, the harmful physiological effects developmental status of the plant. can often be quickly reversed. Thus, as long as no irreversible damage has been caused, grape- 7.4.1. Chilling Stress vines restore root functionality, growth, and water uptake to drain the excess ions from the Damage to plant tissues caused by low but leaves, and growth and gas exchange recover above-freezing temperature (typically in the rapidly (Shani et al., 1993; Walker et al., 1981). range of 0–15 C) is referred to as chilling stress. The photosynthetic cell organelles, the chloroplasts, are particularly sensitive to chill- 7.4. TEMPERATURE: TOO COLD ing stress (Kratsch and Wise, 2000). Swelling OR TOO WARM of chloroplasts, distortion of thylakoid mem- branes, and starch depletion (decrease in num- ber and size of starch granules) inside the Higher temperatures tend to accelerate plant chloroplasts are usually the first microscopi- growth and development so that phenological cally visible signs of chilling injury. At the same stages occur in more rapid succession than time, chilling also decreases phloem loading under cooler conditions (Alleweldt et al., and phloem transport. Accumulation of sugar 1984b; Chuine et al., 2004; Jones and Davis, in the chloroplasts (lowering Cp) due to 2000; Wolfe et al., 2005). In other words, an reduced export and continued starch degrada- increase in temperature accelerates and com- tion may be responsible for chloroplast presses the temporal program of plant develop- swelling by osmotic water influx. Of course, ment—up to an optimum—and provided no damage to the photosynthetic “hardware” usu- other factors (e.g., water deficit, which is often ally has severe consequences for photosynthe- coupled with high temperatures) are limiting sis, although the reduction in photosynthesis growth. Grapevines growing in cool climates could also be caused by feedback inhibition are exposed to a large daily temperature range due to sugar accumulation. Sugar may accumu- and often experience widely fluctuating tem- late in the leaves because cell division, and peratures during spring and autumn. Low hence growth, ceases at low temperature, temperature may limit growth by decreasing which decreases sink demand for assimilates the rate of protein production or cell wall and may result in an oversupply of fixed car- extensibility. Restricting cell wall extensibility bon (Ko¨rner, 2003). With prolonged chilling, 7.4. TEMPERATURE: TOO COLD OR TOO WARM 275 the chloroplasts may disintegrate completely so within a few days. The chilling-induced that the leaves become chlorotic (Kratsch and depression of photosynthesis appears to be Wise, 2000). Leaves also turn chlorotic when similar to the photosynthetic depression due they develop under chilling conditions, but this to water deficit (Ba´lo et al., 1986; Flexas et al., is probably due an inability to produce thyla- 1999a; see also Chapter 7.2), although the clo- koid proteins. Chilling-induced chlorosis is sure of stomata below 15 C is thought to be a normally reversible, and leaves may recover response to the reduced photosynthesis rather and re-green upon exposure to warmer tem- than the reverse (Hendrickson et al., 2004a). peratures if chilling injury is not too severe. Photosynthesis recovers only after several However, the longer plants are exposed to warm nights. Photooxidative stress can trigger low temperatures, the more extensive and irre- anthocyanin accumulation in the exterior versible is the damage (Kratsch and Wise, leaves of red-skinned cultivars so that the 2000). Under prolonged exposure to low tem- leaves may turn red, whereas leaves in the can- perature, vines activate an organized cell sui- opy interior usually do not accumulate antho- cide process (so-called programmed cell cyanins. Because anthocyanins probably act as death) that involves the systematic, energy- photoprotectants, autumn coloration often intensive (i.e., ATP-dependent) dismantling of becomes much more intense during cool the cells accompanied by recovery of cell com- weather, especially if the light intensity ponents (e.g., proteins and DNA). The rescued remains high (Feild et al., 2001; Hoch et al., breakdown products may be exported to other 2003). It also appears that anthocyanins fluctu- plant parts to enable the vine to survive the ate according to changes in the weather; they unfavorable conditions. may be degraded or reformed depending on The more chilling sensitive a grape species the temperature and light conditions (Keskitalo or cultivar, the sooner and the more extensive et al., 2005). is the development of the ultrastructural High humidity protects plant tissues against changes. The most resistant plants may not suf- injury, especially if the low temperatures occur fer injury unless they are simultaneously during the night (Kratsch and Wise, 2000). exposed to some other stress factor, such as Moreover, if temperatures remain relatively high light intensity or water stress. Light low for longer periods of time, the leaves may greatly exacerbates chilling injury due to acclimate to those conditions by re-emitting energy “overload” because light absorption some of the excess energy as fluorescent light decreases less than carbon fixation, which leads (see Chapter 4.1) and increasing the activity of to generation of reactive oxygen species and, enzymes involved in sugar production (see thus, oxidative stress (Kratsch and Wise, Chapter 4.2). Fluorescence emission occurs 2000). Under such conditions, reactive oxygen especially in mature leaves that suddenly expe- species can trigger the degradation of the two rience a “cold shock,” whereas enhanced sugar photosystems, PSI and PSII, and the photosyn- production seems to be more important in thetic enzyme rubisco (Mller et al., 2007). young leaves developing during cool condi- Therefore, symptoms develop sooner and are tions. The acclimation to low temperature, more severe during cold days than during cold termed chilling acclimation, is very similar to nights. Nevertheless, cold nights seem to make the photoacclimation to high light intensity the leaves more susceptible to high light inten- (Ensminger et al., 2006). Both processes aim to sity (Bertamini et al., 2006). When cold nights limit photoinhibition—that is, to balance are followed by bright, warm days, grapevine energy supply and demand. If chilling condi- leaf photosynthesis can be inhibited completely tions persist, the leaves that had grown under 276 7. ENVIRONMENTAL CONSTRAINTS AND STRESS PHYSIOLOGY warm conditions will eventually be shed and Chilling stress during grape ripening may will progressively be replaced by new, accli- lead to carbon shortage in the vine—for exam- mated leaves. These adjustments lead to higher ple, due to photoinhibition induced by cold photosynthetic rates at lower temperatures and nights followed by warm, sunny days. This a downward shift of the optimum temperature can induce a syndrome termed bunch stem for photosynthesis. This response also results in necrosis. This physiological disorder essentially improved water-use efficiency (i.e., a higher constitutes abandonment of some of the vine’s photosynthesis:transpiration ratio), and hence clusters and tends to be more severe on heavily drought tolerance, and accelerates the acquisi- cropped vines. The vascular system in the ped- tion of freezing tolerance (Ensminger et al., uncle and/or rachis, sometimes only on a 2006). Perhaps this is because the canes and shoulder or the tip of a cluster, becomes dys- other perennial parts of the vine act as alterna- functional, the berries on affected clusters stop tive sinks for the surplus assimilates available ripening, and the cluster or its affected portion due to arrested growth. Therefore, repeated is eventually shed by the vine. Thus, reproduc- exposure to chilling temperatures can also tive development is most sensitive to chilling induce adaptive changes in a grapevine that stress during the periods leading up to anthesis result in subsequent tolerance to freezing tem- (i.e., before the vine has invested heavily in peratures. This process is referred to as cold reproduction) and after veraison (i.e., when acclimation or hardening and is discussed later. the seeds are mature). Between fruit set and However, under some cool-climate conditions early ripening, the berries’ sink strength is high the temperature fluctuates too much between enough to attract carbon from remobilized night and day for acclimation to protect the storage reserves when necessary (Candolfi- leaves from inhibition of photosynthesis. Vasconcelos et al., 1994). Chilling during the period leading up to bloom impairs pollen formation in a way that 7.4.2. Cold Acclimation and Freeze is very similar to the effect of drought (see Damage Chapter 7.2); both stresses disturb sugar metab- olism in the anthers, whereas ovule fertility Water freezes at 0 C to form ice crystals, appears to be rather “immune” to low temper- provided no heat is removed from the water. ature (Oliver et al., 2005). The cold-induced If, however, heat is lost as the temperature inhibition of invertase activity in the anthers decreases (e.g., the heat loss from plant tissues interferes with sugar supply (unloading) from to the environment), then water will not freeze the phloem even if the leaves supply abundant unless some nucleation event occurs (e.g., assimilates. This renders the pollen grains vibration of dust or other tiny particles such unable to accumulate starch, and they may as bacterial proteins). This phenomenon is com- instead temporarily accumulate sucrose. If this monly referred to as “supercooling,” which is blockage occurs during meiosis (i.e., several followed by a sudden release of heat at the days before anthesis), it causes pollen sterility point where water finally freezes. At this point, (Koblet, 1966; Oliver et al., 2005). Even brief epi- chemical energy (the so-called latent heat, sodes of chilling during male meiosis, such as which is the energy needed to break the hydro- two or three consecutive cool nights, can irre- gen bonds that hold together individual water versibly inhibit pollen development. This molecules in ice crystals) is converted to (i.e., decreases pollination and fertilization, which released as) sensible heat, which is heat that results in poor fruit set or, in more severe cases, we measure with a thermometer. In the absence abscission of inflorescences. of ice nucleation, highly purified water can be 7.4. TEMPERATURE: TOO COLD OR TOO WARM 277 supercooled to almost 42C, but supercooling can move freely across intact membranes, but does not occur in plants to such an extreme ice crystals cannot, so that the symplast is because water inside plants and on their sur- supercooled (Steponkus, 1984). Thus, ice for- faces is never pure. However, the presence of mation in the apoplast, termed extracellular osmotically active solutes (e.g., sugars and freezing, is not lethal. When the ice crystals nutrient ions) in plant tissues does decrease melt, the water simply diffuses back into the the freezing temperature to several degrees cells and they resume their metabolism. How- below 0C. As a general rule, every mole of ever, the apoplast only contains approximately dissolved solutes per liter of water lowers 10–15% of the total tissue water; thus, extended the melting point of the solution by 1.86C exposure to freezing temperatures dehydrates (Zachariassen and Kristiansen, 2000); thus, the the symplast and causes a water deficit, which extent of this osmotic freezing-point depression interferes with metabolism and puts cells is greater with larger amounts of solutes pres- under physical stress due to shrinkage (Browse ent in the solution or tissue. Plant tissues freeze and Xin, 2001; Mu¨ ller-Thurgau, 1886; Thoma- when they cannot avoid nucleation and pre- show, 2001). Very high solute concentrations vent ice growth (Pearce, 2001). The explosive can be toxic to the cells, and if the cells shrink force of water is considerable because the vol- beyond their minimum tolerated volume, ume of freezing water expands by approxi- osmotic stress and membrane rupture ensue mately 11%. Note that supercooled rainwater (Zachariassen and Kristiansen, 2000). Thus, that freezes instantly when it hits a cold dehydration of the symplast by extracellular (<0 C) surface causes the phenomenon known freezing is the most common cause of freez- as “freezing rain” or “ice storm.” ing-induced injury, and tissue survival During a freezing event, grapevine tissues depends on the extent of the cells’ dehydration lose heat and temporarily supercool due to tolerance (Guy, 1990; Thomashow, 1999). the presence of osmotically active solutes. A tis- In contrast with other tissues, xylem paren- sue is defined as supercooled when it remains chyma cells somehow seem to avoid dehydra- unfrozen below its freezing temperature. As tion during extracellular freezing, at least for a the temperature drops, water freezes first in while. This mechanism, which is termed deep the intercellular spaces, cell walls, and trache- supercooling, enables these xylem cells to ary elements (apoplast), where the concentra- endure lower temperatures than other tissues tion of solutes is much lower than inside the for extended periods (Quamme, 1991). How- cells (symplast) (Mu¨ ller-Thurgau, 1886). The ever, prolonged exposure to freezing tempera- formation of ice (i.e., H2O crystals) leads to tures can lead to the coalescence of small ice exclusion of solutes, which therefore become crystals into large crystals—a process called more concentrated in the apoplast. This concen- recrystallization (Pearce, 2001). This formation tration effect lowers the apoplast water poten- of large ice masses can deform cells and damage tial (by 1.16 MPa C 1), which “pulls” water plant tissues by interfering with their structure out of the symplast (Guy, 1990; Pearce, 2001; (or the structure of entire organs), for example, Steponkus, 1984; Thomashow, 1999). The by separating cell layers. Although this does resulting increase in the cells’ solute concentra- not necessarily kill the vine, the resulting tension tion in turn lowers their freezing point by 2 or in the wood can result in splitting or cracking of 3 C. As a practical aside, the same concentra- woody organs, which may provide infection tion mechanism is exploited in the production sites for crown gall bacteria (see Chapter 7.5). of ice wine, whereby frozen grapes are har- Moreover, the forcing of gases out of solution vested and pressed immediately. Liquid water during ice formation inside the xylem vessels 278 7. ENVIRONMENTAL CONSTRAINTS AND STRESS PHYSIOLOGY can lead to cavitation (i.e., air embolisms; see before the temperature plummets during a clear Chapter 3.3). The vulnerability to such embo- night (Guy, 1990). lisms increases with increasing conduit diame- Individual vine tissues and organs differ in ter and with water stress that decreases xylem their ability to tolerate freezing temperatures. pressure. Therefore, dry soil conditions during Growing organs that have a high water content winter generally result in more severe xylem are very sensitive to frost, and early spring cavitation, especially in vines that had grown growth after budbreak is especially frost sus- vigorously during the preceding years and ceptible (Fuller and Telli, 1999). Ambient tem- hence have large xylem vessels. Although this peratures of 2 or 3 C or lower can damage presents quite a challenge to grapevines experi- leaves, shoots, and green buds (Fennell, 2004). encing repeated freeze–thaw cycles at higher The lethal temperature also depends on the pres- latitudes, they are normally very efficient at ence of moisture and ice nucleating agents repairing such cavitation in spring, unless (especially certain bacteria, such as Pseudomonas unusually dry soil prevents the buildup of root syringae) on the surface of an organ (Luisetti pressure needed to dissolve and push out air et al., 1991). For instance, dry leaves can super- bubbles (see Chapter 2.2). cool to relatively low temperatures, whereas As the temperature drops, the apoplastic wet leaves (e.g., due to condensation of water water initially remains unfrozen, and the tissues during a radiation frost) freeze at just a few cool until they reach their ice nucleation tem- degrees below 0 C due to the presence of perature. Ice formation in the apoplast is accom- nucleating agents on the leaf surface. Ice first panied by a release of heat, called the high- forms on the leaf surface and then penetrates temperature or freezing exotherm, which results through stomata, hydathodes, wounds, or even in a rapid rise in tissue temperature. This tem- the cuticle (although the cuticle normally acts perature is the (nonlethal) freezing temperature as a barrier to ice growth) to initiate freezing in of the tissue. During freezing, the tissue tem- the apoplast (Pearce, 2001). Severe infections by perature remains above ambient until all the mites, insects, or pathogens may create lesions extracellular water is frozen. Once this has through which ice can grow into the leaf. occurred, the tissue temperature returns to Cold hardiness, or freezing tolerance, ambient and continues to drop. As the tempera- remains low during the growing season but ture continues to decline, a second phase of increases in late summer and fall during a pro- heat release occurs when the cell protoplasts cess known as cold acclimation and reaches a freeze, and this is termed the low-temperature peak in midwinter. As a consequence, damage exotherm (Ristic and Ashworth, 1993). Intracel- to buds, canes, cordons, and trunks in midwin- lular freezing kills cells instantaneously due to ter (Figure 7.10) occurs at much lower tempera- the combined effects of membrane damage, ture than during the growing season and symplast dehydration, and protein denatur- depends on the temperature experienced dur- ation. The extent of intracellular ice formation ing the period preceding the cold event. Grape- also depends on the rate of tissue cooling; very vines can survive frosts either by avoiding rapid cooling seems to induce ice formation them (e.g., via late budbreak and early fruit within the cells even at temperatures that would and shoot maturation) or by tolerating them normally only induce extracellular freezing (e.g., by deep supercooling in midwinter). They (Mu¨ ller-Thurgau, 1886). Fortunately, cooling also have “ice sites” or “ice sinks” to accommo- rates are seldom fast enough to favor intracellu- date ice formation. For example, the scales of lar freezing, except in situations in which the buds with their down (see Chapter 1.3) can sun heats southwest-facing trunks and canes cope with ice crystals that grow from water 7.4. TEMPERATURE: TOO COLD OR TOO WARM 279

FIGURE 7.10 Young Merlot cordon (left) with lethal freeze injury to the phloem (brown area), whereas the xylem parenchyma remains viable (greenish color in interior), and frozen primary bud with surviving secondary and tertiary buds (right). Photos courtesy of L. Mills. moving out of the supercooling meristem tissue Shaulis, 1980; Wolpert and Howell, 1985, in the buds’ interior. The formation of ice in the 1986); the brown portion of the shoot is able bud scales is termed extraorgan freezing and to supercool, whereas the green portion is not. may be accompanied by progressive dehydra- Therefore, the green end of the shoots usually tion of the interior tissues as temperature dies back in winter, which is a “self-pruning” decreases (Pearce, 2001). In addition, during effect that can be exploited in lightly (e.g., cold acclimation the buds are thought to mechanically) pruned or nonpruned (so-called become isolated from the subtending cane minimally pruned) vines with high bud and (i.e., disconnected from its vascular tissues) by shoot numbers (Clingeleffer, 1984; Keller and closely packed cells with relatively imperme- Mills, 2007). Cold acclimation is initiated by able cell walls that form a diffusion barrier for decreasing day length in late summer, followed ice forming in the cane and enable the buds to by cool nonfreezing (night) temperatures supercool (Fennell, 2004; Jones et al., 2000). (0–5 C) (Schnabel and Wample, 1987). It is pos- Although the most conspicuous sign of cold sible that the lengthening twilight periods at acclimation in grapevines is the abscission of dawn and dusk are at least partly responsible leaves at the end of the growing season, accli- for the day-length effect that is detected by mation is a gradual process. Leaf abscission is the leaves; the low ratio of red to far-red light preceded by periderm formation and thus in twilight may exert its influence via the vine’s browning of the shoots, entering into dormancy phytochrome system (Franklin and Whitelam, of the winter buds, and withdrawal of nutrients 2007; see also Chapter 5.2). Shorter days alone, from the leaves and redistribution to the per- although inducing dormancy, are insufficient manent organs of the vine—a process termed for grapevines to cold acclimate fully (Fennell resorption. The “wave” of brown periderm and Hoover, 1991); however, day length is moving up the shoot in late summer is asso- thought to be a sufficient signal in many tree ciated with a massive increase in storage carbo- species. Chilling temperatures, or more accu- hydrates (Eifert et al., 1961; Winkler and rately the rate of temperature decline, trigger Williams, 1945) and a decline in water content, a massive temporary redistribution of calcium þ and it correlates very closely with cold acclima- (Ca2 ) from the apoplast to the symplast, and þ tion (Fennell and Hoover, 1991; Howell and it appears to be this Ca2 signal that activates 280 7. ENVIRONMENTAL CONSTRAINTS AND STRESS PHYSIOLOGY the cold acclimation process by which plants fall frosts will still occur in a generally warmer acquire freezing tolerance (Browse and Xin, world, they are likely to become less frequent 2001; Plieth, 2005; Thomashow, 1999, 2001). and less severe. þ This Ca2 signal is strong enough to induce In addition to increasing temporarily in cold acclimation even at high temperatures. response to low temperature, ABA rapidly þ Although it is not known whether foliar Ca2 (i.e., within days) promotes freezing tolerance applications can improve cold hardiness, high even at temperatures that are normally too high þ Ca2 supply in the soil, which is typical of to induce cold acclimation (Guy, 1990; Thoma- many high-pH soils, may indeed be associated show, 1999; Zhu, 2002). This response can be with better freezing tolerance (Percival et al., exploited by applying controlled water deficit 1999). (which triggers ABA production; see Chapter However, although low temperatures lead to 3.2) to vines during an unusually warm cold acclimation, they will not maintain vines autumn. In general, acclimation to water deficit in a very cold hardy condition. In fact, grape- is associated with earlier periderm formation vines gradually lose hardiness when they are (Williams and Matthews, 1990) and predis- exposed to low but nonfreezing temperatures poses vines to hardening against cold, probably for long periods. The process of cold acclima- because the stresses caused by extracellular tion is cumulative; it can be stopped, reversed, freezing and drought are similar. Conversely, and restarted, depending on temperature fluc- factors that promote vigor, such as excessive tuations. Warm episodes during the acclima- soil moisture or nitrogen availability, as well tion period induce rapid deacclimation, and as overcropping, late summer pruning, defolia- the greater the loss of cold hardiness, the longer tion, or damage caused by mechanical harvest- it takes for the vines to reacclimate. Conse- ers, may slow cold acclimation (Currle et al., quently, grapevines seem to be less cold hardy 1983). Nonetheless, application of as much as following a warm autumn than after cool 224 kg N ha 1 rarely compromised bud hardi- autumn conditions (Keller et al., 2008). This ness of Riesling in a study conducted in eastern may cause problems when a sudden freezing Washington, an area known for its cold winters event follows a mild autumn (Keller and Mills, (Wample et al., 1993). It seems that once acti- 2007) and has implications with respect to the vated, acclimation accelerates following brief consequences of global climate change. On the episodes of 0 C, and cold hardiness reaches a other hand, the influence of climate change is maximum after the vine has experienced tem- expected to be more important at the lower lim- peratures around 5C (Fennell, 2004). The rate its of temperature because temperature of cold acclimation is negatively correlated increases will probably be higher at night, at with temperature so that vines harden off faster higher latitudes, and in the winter [Intergov- during cooler autumn temperatures (Keller ernmental Panel on Climate Change (IPCC), et al., 2008). Therefore, cold hardiness increases 2007]. This will continue the trend that has gradually during early autumn and then accel- already occurred since the middle of the 20th erates in late autumn to approach maximum century (Jones, 2005) and could mitigate some freezing tolerance. of the adverse effects of warmer autumn tem- Osmotic adjustment, or accumulation of so- peratures on cold acclimation. Moreover, as called cryoprotectants, especially osmotically the average temperature creeps upward, so will active sugars, is essential for proper acclima- the extremes (i.e., outliers) on either end of the tion because of their effects on freezing-point temperature distribution curve. Therefore, depression and membrane stabilization. These although winter killing freezes and spring and compounds provide cold tolerance by reducing 7.4. TEMPERATURE: TOO COLD OR TOO WARM 281 the extent of cell dehydration and inhibiting the cordon, trunk, and roots is also necessary for nucleation and growth of ice crystals. There- growth and metabolism (e.g., nitrogen assimi- fore, intracellular freezing occurs at much lation in the roots) of these organs. The middle lower temperatures in acclimated tissues than portion of the shoot appears to be “refilled” in nonacclimated tissues. Carbohydrates that first, followed by the basal and apical portion, are produced in the leaves are translocated in the cordon and trunk, and, finally, the roots the phloem as sucrose and accumulated in the (Winkler and Williams, 1945). Whereas the woody parts as starch (Figure 7.11; see also developing grape clusters dominate the hierar- Chapter 5). This accumulation starts soon after chy of photosynthate distribution, partitioning bloom, when the rate of shoot growth slows, to the perennial parts intensifies soon after ver- but peaks only during grape ripening and, aison, when the grape seeds are mature and sometimes, after harvest. Starch accumulation ready to germinate. Thus, toward the end of constitutes a replenishment of nutrient reserves the growing season, the permanent organs that have been remobilized from the vine’s per- become the dominant sink of the grapevine manent parts during budbreak and the initial (Candolfi-Vasconcelos et al., 1994). This period establishment of the canopy (Eifert et al., 1961; is concomitant with the final stages of fruit rip- Winkler and Williams, 1945; Zapata et al., ening and may sometimes interfere with wine- 2004). Of course, partitioning to the shoots, makers’ quest to maximize fruit quality. This

r = −

FIGURE 7.11 Dynamics of grapevine cane carbohydrates with changing winter temperature (left): p, start of periderm formation; LF, leaf fall; B, start of bleeding; BB, budbreak (modified with permission from Eifert et al., 1961). Association between cane sugar concentration and the mean temperature over the preceding 5 days (top right, calculated from data in Eifert et al., 1961), and starch grains (St) deposited in the cane xylem (bottom right; reproduced with permission from Plank and Wolkinger, 1976). 282 7. ENVIRONMENTAL CONSTRAINTS AND STRESS PHYSIOLOGY is especially so when environmental stress fac- organs decreases only slightly (primarily due tors limit the amount of sugar available for dis- to maintenance respiration) during the dor- tribution, which can occur due to even a few mant period, but the sugar:starch ratio unusually cool or cloudy autumn days during increases as mean daily temperatures decline and after which ripening can stall. The change (Eifert et al., 1961; Wample et al., 1993; Winkler in relative sink priorities is also the reason and Williams, 1945). why harvest date is not a significant factor in Once sugar translocation ceases, the pores of reserve carbohydrate accumulation and cold the sieve plates separating the phloem’s sieve hardiness (Hamman et al., 1996; Wample and tubes are sealed by callose (see Chapter 1.3), Bary, 1992), which permits the recurring and the cork cells are waterproofed by suberization, often extreme delay in harvest of grapes des- and the perennial organs are drained of any tined for ice wine production near the latitudi- water that is not absolutely essential to main- nal and altitudinal margins of grape growing. tain cell function (e.g., maintenance respira- Starch does not increase cold hardiness of tion). This tissue dehydration reduces the the canes, cordons, and trunks because it is water content to 42–45% and is necessary dur- osmotically inert. Acclimation also requires ing acclimation because it is the rupture of cell conversion of the starch stored in phloem and membranes by ice crystals that causes freezing xylem parenchyma cells back to sugars (mainly injury in nonacclimated plants. There is a sucrose and some glucose and fructose), which strong association between cold acclimation decrease the cells’ osmotic potential and act as and declining water content in the buds and cryoprotectants (Currle et al., 1983; Fennell, woody parts of the vine (Wolpert and Howell, 2004; Guy, 1990). This conversion seems to be 1985, 1986). initiated by short days in mid-September (and In addition to sugars and tissue water, pro- thus before grapes are usually harvested in teins and amino acids are also involved in cold cooler climates), then follows the decreasing acclimation. Soluble proteins (especially glyco- temperatures in late autumn (below 5 C) proteins; i.e., proteins attached to sugar mole- and continues until midwinter (Eifert et al., cules) are secreted to the apoplast, particularly 1961; Hamman et al., 1996; Mu¨ ller-Thurgau, in the bark during autumn, with the nitrogen 1882; Wample and Bary, 1992; Winkler and for their synthesis being provided from remobi- Williams, 1945; see also Figure 7.11). Accumu- lization in the senescing leaves (Griffith and lation of soluble sugars also accelerates because Yaish, 2004; Guy, 1990; Pearce, 2001). In addi- cool temperatures reduce respiration rates (see tion to reinforcing the cell walls and making Chapter 5.2). Nevertheless, the simultaneous them more elastic, the high viscosity of these decline of photosynthesis due to the cooling so-called cryoprotective or “antifreeze” pro- temperatures and shortening days also requires teins prevents the growth of ice crystals and sugars produced from stored starch to be used depresses the freezing point. Incidentally, polar to generate energy in addition to providing fishes use the same strategy to avoid freezing the substrates for the formation of cryoprotec- in subzero saltwater temperatures, and many tive components (Druart et al., 2007). Moreover, hibernating insects add glycerol or even ethyl- starch is also needed to produce fatty acids ene glycol to their supercooling arsenal, because during the transition to dormancy the enabling them to survive temperatures as low vacuole in the cambium cells splits into several as 50 C (Zachariassen and Kristiansen, 2000). smaller vacuoles, which requires synthesis of Antifreeze proteins, which are particularly new membrane lipids (Druart et al., 2007). The strong inhibitors of ice recrystallization, are very total amount of carbohydrate in the perennial similar to the so-called pathogenesis-related 7.4. TEMPERATURE: TOO COLD OR TOO WARM 283

(PR) proteins that grapevines secrete into the (Du¨ ring, 1997; Mills et al., 2006 ). After a mini- apoplast upon fungal attack during the growing mum of approximately 10 C has passed, rising season (see Chapter 7.5). In fact, the antifreeze temperatures induce a reversal of the starch to proteins have probably evolved from PR sugar conversion so that starch again accumu- proteins, and they enhance the resistance of lates (Currle et al., 1983; Eifert et al., 1961; Wink- overwintering plant tissues to cold-tolerant ler and Williams, 1945). This change may be (psychrophilic) pathogens as well as to the freez- associated with the switch from endodormancy ing temperatures (Zachariassen and Kristiansen, to ecodormancy of the cambium (see Chapter 2000). Nevertheless, the PR proteins accumu- 2.2) and goes along with a concomitant decrease lated by nonacclimated tissues (e.g., in shoots in cold hardiness as the vine deacclimates and following powdery mildew infection) lack anti- prepares for budbreak in spring. Although the freeze activity so that late-season fungal infec- sugar:starch ratio also reflects temperature fluc- tions do not enhance cold tolerance (Griffith tuations during winter, the conversion of starch and Yaish, 2004). back to sugar following colder temperatures It appears that free amino acids (particularly gradually slows toward the end of winter proline, arginine, alanine, asparagine, and ser- (Currle et al., 1983; Wample and Bary, 1992). ine) may also be accumulated in parallel with Moreover, the concentration of antifreeze pro- sugars during acclimation, but little is known teins also declines rapidly upon exposure to about their contribution to cold hardiness warm temperatures. Unfortunately, deacclima- (Currle et al., 1983). It could be that the cryopro- tion and loss of freezing tolerance with warm tective properties of proline may be due to its temperatures seem to be more rapid processes function as protein and membrane stabilizer than cold acclimation. Thus, cold hardiness can during water stress (Thomashow, 2001; see also be partially lost within a few days during unusu- Chapter 7.2). Deep-supercooling xylem paren- ally warm periods in winter (Guy, 1990; Mills chyma cells also seem to accumulate flavonols et al., 2006). Southwest-exposed sides of trunks (glycosides of quercetin and ka¨mpferol) that are particularly vulnerable when they warm up help them suppress ice nucleation (Kasuga due to reflection of solar radiation from snow et al., 2008). In addition to the effect of osmotic covers. Although hardiness is restored when potential, the extent of cold tolerance is also cool temperatures return, the deacclimation modified by adapting the size of individual behavior predisposes grapevines for cold injury cells, thickening of cell walls, and changes in when the temperature drops suddenly after a membrane properties that reduce the sensitiv- warm episode. Moreover, the sensitivity to tem- ity of the membranes to mechanical stress due perature fluctuations increases and deacclima- to contraction and expansion (Steponkus, tion occurs more readily as spring approaches 1984). Finally, activation of the antioxidant sys- (Wolf and Cook, 1992), making vines more tem during the cold-acclimation phase vulnerable to sudden cold spells. Once root pres- enhances the protection of cell membranes sure begins to push water up the trunk before from damage due to oxidative stress associated budbreak, the rapid rehydration of the vine with low temperatures (Druart et al., 2007). The tissues is associated with an equally rapid loss combination of all of these processes can either of cold hardiness (see Figure 7.12). delay the onset of freezing or diminish its Many growers in particularly vulnerable adverse consequences. areas (e.g., New Zealand, Washington, and During the winter, grapevines continually Ontario) use wind machines in an attempt to adjust their degree of cold hardiness in response increase the temperature around the vines to fluctuating temperatures (Figure 7.12) (Atwell et al., 1999). Wind machines are most 284 7. ENVIRONMENTAL CONSTRAINTS AND STRESS PHYSIOLOGY

FIGURE 7.12 Dynamics of Merlot cold hardiness with changing winter temperature (left): B, start of bleeding; LF, leaf fall; LT, lethal temperature; lines indicate LT for 10% of the phloem and xylem and 50% of the buds with gray area indicat- ing the 10–90% window. Association between bud, cane phloem, and cane xylem hardiness and the mean daily temperature (right). L. Mills and M. Keller, unpublished data. effective during clear, calm nights, when heat is in midwinter bud damage occurs at somewhat lost to the sky by long-wave radiation. Due to higher temperature than damage to the woody the resulting temperature inversion, the tem- tissues (Mills et al., 2006). However, the temper- perature 20 m above the ground can be up to ature near the ground is usually lower than at 10 C warmer than that at ground level because that at cordon level, which can sometimes cold air settles near the ground after sunset. At result in trunk damage when canes and buds wind speeds greater than 1.5 m s 1, however, may just escape the critical temperature. Within the temperature gradient collapses so that arti- the compound winter bud, the primary bud is ficial mixing of air is useless. generally less hardy than the secondary bud, Survival of a grapevine following cold injury which in turn is less hardy than the tertiary depends on the type of tissue that is damaged, bud (see Figure 7.10). This is probably a reflec- the extent of damage, and the ability to recover tion of the degree of bud differentiation; enough to (at least partially) resume proper increasing differentiation leads to increased function of the affected organs. Note that the susceptibility to ice nucleation. Nonetheless, term crop recovery refers to the crop produced even primary buds in close proximity are not from new growth following injury by spring all killed at exactly the same temperature: The frost. During the cold acclimation and deaccli- difference between 10% bud damage and 90% mation phases, grapevine buds are usually damage can be up to 7 C during the acclima- slightly hardier than canes or trunks, whereas tion period in fall and the deacclimation period 7.4. TEMPERATURE: TOO COLD OR TOO WARM 285

in spring, but this range is often smaller (3 C) buds on the cordon or trunk that may break in midwinter when the buds are fully accli- even when all the dormant buds on the canes mated (Mills et al., 2006). Swelling buds in have been killed (Pratt and Pool, 1981). spring lose cold hardiness very rapidly, possi- In contrast to the phloem, the xylem can bly because their water content increases from accommodate the presence of ice, which can approximately 40% to 75–80% by the woolly expand inside the vessels. Damage to the xylem bud stage (Fuller and Telli, 1999). is not by itself detrimental to the vine, but it Within woody organs, the phloem is usually normally only occurs after the phloem and the first tissue to be damaged by a severe cold cambium have already been killed so that event (see Figure 7.10). Phloem injury can inter- xylem injury is often a sign of disaster. Remark- rupt the transport of assimilates from the per- ably, however, as long as a sufficient number of manent parts of the vine to the developing immature (i.e., relatively undifferentiated) buds in spring. However, even when the xylem parenchyma cells survive, auxin can phloem and vascular cambium are completely induce these cells to dedifferentiate to directly destroyed, starch can also be remobilized from form new phloem cells, complete with sieve the xylem parenchyma cells and released as elements and companion cells (Pang et al., sugars into the xylem conduits along with 2008). At the same time, surviving ray cells water (Ame´glio et al., 2004; Sakr et al., 2003; form callus, which will produce new cambium, Winkler and Williams, 1945). This generates so the vascular tissues are progressively reacti- root pressure that drives out air bubbles and vated. Thus, restoration may occur via the divi- reactivates the buds (see Chapters 2.2 and 3.3). sion of surviving xylem cells, provided some Moreover, although dead tissues cannot be bud-derived auxin can diffuse down to the repaired, they can be replaced by new tissues. “site of action.” However, although more Thus, phloem can be readily restored if the parenchyma cells may differentiate into vascu- cambium survives. Death of the cambium is lar cells when auxin transport is interrupted more serious, although the extent of damage (especially near surviving buds acting as auxin depends on the position of the dead cambium. sources and above log jams of auxin flow), Dead cambium surrounding the trunk effec- these vascular tissues may remain discontinu- tively “girdles” the trunk and can be lethal to ous because they cannot become properly the vine because any surviving buds will break aligned in the absence of polar auxin flow. and begin to grow in spring. Girdling disrupts Because the production of new cells depends the communication between buds/leaves and on the supply of sucrose and other nutrients roots because it blocks movement of sucrose stored in the parenchyma cells, it seems proba- and auxin. If the cambium and phloem cannot ble that a high reserve status promotes recov- be restored rapidly in spring and communica- ery from cold injury. tion reestablished, the roots will starve from The maximal extent to which vines become photosynthate deficiency, and the vine can wilt cold hardy varies with species and cultivar and collapse. By producing and releasing (Williams et al., 1994). Hardier species or culti- auxin, the swelling and breaking buds in vars ordinarily can survive with more of their spring are responsible for reactivation of the water frozen than can less hardy ones. The phloem and, subsequently, renewed cambium eastern Asian species V. amurensis and the activity (i.e., cell division) and production of northeastern American species V. riparia can new vascular tissues (see Chapter 1.3). This survive midwinter temperatures as low as implies that at least some buds must be alive 40 C, whereas the American species M. rotun- for recovery to occur, and this includes latent difolia, at the other end of the spectrum, suffers 286 7. ENVIRONMENTAL CONSTRAINTS AND STRESS PHYSIOLOGY severe damage at 12C (buds even at 5C). assimilation are reversible over the physiologi- The majority of the other American species cal range of approximately 10–35C, but higher are not as hardy as V. riparia, but they are har- (and lower) temperatures can cause injury to dier than V. vinifera. Most V. vinifera cultivars the photosynthetic apparatus. Excessive heat are killed at midwinter temperatures below (and cold) tends to result in symptoms that approximately 25C, but individual cultivars are similar to those caused by drought stress vary by several degrees (Davenport et al., (see Chapter 7.2). This is not surprising because 2008a; Mills et al., 2006). For example, Riesling, heat (and cold) stress has a major effect on Gewu¨ rztraminer, Cabernet franc, and Cabernet plant water relations, decreasing the relative Sauvignon are ordinarily regarded as more water content of tissues and inducing oxidative cold tolerant than Se´millon, Sauvignon blanc, stress. Very high temperatures, above appro- Merlot, and Syrah, which are hardier than Gre- ximately 40C, cause a sharp decline in nache or Mourve`dre. However, the relative photosynthesis due to the disruption of the order of cultivar differences can be modified functional integrity of the photosynthetic by location, cultural practices, and timing of a machinery in the chloroplasts (Zso´fi et al., cold event. Concord (V. labruscana) is consid- 2009). Heat increases the physical distance ered to be significantly hardier than Riesling between the light-harvesting antenna pigments (V. vinifera), and Chardonnay (V. vinifera)is and the reaction center in PSII (see Chapter considered to be almost as hardy as Riesling, 4.1). In addition, the water-splitting system of but the early budbreak and, consequently, early PSII is very heat sensitive; thus, heat inhibits deacclimation of Concord and Chardonnay in PSII-driven electron transport (Berry and Bjo¨rk- late winter induce a substantial loss in hardi- man, 1980). The activity of PSI, on the other ness. As a general rule, species and cultivars hand, is much more heat-stable. The same with a low temperature requirement for bud- applies to the photosynthetic enzymes involved break (see Chapter 2.2) deacclimate earlier than in CO2 assimilation, which are stable to over species and cultivars with a higher temperature 50 C, although heat stress leads to protein threshold and are more cold sensitive toward denaturation and misfolding of newly assem- the end of winter. bled proteins (Berry and Bjo¨rkman, 1980). For instance, rubisco inactivation increases expo- 7.4.3. Heat Acclimation and Damage nentially with increasing temperature, and rubisco activity strongly declines above 35C. Although the majority of the world’s grape Therefore, although stomata start closing above crop is produced in temperate climates, pro- approximately 35C, this seems to be a duction, especially of table and raisin grapes, response to the reduced photosynthesis rather extends to some hot areas where summertime than the cause of it. Leaf temperatures exceed- afternoon temperatures often rise above 35C ing 45C for prolonged periods will kill grape- or even 40C (Williams et al., 1994). The optimal vine leaves (Gamon and Pearcy, 1989). daytime temperature for grapevine growth, Moreover, high temperatures are often related photosynthesis, yield formation, and fruit rip- to low air humidity so that stomata close in ening is below 30C, and heat stress is usually response to the high vapor pressure deficit defined as temperatures exceeding 5C above (see Chapter 3.2) and not because of heat. The those associated with optimal growing condi- influence of temperature on photosynthesis is tions. Temperature has a strong effect on pho- also much more pronounced at high light tosynthesis and respiration (see Chapter 5.2). intensity (Gamon and Pearcy, 1989, 1990). Changes in the rate of photosynthetic CO2 Thus, leaves are more susceptible to heat waves 7.4. TEMPERATURE: TOO COLD OR TOO WARM 287 under a blue sky or during periods of water case with other stresses, the capacity of grape- deficit. vines to recover from heat stress depends on Heat-stressed plant cells seem to accumu- the intensity and duration of the stress and þ late calcium ions (Ca2 ) in the cytosol, which the growth stage at which it occurs. Brief epi- helps to reduce the permeability (and thus sodes of extreme heat are worse than longer leaking) of membranes and to maintain mem- periods of moderately high temperature. On brane integrity. Maintenance of membrane the other hand, photosynthetic acclimation to integrity occurs at least in part through changes high temperatures leads to decreased perfor- in the fatty acid composition of the membranes: mance at low temperatures and vice versa. Warm temperatures lead to higher contents in Therefore, although vines can adapt to seasonal saturated and monounsaturated fatty acids changes in temperature regimes (Zso´fi et al., (Penfield, 2008; Sage and Kubien 2007). On the 2009), short-term temperature fluctuations over other hand, however, these changes in mem- a wide range can result in insufficient photo- brane composition may increase the tissue sus- synthesis for growth and/or fruit ripening. ceptibility to chilling (Iba, 2002). Calcium also Moreover, heat stress also accelerates leaf aging enhances heat tolerance by stimulating the and senescence so that chlorophyll content plant’s antioxidant system in an attempt to pro- often decreases along with photosynthesis tect the photosynthetic machinery from oxida- (Thomas and Stoddart, 1980). tive damage. Heat stress moreover appears to There is variation among Vitis species and result in breakdown of starch to sugars and cultivars in terms of heat sensitivity. Even under accumulation of amino acids (perhaps because relatively modest light intensities, photosynthe- they are not being used for protein production sis in heat-stressed (40 C) V. aestivalis leaves or because proteins are being degraded), espe- may decline more than in V. vinifera, and this cially glutamine, but not the “normal” compat- decrease is associated with damage to PSII and ible solute proline (Guy et al., 2008; Rizhsky subsequent chlorophyll degradation and leaf et al. 2004). In addition, emission of volatile senescence (Kadir, 2006). Photosynthetic recov- isoprenoids, such as isoprene (which is a liquid ery of the leaves that survive a heat episode at room temperature but vaporizes above 34 C) may be slow, extending over several weeks, or monoterpenes, helps the leaves to recover and is incomplete (Gamon and Pearcy, 1989; from brief episodes of excessive temperatures Kadir et al., 2007). When heat-susceptible culti- (>40C) by stabilizing the thylakoid mem- vars such as Concord are grown in warm cli- branes and by scavenging reactive oxygen spe- mates or hot growing seasons with intense cies (Pichersky and Gershenzon, 2002; Sharkey solar radiation, they often develop a condition et al., 2008). This antioxidant activity provides termed “blackleaf,” whereby the adaxial epider- thermotolerance against heat spikes and mis cells of sun-exposed leaves become disco- strongly decreases oxidative damage in the lored, turning bronze-brown, purple, or black leaves. Therefore, despite the injury to the pho- (Figure 7.13). The discoloration usually begins tosynthetic system, leaves can recover (i.e., the in midsummer on the leaves’ sun-facing ridges, inactivation is reversible) within a few days especially in water-stressed vines, and is after temperatures return to physiological accompanied by chlorophyll degradation and a levels, as long as the heat stress was not so decline in photosynthesis and may result in severe as to cause membrane leakage and necrosis and leaf abscission (Smithyman et al., necrosis; grape leaves appear to be killed when 2001). These blackleaf symptoms look suspi- the leaf temperature exceeds approximately ciously like the UV damage symptoms described 45 C (Abass and Rajashekar, 1991). As is the in other plants and may be a consequence of the 288 7. ENVIRONMENTAL CONSTRAINTS AND STRESS PHYSIOLOGY

FIGURE 7.13 Blackleaf symptoms initially appear on south-facing ridges of Concord leaves (left) and eventually cover most of the exposed leaf surfaces (right). Photos by M. Keller. polymerization of phenolic compounds in the probably increase in both frequency and sever- epidermal cells that results from oxidative stress ity. An analysis for southern Australia shows (Kakani et al., 2003; Yamasaki et al., 1997). In that the probability of a run of 5 consecutive days other words, blackleaf symptoms probably arise over 35 C doubles for a 1 C warming and from injury caused by UV radiation rather than increases by a factor of five for a 3C warming from heat damage per se. Growth and fruit rip- (Hennessy and Pittock, 1995). As model calcula- ening are suppressed as a consequence of the tions for the United States demonstrate, such decrease in photosynthesis. In addition, high heat episodes could be detrimental to fruit qual- temperature shifts carbon partitioning to favor ity in areas that already experience warm or hot vegetative growth at the expense of fruit growth climates (White et al., 2006), which encompass and ripening. Thus, the reduction in photosyn- many of the current grape production regions thesis due to postveraison heat stress can mark- in the world. edly delay or even inhibit fruit ripening by Evapotranspiration at the leaf surface cools decreasing the amount of assimilates available the leaf tissues so that the leaf temperature is for export to the clusters. Because this may lead generally close to air temperature (see Chapter to uneven ripening or an overall delay of fruit 3.2). Evaporative cooling of the leaves thus pro- maturation (Cawthon and Morris, 1982), it has vides some heat resistance. Because high stoma- implications for grape production in an increas- tal conductance favors rapid transpiration rates, ingly warmer climate. In addition to a rise in it lowers leaf temperature and appears to reduce average temperatures and an associated increase deleterious effects of heat stress. This is particu- in cumulative heat units and growing-season larly important during the critical flowering length, meteorologists and climate modelers and fruit development stages, in which heat predict that shifts will also occur in the number stress would result in lower crop yields. Higher and extent of extreme weather events (IPCC, stomatal conductance also increases CO2 2007). This conclusion follows directly from the diffusion into the leaf and favors higher photo- statistical normal distribution of temperature synthetic rates. Higher photosynthetic rates data around a mean, which follows a so-called could in turn lead to more biomass and higher bell curve. Therefore, summer heat waves will crop yields. However, higher stomatal 7.5. LIVING WITH OTHER ORGANISMS: DEFENSE AND DAMAGE 289 conductance appears to favor higher yields by a denaturation of membrane proteins. The disrup- mechanism not directly related to photosynthe- tion of membrane structure is irreversible and sis, perhaps by reducing respiration. Respiration leads to leakage of cell solutes and breakdown is also strongly stimulated by increasing temper- of cell compartmentation, which is normally ature and results in a net loss of assimilated car- lethal for the cell. The poor ability of grape bon. High yields are generally an important goal berries for transpirational cooling makes them for the production of table, raisin, or juice relatively susceptible to overheating. Indeed, grapes, whereas moderate to low yields are pre- sun-exposed berries can heat up to 15C above ferred in wine grape production to maximize air temperature, whereas shaded berries are fruit quality. Nevertheless, in areas where vege- usually at approximately ambient temperature. tative growth can be controlled by deficit irriga- This has important implications for vineyard tion and crop load can be adjusted manually, row direction, trellis design, and canopy man- management practices (including varying the agement. Because during a sunny day the air timing of deficit irrigation) aimed at favoring temperature in the early afternoon is generally high stomatal conductance could be used to higher than in the morning, exposed grape improve quality (e.g., see the discussion of berries on the “afternoon side” of the canopy organic acid synthesis in Chapter 6.2). Because can reach considerably higher absolute tempera- water deficit increases a vine’s susceptibility to tures than their counterparts on the “morning heat stress, drought during the bloom period is side.” Therefore, whereas cultural practices that especially detrimental to fruit set if it coincides maximize sun exposure are usually beneficial in with a heat episode. a cool climate (where they can accelerate ripen- Just like other developmental processes, ing), they may be detrimental in a warm, sunny grape ripening is accelerated by high tempera- climate (see Chapter 6.2). Symptoms of sunburn tures. This is especially true for sugar accumula- on clusters include initial reddening or brown- tion and malate degradation. However, because ing of the exposed portion of the skin (on both grape berries are designed to minimize water red and white grapes), followed by sinking in loss by transpiration, especially after veraison of the affected area or dehydration (shriveling) (see Chapter 6.2), they cannot take advantage of of the whole berry (Figure 7.14). Sunburn symp- the evaporative cooling mechanism that usually toms can become particularly acute following protects leaves from overheating. Too much heat leaf removal late in the growing season because, can inhibit or even denature proteins in the in contrast to berries that have been exposed berries. When plant organs, such as previously since the beginning of the season, the previously shaded clusters following leaf removal, are sud- shaded skins have not accumulated sufficient denly exposed to high temperature (38–40 C), amounts of “sunscreen” such as flavonols and they produce so-called heat shock proteins that xanthophylls (Du¨ ring and Davtyan, 2002; Kolb function as chaperones by helping to fold other et al., 2003). proteins into a suitable structure, preventing accumulation of proteins that have been dena- 7.5. LIVING WITH OTHER tured by heat, and helping to reactivate them ORGANISMS: DEFENSE AND (Iba, 2002). Nevertheless, sunburn symptoms DAMAGE can develop as an expression of oxidative dam- age that usually results from a combination of high light intensity and high temperature. Heat Grapevines share their living quarters, both injury usually results from the disintegration of above- and belowground, with a myriad of cell membranes due to lipid phase changes and other organisms, mainly arthropods (spiders, 290 7. ENVIRONMENTAL CONSTRAINTS AND STRESS PHYSIOLOGY

FIGURE 7.14 Delayed ripening of the sun-exposed side of a Cabernet Sauvignon cluster in a warm climate (left), symp- toms of sunburn on grape berries (center), and shriveling of sunburnt berries (right). Photos by M. Keller. mites, and insects) and microorganisms (fungi, and humid climates can be remarkably similar oomycetes, bacteria, and viruses) in addition to that often observed under deficit irrigation to some nematodes, birds and mammals, and (see Chapter 7.2) in warm and dry climates; other plants. The majority of these normally vines have diminished vigor, smaller berry leave grapes alone, do little damage, or are size, improved fruit composition, and reduced unable to overcome the grapes’ defense strate- bunch rot (Keller et al., 2001a,b; Valde´s-Go´mez gies (the latter is called an incompatible interac- et al., 2008). Vineyard floor vegetation is desir- tion). For example, grape berries are typically able for many reasons but may compete with heavily colonized by yeasts, especially in the vines for resources if not managed carefully, vicinity of stomata and small cracks in the cuti- especially in warm and dry climates (Celette cle that allow exudation of sap from the meso- et al., 2009; Tesic et al., 2007). Due to preferential carp (Belin, 1972). Yet this is very rarely a growth of vine roots beneath emitters in drip- concern, unless harvested fruit is left standing irrigated vineyards in dry climates (Stevens too long before processing so that uncontrolled and Douglas, 1994), even if interrow cover fermentation sets in. Some organisms, however, crops are mowed or incorporated into the soil compete with the vines for resources (espe- by tilling, the nutrients released during their cially other plants, which are therefore called microbial decomposition may be mostly avail- weeds) or make a living feeding on various able to cover crop regrowth rather than to the grapevine structures, which makes them pests grapevines. However, even in humid climates, (in the case of arthropods) or pathogens (in competition from cover crops may increase the case of microorganisms, when we speak of vineyard fertilizer requirements if vine produc- a compatible interaction). tivity is to be maintained (Keller et al., 2001a,b). Plants sharing a vineyard with grapevines, Nitrogen, at least, can also be supplied using whether they are called weeds or resident veg- leguminous cover crops that employ symbiotic etation or are planted deliberately as cover Rhizobium bacteria to fix atmospheric nitrogen ! þ crops or green floor covers, use water and tend (N2 NH4 ), which mitigates the need for to reduce nutrient availability for the vines synthetic fertilizers ( Jackson et al., 2008; Patrick (Celette et al., 2009; Keller, 2005; Morlat and King and Berry, 2005). Consequently, cover Jacquet, 2003; Tesic et al., 2007). Performance crop management should aim to synchronize of vines with a permanent floor cover in cool nutrient availability in the soil to the seasonal 7.5. LIVING WITH OTHER ORGANISMS: DEFENSE AND DAMAGE 291 demand by the vines (Keller, 2005; Perret et al., short generation times. Large numbers, varia- 1993). tion, and rapid reproduction mean rapid evolu- Organisms also interact in other ways. In tion. Because random mutations happen some instances, pathogens rely on pests (so- every so often, the laws of probability demand called vectors) to carry them from one plant to that in a sufficiently large or sufficiently fast- another. For example, certain viruses are trans- reproducing population even unlikely changes mitted by nematodes or insects, and phytoplas- are bound to occur. Nonrandom natural mas (specialized bacteria living in the phloem) selection does the rest, quickly weeding out require phloem-sucking insects (e.g., leafhop- unfavorable mutations, ignoring irrelevant pers) for their transmission (Bovey et al., changes, and favoring material that benefits 1980). Of course, vegetative propagation is also reproduction. Consequently, even the most a very effective means to disperse such patho- unthinkable mutants are bound to arise, and gens (which is why they are often collectively any such fungal, bacterial, or viral strain with termed graft-transmissible diseases). Until just a minuscule advantage over the others will recently, the evolution of grapevines was soon dominate the population. These traits tightly linked to the evolution of other organ- enable microbes to evolve extremely rapidly, isms that happen to like fruits and leaves. under sufficient selection pressure sometimes However, such coevolution has been greatly within mere days. In addition, bacteria have hindered since the introduction of vegetative neither true biological species nor sexual repro- propagation that deprived cultivated vines of duction (Mayr, 2001). Instead, they readily the opportunity to adapt by sexual reproduc- exchange DNA with one another across tion, leaving open only the avenue of somatic apparent “species” boundaries, when two not mutations (see Chapter 2.3). necessarily related bacteria meet and connect Because mutations in somatic cells during with each other by growing a temporary tube cell division or mitosis (Greek mitos ¼ string, between them across which genes flow from loop) occur several orders of magnitude less one bacterial cell to its neighbor. This so-called frequently than in germ cells during meiosis, conjugation is analogous to copying systems there is very little genetic variation within most data files directly from one computer to modern grape cultivars compared with their another. Vegetatively propagated grapevines, wild relatives. Consequently, the influence of by comparison, evolve only very slowly, often novel environments or newly introduced remaining essentially unchanged for hundreds pathogens will cause more problems for some or even thousands of years (see Chapter 1.2). cultivars than for others, depending on their This puts microorganisms at a tremendous specific genotype. Moreover, a population that competitive advantage over cultivated plants comprises many genetically identical indivi- and is a major cause of disease epidemics and duals, such as is typical of the monocultures of the rapid emergence of strains that are that are modern vineyards, is vulnerable to resistant to pesticides. any pathogen that discovers the key to exploit- However, even insects can multiply at aston- ing this susceptibility. This puts cultivated ishing rates; a single phylloxera egg hatching in grapevines at great risk from organisms, espe- spring may result in 5 billion parthenogeneti- cially microorganisms, that like to eat their cally generated descendants by midsummer fruit, leaves, or roots. Microorganisms such as (Battey and Simmonds, 2005). A pathogen or fungi, bacteria (especially bacteria), and viruses pest introduced to a new area or made virulent are extremely abundant in the environment, are through a mutation to which local cultivars exceedingly diverse, and have unnervingly have no defense could have devastating 292 7. ENVIRONMENTAL CONSTRAINTS AND STRESS PHYSIOLOGY consequences from which wild and diverse unripe grapes could be another defense against plant communities may be somewhat protected fruit-eating birds and mammals. because they have coevolved with the patho- A variety of insects and mites (along with gens. Such coevolution essentially constitutes the crabs collectively grouped into the arthro- a genetic “arms race,” in which mutations pods or the phylum Arthropoda from Greek occur in and natural selection acts on both the arthron ¼ joint, podos ¼ foot) feed on grapevine pathogens and the hosts. Moreover, mixing organs. Among them is the most devastating of genes in new combinations during sexual grapevine pests—the root-feeding, aphid-like reproduction (see Chapter 2.3) presents a phylloxera, Daktulosphaira vitifoliae Fitch (also moving (and, it is hoped, elusive) target for called Viteus vitifolii), which can multiply in would-be pathogens and pests. Coevolution all but very sandy soils and spreads rapidly and sexual reproduction, of course, can do except in very dry climates. Phylloxera (Greek nothing to improve plant resistance if a patho- phyllon ¼ leaf, xeros ¼ dry) is indigenous to gen and its potential host evolved in geo- the eastern and southwestern United States graphic isolation from one another. Both and central America and was introduced to cultivated and wild forms of the European Europe on infested plant material (probably as V. vinifera, when confronted with phylloxera eggs on rooted cuttings) in the middle of the and the mildew-causing pathogens introduced 19th century. The subsequent epidemic across from North America in the second half of the the continent nearly destroyed the European 19th century, quickly succumbed for precisely wine industry, before grafting to resistant the same reason native American peoples suc- (more accurately tolerant) American rootstocks cumbed to European diseases after the Spa- was introduced and accepted as the only feasi- niards had inadvertently introduced the ble remedy. On American Vitis species, phyl- pathogens to the Americas: They had not coe- loxera has a complex life cycle, alternating volved with the pathogens and therefore had between root-feeding and leaf-feeding forms not had a chance to “invent” effective genetic (Galet, 1996; Granett et al., 2001). The leaf- resistance strategies. At the beginning of the feeding form usually cannot multiply on the 21st century, history seems to be repeating leaves of European V. vinifera, but the root- itself in China, where phylloxera is wreaking feeding form is extremely damaging. Once havoc among native populations of wild and established, the insects and secondary infec- cultivated grapes (Du et al., 2009). tions by fungal pathogens can destroy the roots Birds can be important pests on wine grapes of susceptible V. vinifera cultivars. Infested because grapes have evolved to have their vines gradually decline, often displaying symp- seeds dispersed by birds (see Chapter 2.3), toms of potassium deficiency, and eventually which often interferes with a grower’s desire die as they run out of water and nutrients. to mature the fruit well beyond the stage at The decline is rapid on heavy clay soils but which seeds are ready to germinate. The pres- can be slow on sandy soils, especially in very ence in the green, immature berry of malic acid, cool or hot climates (Granett et al., 2001). which is an effective bird deterrent, and its Because damage to the roots of American Vitis degradation during ripening probably is an species is very limited, these phylloxera- adaptive mechanism evolved by grapes to tolerant species or interspecific crosses are used ensure protection of the developing seeds and throughout the world as rootstocks that protect their dispersal once they have reached matu- vineyards from succumbing to phylloxera rity. The astringent tannins and the toxic, (Galet, 1996; Granett et al., 2001). The mechan- needle-like calcium oxalate crystals present in isms of resistance or tolerance are not well 7.5. LIVING WITH OTHER ORGANISMS: DEFENSE AND DAMAGE 293 understood, but many grape species may be shoots and are dispersed mainly by movement able to form a cork layer around areas where of shoots (e.g., due to wind or machinery). Rust phylloxera feeds on the roots, which may cut mites are particularly destructive when cool off the food supply to the insects. Despite this spring temperatures slow shoot growth because knowledge gap, the breeding and use of root- rapidly growing shoots quickly outgrow the stocks, many of which continue to prevent feeding mites. In contrast, bud mites live and damage after many decades of use, is an out- feed inside the developing and dormant buds, standing example of a highly successful which leads to stunted shoots (often with zig- biological pest control strategy. zag growth) that are missing one or all of their All arthropods have natural enemies (preda- inflorescences or the apical meristem (Bernard tors or parasites), which we consider beneficial et al., 2005). The bud mites migrate to newly organisms; many of these are small wasps that forming buds after budbreak; they are spread parasitize insect eggs, and others are bugs, lady mainly via infested planting material. In extreme beetles, or spiders that eat other insects or mites cases, bud necrosis (i.e., death of the primordia (Mullins et al., 1992). However, one insect, the inside the bud) from mite feeding can prevent multicolored Asian lady beetle Harmonia axyri- budbreak altogether. In biologically diverse dis Pallas, which was originally introduced into vineyards, both of these (as well as other damag- the United States as a biological control agent, ing mite species) are usually kept in check by may directly influence wine and grape juice predatory mites. aroma. When the beetles hiding in grape clus- When leaves are damaged by arthropod ters are disturbed or crushed during juice pro- feeding or other mechanical injuries, they cessing, they release a yellowish fluid from release a blend of volatiles that is composed of their legs in a process termed “reflex bleeding.” ethylene, terpenoids, and other compounds The methoxypyrazines in this fluid appear to derived from fatty acids in the cell membranes, lead to off-flavors, termed “ladybug taint,” such as methyl salicylate or methyl jasmonate. due to their extremely potent impact on vege- The emission of such volatiles is an indirect tal, green pepper, and herbaceous aromas defense response and serves to attract benefi- (Pickering et al., 2004, 2005; see also Chapter cial arthropods that are predators or parasites 6.2). This lady beetle is therefore now consid- of the herbivorous pests but do not feed on ered an important contaminant pest. grapevines (James and Price, 2004; Pichersky In addition to insects, several mite species and Gershenzon, 2002; Poelman et al., 2008; (mites are related to spiders rather than insects) Takabayashi and Dicke, 1996). In addition to also can be potent grapevine pests. For instance, this indirect effect, some volatiles also appear grape rust mites (Calepitrimerus vitis Nalepa) and to have a direct defensive function. Hexanals grape bud mites (Colomerus vitis Pagenstecher) and hexenals, for instance, may be toxic to are responsible for a phenomenon termed insects, such as certain aphids (which imbibe “restricted spring growth” (Bernard et al., phloem sap), whereas other volatiles deter 2005). Symptoms include retarded budbreak, some insects (especially Lepidoptera, whose stunted shoots and distorted leaves in spring caterpillars chew on the leaves) from laying until fruit set (with poor fruit set or loss of eggs on the leaves. Moreover, grapevines that inflorescences), intense growth of lateral shoots, have been attacked by pests may also be able and bronze discoloration (russetting) of mature to use volatile emission as a signal of imminent leaves later in the growing season. The damage threat to nearby vines, which can detect this is caused by overwintering populations of signal and respond to it by activating their rust mites, which feed on the newly emerging own defenses. There is even evidence of plants 294 7. ENVIRONMENTAL CONSTRAINTS AND STRESS PHYSIOLOGY under herbivore attack releasing root exudates as reinforcement of cell walls by deposition of that then induce neighboring plants to release callose, lignin glycoproteins, and phenolics and volatiles from their leaves (Bais et al., 2006), production of so-called pathogenesis-related but it is unknown whether grapevines can do (PR) proteins such as chitinases and glucanases. this. Such plant-to-arthropod and plant-to- Cell separation is also accompanied by accumu- plant communication by use of air-transmissi- lation of PR proteins to prevent infection; for ble signals may be very important in minimiz- example, these proteins accumulate at the base ing herbivore-induced damage because plants of anthers and calyptras at the time of shedding cannot hide or run away from pest attack. The (Roberts et al., 2002). If this defense response is number and relative abundance of beneficial unsuccessful and pathogens penetrate into the arthropods often increases as the botanical tissues, the vine can shed the entire infected diversity increases in a vineyard and its vicinity organ to prevent the spread of the pathogen (e.g., due to diverse cover crops, resident throughout the plant. However, plants have floor vegetation, or adjacent brush or hedgerow evolved an array of strategies to resist fungal vegetation), and so does the number of indif- infection. These strategies can be constitutive or ferent or neutral arthropod species (i.e., those induced. Constitutive resistance strategies are that are neither harmful nor beneficial), which passive and are present regardless of infection results in an overall increase in biodiversity (i.e., preinfection). They include physical and ecosystem stability and keeps pest pop- barriers (e.g., cuticle and cell walls) and chemi- ulations in check (Altieri et al., 2005; Remund cals with antimicrobial activity (so-called et al., 1989). An additional benefit of cover phytoanticipins such as phenolics), which are crops is their ability to significantly reduce the generally accumulated in the cell vacuoles (often splash dispersal of fungal spores during rain- as inactive precursors) and provide nonspecific fall compared with bare soil (Ntahimpera protection against a wide range of would-be et al., 1998). invaders. Induced strategies, on the other hand, Any prospective pathogen that attempts to are actively initiated in response to pathogen penetrate the epidermis first has to overcome invasion (i.e., postinfection) and specifically the waxy cuticle and thick outer cell walls on target pathogens that have overcome the consti- plant organs before it can cause disease. Accord- tutive barriers. They include the production of ingly, there seems to be an inverse association reactive oxygen species, fortification of cell walls between the thickness of the cuticle and outer (lignification, suberization, or incorporation of cell wall of different Vitis cultivars and their sus- callose, proteins, silicon, and calcium), and ceptibility to powdery mildew (Heintz and production of antimicrobial compounds (e.g., Blaich, 1989). Wounding of leaves and grape proteins and so-called phytoalexins). Active berries caused by herbivore, bird, or arthropod defenses are usually restricted to the site of feeding or by other mechanical damage (e.g., invasion. The infected and neighboring cells from wind or machinery) can provide possible accumulate the antimicrobial chemicals to high “enhanced-access” points for pathogens. concentrations in an attempt to restrict the In addition, shedding of plant organs (e.g., of spreading of the invading pathogen. Cell wall flower caps during anthesis) leads to exposed reinforcement also requires the release of fracture surfaces that provide ideal sites for metabolic precursors to the apoplast, before they pathogen invasion (Roberts et al., 2002; Viret can be incorporated in the cell walls, and is et al., 2004). This is why plants respond to usually accompanied by the production of physical damage by mechanisms that aim at reactive oxygen species that induce cross-linking healing wounds and preventing invasion, such of the cell walls (Field et al., 2006). 7.5. LIVING WITH OTHER ORGANISMS: DEFENSE AND DAMAGE 295

Grapevines, like other plants, have special even activate defenses in distant healthy tissues receptor proteins that recognize invading (Heil and Ton, 2008; Thatcher et al., 2005). In pathogens by some of the microbial enzymes other words, the secondary signals exert a kind or complex carbohydrates, proteins, and lipids, of remote control; that is, they act systemically. especially those in the fungal cell walls such as Although salicylic and jasmonic acid are not chitin (Shibuya and Minami, 2001). In addition, volatile, their methylated forms—methyl salic- they also seem to be able to interpret as ylate and methyl jasmonate—can be emitted intruder signals the breakdown products of by the infected leaves within hours of an infec- their own cuticle and cell walls. These com- tion and taken up by distant uninfected leaves, pounds are collectively termed elicitors because where they may be transformed back into the they elicit a defense response by the plant. In nonmethylated forms to prime or sensitize fact, the defense response results from activa- those distant leaves for defense. The salicylic tion of various biochemical pathways by a and jasmonic acids arriving days to weeks after series of signaling cascades that are triggered an infection via the vascular pathway may then by the detection of a pathogen. One of the first amplify the induced defense response in those signals is the so-called oxidative burst on the leaves (Heil and Ton, 2008). In some instances, cell membrane (Apel and Hirt, 2004; Jones H2O2 in concert with secondary signals induce and Dangl, 1996; Mittler, 2002; Smith, 1996; the infected cells and those surrounding the Somssich and Hahlbrock, 1998). Within min- infection site to commit suicide in a process utes of an attempted infection by a foreign termed hypersensitive response. This limits invader, there is a rapid rise in reactive oxygen food supply to the pathogen, starving and con- • species (mainly superoxide, O2 ) in the apo- fining the invaders in a localized area (Apel plast. The superoxide is rapidly converted to and Hirt, 2004; Jones and Dangl, 1996). In addi- hydrogen peroxide (H2O2), which diffuses into tion, nearby vascular tissues may be occluded the cells and is perceived by the vine as a signal by the deposition of callose, which limits the of an imminent threat. In contrast to the situa- spread of the invader or its toxins. tion during abiotic stress (see Chapter 7.1), this A necrotroph (Greek nekrois ¼ death, trophe´ • ¼ oxidative burst is premeditated (O2 is nutrition) is defined as a pathogen that kills produced enzymatically by NADPH-oxidase, its host’s cells and then obtains its food supply peroxidase, and amine oxidase), and the anti- from these dead cells. The typical necrotroph oxidant systems remain silent to boost the attacking grapevines is B. cinerea Pers.:Fr., amount of H2O2. In response to this signal, which can infect all green plant organs but is the surrounding plant cells mount structural best known for causing gray mold in grapes barriers; they reinforce (by callose deposition) and many other plant species (Williamson et al and often lignify (H2O2 stimulates lignification ., 2007). In a devious twist, rather than by peroxidase and also has antimicrobial being killed by the oxidative burst inside the effects) their cell walls at the sites of attempted host tissues, such necrotrophs may actually entry and produce PR proteins. These proteins exploit (and thus subvert) the plant’s defense owe their antifungal activity to their ability to response by promoting tissue senescence, degrade chitin and glucans, which are impor- which in turn favors disease progression (Elad tant components of the cell walls of fungi and Evensen, 1995; von Tiedemann, 1997). (Selitrennikoff, 2001). Necrotrophs, as their name suggests, also Secondary signaling molecules, including benefit from rather than being inhibited by the salicylic acid, jasmonic acid, and ethylene, then cell death induced during a hypersensitive augment the early defense response and may response, which, in contrast, is very effective 296 7. ENVIRONMENTAL CONSTRAINTS AND STRESS PHYSIOLOGY against the spread of biotrophs, again as their haustoria in the case of the mildews or arbus- name suggests (Poland et al., 2009). Therefore, cules in the case of mycorrhiza; the host cell although killing the cells around sites of membrane becomes wrapped around the attempted infection is indeed a useful strategy mycelium but is not penetrated by it (Hall to contain biotrophs, this approach comes at and Williams, 2000; Pearson and Goheen, the cost of increased vulnerability to invasion 1998; Smith et al., 2001). This maximizes the by necrotrophs, whose mode of life evolved intruder’s surface area inside the host for active on a platform of thriving on dead tissue and nutrient absorption, which is a one-way street that is only encouraged by suicidal plant cells. for mildews and a two-way street for A biotroph (Greek bios ¼ life) is an organism mycorrhiza. that can live and multiply only on another Induced structural barriers may fail to con- living organism (i.e., it cannot be grown in tain a pathogen or may work for some but not culture), making it an obligate parasite or sym- other pathogens. Several hours after an infec- biont, depending on whether its presence is tion, the vine therefore also activates a second damaging or beneficial to its host. Examples line of defense, this time of a more direct, bio- of biotrophs include most viruses, the powdery chemical nature. This induced biochemical mildew fungus E. necator [Schwein.] Burr. (for- defense includes accumulation of antimicrobial merly Uncinula necator), and the downy- compounds, including the phytoalexins and PR mildew-causing oomycete Plasmopara viticola proteins. Another defense strategy, especially (Berk. & Curt.) Berl. & de Toni, both of which in response to xylem-invading fungal and develop on green plant parts, but also mycor- bacterial pathogens, is the accumulation of ele- rhizal fungi that enter in a symbiotic relation- mental sulfur in the vessel walls and xylem ship with the roots of their hosts (see Chapter parenchyma cells (Cooper and Williams, 7.3). Biotrophs typically tap into their host’s 2004). Unfortunately, most bacteria (e.g., the epidermis cells to extract sugar (mainly glu- Pierce’s disease-causing Xylella fastidiosa) are cose) and other nutrients (especially amino not sensitive to S. In contrast, the nonvascular acids) to support their hyphae growing on the pathogen E. necator and other fungi are very surface and act as sinks altering carbon distri- sensitive. Accordingly, S dust and S-lime mix- bution in the host to divert sugars and amino tures have been in use as the world’s first fun- acids for their own metabolism (Brem et al., gicides for thousands of years, and their 1986; Hall and Williams, 2000; Smith et al., effectiveness against powdery mildew was dis- 2001). In other words, biotrophs compete with covered soon after the introduction of the fun- the sinks of their hosts for carbohydrate supply. gus in Europe. Moreover, applications of They rely on a strategy that attempts to invade S fertilizers can enhance the resistance to host tissues with minimal damage to their E. necator (see Chapter 7.3). The disadvantage host’s cells and suppress or bypass its defense of S application, however, is the risk of spider system, and infection is usually limited to the mite infestations, especially when S is applied epidermis. In fact, after breaking through the as dust: Mites like dust. cell wall, using a high-precision chemical dril- Phytoalexins (Greek phyton ¼ plant, alexein ¼ ling approach (i.e., they penetrate a cell by to ward off) are a diverse group of fungitoxic digesting a pore through its wall; see Cantu (i.e., antimicrobial), low-molecular-weight et al., 2008), they may even exploit the host’s metabolites that plants produce in response to defense strategies to accommodate the estab- various kinds of stresses, such as attack by lishment of elaborate intracellular feeding pathogens or wounding (Kuc, 1995; Smith, structures. These structures are termed 1996). These “natural fungicides” include more 7.5. LIVING WITH OTHER ORGANISMS: DEFENSE AND DAMAGE 297 than 300 chemically diverse compounds (e.g., pterostilbene (Breuil et al., 1999; Hoos and phenolics, terpenoids, polyacetylenes, fatty Blaich, 1990; Pezet et al., 1991), whereas fungi acid derivatives, and others) and have been regarded as not normally pathogenic for grape found in more than 100 plant species from berries ostensibly lack this ability. Moreover, it more than 20 families. The phytoalexins charac- is possible that accumulation of the fungitoxic teristic of grapevines are phenolic compounds viniferins comes too late: They may be formed named stilbenes and include resveratrol and only after the infected tissues have already its glucoside piceid, pterostilbene, and several become necrotic (Keller et al., 2003b). Despite resveratrol oligomers, the viniferins (Jeandet their inducible nature in green plant organs, et al., 2002; see also Chapter 6.2). They are pro- stilbenes also seem to be present as constitutive duced at or near sites of infection and can compounds in the vine’s woody organs, includ- inhibit spore germination and mycelium ing canes, trunks, and roots, but also pedun- growth of a variety of fungi and oomycetes. cles. Also, in concert with other phenolics and The production of these phytoalexins is trig- even terpenoids, they may contribute to the gered by high-molecular-weight microbial general pathogen resistance and durability of compounds and components of plant cell walls wood (Kemp and Burden, 1986; Langcake and that are released during infection and are called Pryce, 1976; Pool et al., 1981). elicitors (Blaich and Bachmann, 1980; Darvill Similar to stilbenes, the PR proteins, which and Albersheim, 1984). In addition, stilbenes are mostly accumulated in the cell walls, also also accumulate, along with PR proteins, in have the capacity to directly inhibit spore ger- response to the air pollutant ozone (O3) that mination and/or hyphal growth of certain forms as part of photochemical smog and is pathogens (Giannakis et al., 1998; Jacobs et al., highly phytotoxic, inducing leaf symptoms 1999; Monteiro et al., 2003). These antimicrobial such as numerous yellowish to reddish spots proteins include b-1,3-glucanases (members of (“stipple”) or blotches (“mottling”), bleaching, the so-called PR-2 protein family), chitinases bronzing, or, in severe cases, premature senes- (PR-3 family), osmotin/thaumatin-like proteins cence (Sandermann et al., 1998; Schubert et al., (PR-5 family), and perhaps some nonspecific 1997; Williams et al., 1994). lipid transfer proteins (nsLTPs; PR-14 family). The rate at which phytoalexins accumulate Some of these antifungal proteins are active at the site of infection determines whether or constitutively (i.e., present at some base con- not the pathogen attack is successful (Kuc, centration), and all accumulate to high concen- 1995; Smith, 1996). If accumulation is too slow, trations in response to fungal attack of both the intruder has grown far beyond the inocula- leaves and berries. Whereas the PR-5 proteins tion site by the time phytoalexin concentrations are not produced constitutively in preveraison are high enough to inhibit its growth. In this grape berries and other organs, they and the natural arms race, many microorganisms have PR-3 (but not PR-2) proteins begin to accumu- found ways to degrade and thus detoxify phy- late in the berries at veraison (Negri et al., toalexins. This detoxification is an important 2008; Robinson et al., 1997; Salzman et al., trait conferring pathogenicity (also termed vir- 1998; Tattersall et al., 1997). This induction is ulence), enabling the fungus to infect the plant probably a consequence of the osmotic stress tissue (Darvill and Albersheim, 1984; Jeandet caused by sugar accumulation and makes the et al., 2002; Sbaghi et al., 1996; Smith, 1996; ripening berries increasingly resistant to pow- VanEtten et al., 1989). For instance, employing dery mildew (E. necator), downy mildew the enzyme laccase (aka stilbene oxidase), (P. viticola), and black rot [Guignardia bidwellii B. cinerea is able to degrade resveratrol and (Ellis) Viala & Ravaz]. In addition, whereas 298 7. ENVIRONMENTAL CONSTRAINTS AND STRESS PHYSIOLOGY moderate amounts of sugars can boost the colo- the intensive use of costly pesticides to optimize nization by some fungal pathogens (seemingly control and to manage pesticide resistance of because sugars are important carbon sources pathogens. Nonetheless, although such strate- for the microbes), higher sugar concentrations gies usually work reasonably well for fungal may reverse this effect, leading to lower sus- and fungi-like pathogens, they fail completely ceptibility. Indeed, E. necator appears to be for most bacteria and viruses that insert them- unable to establish new infections on grape selves within their hosts’ cells. Once present in berries exceeding approximately 7 Brix, a a victim, vegetative propagation ensures their threshold that is normally reached just before dissemination with new planting material veraison (see Chapter 6.2). It is possible that (typically cuttings or buds) taken from the host. this phenomenon, which is termed “high-sugar Some viruses are further distributed to healthy resistance,” may be at least partly due to the plants (e.g., in neighboring vineyards) by insect osmotic stress the high sugar concentration or nematode vectors. In contrast with other imposes on the fungal cells. Nevertheless, the disease agents, bacteria and viruses cannot be rachis supporting the ripening berries remains eradicated or contained with any curative or susceptible as long as it remains green. therapeutic control measures short of destroying Some members of the PR-3, PR-5, and PR-14 infected plants (Rowhani et al., 2005). families also seem to inhibit spore germination The remainder of this chapter discusses and growth of Phomopsis viticola Sacc., which examples of some of the major pathogens causes cane and leaf spot disease, and Elsinoe afflicting vineyards throughout the world, ampelina (de Bary) Shear, which causes grape- their disease symptoms, and interaction with vine anthracnose (Monteiro et al., 2003). The their hosts. V. labrusca-derived Concord grapes accumulate very high concentrations of these or closely 7.5.1. Bunch Rot related PR proteins, which also contributes to their strong B. cinerea resistance compared with The term bunch rot refers to the disintegra- the susceptible V. vinifera cultivars (Salzman tion of ripening grape clusters due to infection et al., 1998). Although the latter produce much by a range of different pathogens. The most lower amounts, their PR-3 and PR-5 proteins important of these is the fungus B. cinerea, also can make up half of the total soluble protein known by the name of its sexual form Botryoti- in mature grape berries and are major contribu- nia fuckeliana. In fact, B. cinerea is one of the tors to protein instability in white (but usually most ubiquitous plant pathogens in the world, not red) wines (see Chapter 6.2). with a wide range of host species. Its dry dis- Because of the immense economic impor- persal spores called conidia can survive tem- tance of grapevine diseases, stringent phyto- peratures as low as 80 C for several months, sanitary measures are generally imposed to can be blown by the wind over enormous dis- control disease outbreaks. Cultural practices tances, and can germinate (making them infec- aimed at reducing disease inoculum and spread tive) over a temperature range of 1–30 C, as of infections include canopy management (open long as the relative humidity exceeds 90% canopies facilitate rapid drying after rain, mini- (Pearson and Goheen, 1998; Pezet et al., 2004). mize shade, and maximize spray penetration Most vineyards, even in isolated areas, are and coverage) as well as nutrient and irrigation under permanent pressure from wind-dis- management (judicious fertilizer and water persed conidia, so B. cinerea can be regarded application avoids excess vigor). In addition, as part of a vineyard’s environmental micro- successful disease management often requires flora. Nevertheless, its requirement for high 7.5. LIVING WITH OTHER ORGANISMS: DEFENSE AND DAMAGE 299 humidity provides some protection to many development of noble rot during ripening, but vineyards, as long as they are not compromised rain can quickly result in the detrimental bunch by overly dense canopies (Valde´s-Go´mez et al., rot (Mullins et al., 1992). Winemakers inadver- 2008; see also Chapter 5.2). tently discovered the benefits of noble rot for The B. cinerea fungus is responsible for one wine quality when a delay in the harvest per- of the worst fungal diseases of grapes, namely mission in the German Rheingau in 1775 and gray mold, which can cause serious crop losses the invasion of France by the German–Russian and reductions in fruit quality. The fungus is armies in 1815 both led to late harvests able to use the otherwise stable tartrate as a car- (Dittrich, 1989; Pezet et al., 2004). bon source (in addition to grape sugars), con- The fungus opportunistically invades all verting some of the acid’s degradation green organs of the vine through wounds or products into small amounts of malate and via senescent or dead tissues and also invades other organic acids. Enzymes such as the poly- young organs directly through the cuticle and phenol oxidase termed laccase secreted by the outer cell wall, both of which it degrades in fungus can readily oxidize phenolic com- the process, causing necrosis (Elad and Even- pounds in grapes and continue to do so in the sen, 1995; Salinas et al., 1986). Whereas pre- fermenting juice and wine made from these bloom flowers are relatively well protected by grapes (Dittrich, 1989; Dubernet, 1977; Macheix the calyptra, grape flowers immediately after et al., 1991; Pezet et al., 2004; Ribe´reau-Gayon, anthesis are particularly vulnerable to infec- 1982). When phenolics are oxidized, they turn tion, especially when senescent anthers and into quinonens, which in turn can form brown calyptras remain stuck in the inflorescence polymers; this leads to discoloration of red (Bulit and Dubos, 1982; Pearson and Goheen, wines and browning of white wines. Moreover, 1998). The fungus can penetrate flowers at the the intruder also reduces amino acid concentra- receptacle end, possibly through the scar left tions and degrades aroma compounds (e.g., behind after capfall, and may remain as latent terpenoids), which shifts a wine’s aroma from mycelium inside the developing berries with- fruity to “phenol” or “iodine.” Ironically, how- out causing disease symptoms until the berries ever, some of the world’s most highly prized begin to ripen (Keller et al., 2003b; Pezet et al., dessert wines, particularly the French Sau- 2003, 2004b; Viret et al., 2004). Many other fungi ternes, German Trockenbeerenauslesen, and also seem to be able to establish latent infec- Hungarian Tokajis, are produced from grapes tions in grape berries, some of which (espe- infected with B. cinerea. This so-called “noble cially Alternaria, Cladosporium, and Penicillium rot” appears to be due to B. cinerea growing species) may also cause postharvest rot in table mainly in the epidermis of grape berries, which grapes (Dugan et al., 2002). Damage to the leads to desiccation due to increased water per- berries by cracking of the cuticle from pressure meability of the skin and to concentration of within the berry and physical damage from sugars (especially fructose due to the “taste” insects, hail, and wind predispose clusters to of the fungus for glucose) and, to a lesser berry infection (Kretschmer et al., 2007) and, extent, acids in the berry (Dittrich, 1989; Pezet together with wet conditions, lead to disease et al., 2004). One additional beneficial change expression and fruit loss (Figure 7.15). Cracking induced by the fungus is the accumulation of of berries in susceptible cultivars can ensue glycerol in the berries, which contributes to during rainfall or during humid nights, partic- the sweetness of the resulting wine (Ribe´reau- ularly in combination with high soil moisture Gayon et al., 2006). Morning fog followed by (Keller et al., 2006). Once a disease outbreak warm, dry days is thought to favor the has occurred, conducive conditions (especially 300 7. ENVIRONMENTAL CONSTRAINTS AND STRESS PHYSIOLOGY

FIGURE 7.15 Botrytis cinerea infection in ripening grape cluster (left), Erysiphe necator blotches on abaxial side of leaf (center), and Plasmopara viticola “oilspots” on adaxial side of leaf (right). Photos by M. Keller. rainfall during the ripening phase) will quickly the necrotroph, which secretes a whole arsenal lead to secondary infections via sporulating of wall-degrading proteins to digest the pectins conidia. Such secondary infections spread and other polysaccharides in its passing host’s much more readily throughout tight clusters cell walls (Cantu et al., 2008). Such cell wall whose berries touch each other, retaining sur- degradation appears to be associated with the þ face water and rubbing off epicuticular wax, release of bound Ca2 , which, due to its toxic- than through loose clusters whose berries are ity, may help the invader to kill the host cells separated by gaps and dry rapidly after rain (Kaile et al., 1991). Although the constitutively (Marois et al., 1986; Rosenquist and Morrison, produced glycolate and phenolic compounds, 1989; Vail and Marois 1991; Vail et al., 1999). especially tannins and their subunits but also The importance of surface water for the spread hydroxycinnamic acids and flavonols, provide of the fungus is a major reason why removal of a protective barrier by inhibiting the macerat- leaves around the fruit clusters is a critical can- ing fungal enzymes as well as laccase (Goetz opy management practice in regions that are et al., 1999; Tabacchi, 1994), the increasing prone to rainfall during the ripening period. degree of polymerization of skin tannins The increased air flow and, once the sun shines toward fruit maturity (see Chapter 6.2) may again, the higher berry surface temperature decrease their ability to bind and denaturate favor speedy drying of the clusters after a rain- the fungal proteins (Jersch et al., 1989). The fall event. continued accumulation in ripening grape In contrast with open flowers, young, unripe berries of oxalate may also contribute to the grape berries are highly resistant to B. cinerea, decline of resistance because oxalate is sus- whereas ripening fruit again drop their pected to facilitate degradation of cell walls defenses against fungal attack and become and repress plant defenses; in fact, B. cinerea increasingly susceptible to infection (Blaich secretes oxalate during infection (Williamson et al., 1984; Bulit and Dubos, 1982). This relaxa- et al., 2007; van Kan, 2006). Plants apparently tion of defenses is partly related to the gradual retaliate by producing oxalate oxidase to partly modification of the grape cuticle and cell walls disarm this fungal weapon (Walz et al., 2008). and the disappearance or modification of sev- In addition to these constitutive barriers, green eral preformed (i.e., constitutive) defensive berries produce stilbene phytoalexins after substances. Once the berries have begun to dis- infection, which may slow the spread of the mantle their cell walls, they are no match for fungus, although resveratrol is a rather 7.5. LIVING WITH OTHER ORGANISMS: DEFENSE AND DAMAGE 301 ineffective botryticide and is easily degraded when a grape berry rots away, its seeds gener- by fungal laccase (stilbene oxidase) enzymes ally remain free of infection and simply drop (Hoos and Blaich, 1990; Pezet et al., 1991). The to the ground, where they may germinate. It berries’ ability to produce stilbenes also seems seems at least plausible that this might consti- to decline over the course of berry development tute something like an insurance policy in case and ripening (Bais et al., 2000; Jeandet et al., there are no birds or mammals present to eat 1991). the fruit. If this explanation is correct, B. cinerea Accumulation of stilbenes in response to may be regarded as a mutualistic symbiont in infection by B. cinerea might occur at the cost grape berries rather than as a pathogen, or at of reduced production of flavonoids such as least as a fungus able to switch its lifestyle from tannins, anthocyanins, and flavonols. After all, pathogenic (in flowers and young leaves) to healthy, nonstressed leaves and berries do not mutualistic (in developing berries). produce stilbenes, which is why these com- In contrast to grape berries, expanding pounds are also termed stress metabolites. leaves gradually become more resistant to fun- Conversely, it is possible that the accumulation gal infection (Langcake, 1981; Langcake and of anthocyanins in red grape berries at veraison McCarthy, 1979). This may partly be caused decreases the berries’ capacity to produce stil- by the accumulation of the two major phenolic benes. Like anthocyanins, resveratrol is accu- biopolymers—lignin and tannin. Lignin is mulated predominantly in the berry skin, deposited in mature cell walls, resulting in although it also seems to be present in the physical strengthening similar to the role of seeds. Induction of stilbene synthase can inhibit steel rebars in ferroconcrete. Tannins are also the activity of chalcone synthase, and it has produced at variable concentrations and inhibit been argued that the two enzymes compete the cell wall-degrading enzymes secreted by for substrates (Fischer et al., 1997; Gleitz et al., B. cinerea (Bachmann and Blaich, 1979; Goetz 1991). However, this does not explain why et al., 1999). In addition, mature leaves also con- white grapes also produce lower amounts of tain high concentrations of an array of flavo- stilbenes (Bais et al., 2000) and become more nols, hydroxycinnamic acids, and phenolic susceptible after veraison, nor the fact that vul- acids (Bachmann, 1978; Egger et al., 1976; Rapp nerability remains high after the cessation of and Ziegler, 1973), many of which inhibit fun- pigment accumulation in red grapes and is a gal laccases (Goetz et al., 1999; Tabacchi, 1994) major cause of damage from storage rot in table and produce PR proteins in response to infec- grapes (Pearson and Goheen, 1998). If one con- tion (Renault et al., 1996). Furthermore, stil- siders this phenomenon from a plant reproduc- benes accumulate much more rapidly in tion standpoint, the picture becomes clearer. leaves, especially older leaves, than in fruit. The pigmentation of grapes provides visual Leaves of the B. cinerea resistant American Vitis cues that attract seed dispersers to ripened species (e.g., V. riparia and V. rupestris) and fruit, which become edible after veraison. interspecific hybrids generally have a greater Simultaneously, the barriers erected against capacity for stilbene synthesis than the suscep- microbial intruders, which are detrimental dur- tible European V. vinifera cultivars (Stein and ing seed development, are dismantled when Blaich, 1985). Different cultivars, and perhaps the seed is viable and ready to be dispersed. different clones of the same cultivar, also vary Therefore, both the increase in anthocyanins in the amount of resveratrol they accumulate and the decrease in stilbene-synthesizing following fungal attack. For instance, Cabernet capacity during ripening may serve the same Sauvignon leaves appear to be able to produce purpose—to ensure seed dispersal. After all, twice the amount of stilbenes that accumulate 302 7. ENVIRONMENTAL CONSTRAINTS AND STRESS PHYSIOLOGY in Pinot noir or Chardonnay. However, it In addition to gray mold, grapes can also seems unlikely that their greater capacity for succumb to sour rot, which is caused by a com- stilbene production is the only reason Cabernet plex of various pathogens that include bacteria, Sauvignon, Merlot, Mourve`dre, or Sauvignon fungi, and yeasts. Most of these are secondary blanc have relatively resistant berries, whereas pathogens that can only penetrate into grape the Pinots and Muscats, Cabernet franc, berries through preformed wounds (e.g., from Grenache, or Se´millon are among the most sus- powdery mildew infection or hail damage). ceptible cultivars. Part of the variation in Fungi of the genera Penicillium, Aspergillus, susceptibility among cultivars and clones can Alternaria, and others participate in the infec- also be attributed to differences in cluster com- tion, in addition to yeasts (which are single- pactness rather than stilbenes; more compact celled fungi) belonging to Hanseniaspora, clusters with berries rubbing against each other Candida, Kloeckera, and others. These yeasts in are more easily infected by B. cinerea than loose turn attract fruit flies (genus Drosophila) that clusters because rubbing may compromise the are vectors of Acetobacter (acetic acid bacteria), integrity of the cuticular wax on the berries. which causes the typical “vinegar” smell of Such variation among cultivars in a variety of grapes infected with sour rot. developmental, morphological, and biochemi- cal traits, each conferring partial resistance, 7.5.2. Powdery Mildew demonstrates the existence of quantitative (or incomplete), rather than qualitative (or com- The fungus causing powdery mildew (also plete), disease resistance (Poland et al., 2009) termed oidium) on grapes, E. necator, is argu- within the species V. vinifera, which results in ably the most widespread and most consis- a wide range of disease severity among culti- tently damaging pathogen of grapevines. This vars under similar environmental conditions. is because, in contrast to B. cinerea or P. viticola, Even the rootstock seems to be able to influ- it requires only 40% relative humidity to germi- ence how much resveratrol-infected scion tis- nate, a threshold that is easily reached on the sues produce (e.g., Teleki 5BB > 1103 Paulsen). lower surface of transpiring leaves, even if the It is possible that rootstocks exert their effect surrounding air is much drier (Keller et al., via differences in nutrient uptake and transport 2003a; Pearson and Goheen, 1998). The rate of because plant nutrition also plays a role in dis- development of the fungus accelerates up to ease resistance. However, although vines with an optimum of approximately 85% humidity high nitrogen status tend to have a higher and 25C, but frequent rainfall, especially incidence of bunch rot (Delas et al., 1982; Keller heavy rain, and temperatures outside the range et al., 2001a), research failed to establish a corre- 10–32 C limit its development, with tempera- lation between the nitrogen content of grape tures greater than 35 C killing it outright berries and their susceptibility to B. cinerea (Carroll and Wilcox, 2003; Gadoury and (Delas, 1972). On the other hand, the production Pearson, 1990; OEPP/EPPO, 2002). Mild rain- of both constitutive phenolics, especially flavo- fall, in contrast, seems to benefit E. necator by nols and hydroxycinnamic acids, and inducible enhancing spore dispersal. The fungus can col- stilbenes declines as soil nitrogen availability onize all green plant surfaces (see Figure 7.15) rises, which may render leaves and berries less but thrives in shade and often develops in the able to resist fungal invasion. Of course, the interior of dense canopies (Nicholas et al., unfavorable canopy microclimate that is often 1998). In addition to an influence of air humid- associated with high vine nitrogen status also ity, this shade effect seems to be related to the favors disease development. absence of UV light, which is effectively filtered 7.5. LIVING WITH OTHER ORGANISMS: DEFENSE AND DAMAGE 303 out by the leaves’ epidermis (Keller et al., sinks, such as fruit, roots, and storage reserves. 2003a). The environmental factors that favor Although the fungus-derived cytokinins may disease development are of particular concern also delay leaf senescence (Walters and for young grapevines growing inside plastic McRoberts, 2006), the vine may shed severely sleeves or “grow tubes,” which are used to infected leaves, which can further depress yield accelerate growth and facilitate weed control. formation, fruit ripening, replenishment of These tubes create a microclimate that is char- storage reserves, and cold acclimation. The acterized by reduced light, especially UV light, close relationship between leaf sugar and fun- and by elevated relative humidity and daytime gal growth suggests that events that lead to temperature. higher sugar concentration in leaves, such as The typical whitish “powdery” symptoms intense fruit removal or girdling, might also on leaf surfaces are due to mycelium and spor- increase the leaves’ vulnerability to infection. ulating bodies of the fungus, whereas infected Infection of flowers very early in the growing shoots become necrotic, and petioles or pedun- season can result in poor fruit set and conse- cles become brittle and break easily later in the quently low yield, whereas infection after fruit growing season. Spores germinate on the sur- set may reduce berry size, perhaps because face of plant organs, invade the cuticle and cell imported sugar is diverted to and consumed by walls, and rapidly establish haustoria inside the the fungus instead of being available for berry epidermis cells (Pearson and Goheen, 1998). growth. Moreover, berries that are heavily Like all biotrophic pathogens, E. necator infected early in development usually shrivel depends on the living host plant for assimilate up or drop off, whereas later infections damage supply; that is, it acts as a sink. Therefore, the the epidermis so that the berries may split fungus does not kill its host’s cells but sup- (crack) upon expansion during ripening. In presses their defense responses in susceptible addition, this opens the door for secondary cultivars. Infected leaves generally have infections by B. cinerea and other bunch rot increased concentrations of sugars (especially opportunists. Also, the characteristic “mush- hexoses, which are the preferred carbohydrates room”’ (from 1-octen-3-one) and “geranium- for uptake by the fungus) due to import of like” (from 1,5-octadien-3-one) odor of powdery sucrose from uninfected plant parts and mildew renders infected grapes unpalatable and subsequent breakdown by invertase in the cell can impart off-odors in wine, even though yeasts walls (Brem et al., 1986; Hall and Williams, degrade most of these compounds during 2000). This is a rare example of the conversion fermentation (Darriet et al., 2002; Stummer of source leaves to sinks and a demonstration et al., 2005). However, even less severe infections of the pathological nature of this conversion, may blemish the berry surface, which can render which appears to be caused by an “injection” table grapes unmarketable. of cytokinin from the pathogen (cytokinins Whereas American Vitis species are rela- induce invertase activity) and also involves tively resistant to the fungus, the European amino acid import (Walters and McRoberts, V. vinifera cultivars (i.e., most wine and table 2006). However, infection—or the sugar accu- grape cultivars) are readily infected because, mulation resulting from it—will decrease unlike their American relatives, they did not photosynthesis and starch storage in and coevolve with the pathogen and produce lower assimilate export from infected leaves (Hall amounts of PR proteins (Eibach, 1994; Fung and Williams, 2000). Consequently, this power- et al., 2008). Nevertheless, there is some varia- ful extra sink (i.e., the fungus) alters assimilate tion among V. vinifera cultivars in the degree partitioning in the vine at the expense of other of susceptibility (Doster and Schnathorst, 1985; 304 7. ENVIRONMENTAL CONSTRAINTS AND STRESS PHYSIOLOGY

Roy and Ramming, 1990). For instance, Char- made from such grapes, which has the undesir- donnay and, to a somewhat lesser degree, able consequence of reducing protein stability Cabernet Sauvignon are among the most sus- (i.e., increasing haze formation) in white (but ceptible cultivars, whereas Pinot noir, Riesling, probably not red) wines (Girbau et al., 2004). or Malbec seem to be much better able to resist or tolerate infection. Although stilbene phytoa- 7.5.3. Downy Mildew lexins are also effective against E. necator (at least in vitro), infection of V. vinifera tissues Although usually regarded as a fungus does not normally trigger their production because it looks like one and produces spores, (Keller et al., 2003a), perhaps because the bio- the causal agent P. viticola is in fact more trophic fungus avoids cell damage so as not to closely related to certain algae and kelps and threaten its own survival (Mendgen and Hahn, diatoms with which they are placed in the 2002). In contrast, the resistant American Vitis kingdom Protista. Also, in contrast to “true” species do accumulate stilbenes in response to fungi, its cell walls contain cellulose instead of infection. In addition, flavonols, which accu- chitin, and its cell nuclei are diploid, not hap- mulate in the epidermis and cuticular wax in loid like those of fungi. It belongs to the class response to UV (especially UVB) radiation of Oomycetes (also called water molds) and is and provide a “sunscreen” for plant tissues, not related to the powdery mildew fungus. may be involved in V. vinifera resistance against Because its spores can germinate at greater E. necator. It is conceivable that enhanced syn- than 95% relative humidity, it thrives under thesis of such constitutive phenolic compounds humid, shady conditions, especially with fre- in sun-exposed leaves and berries contributes quent rainfall and temperatures of approxi- to an unfavorable environment for powdery mately 20–25 C, which is common in coastal mildew colonization. Moreover, flavonol pro- areas and other regions with summer rainfall duction is strongly reduced by high soil nitro- (Galet, 1996; OEPP/EPPO, 2002; Pearson and gen availability, and high plant N status Goheen, 1998). Although Mediterranean cli- makes vines more susceptible to colonization mates are less favorable for the pathogen native by E. necator (Bavaresco and Eibach, 1987; to the southeastern United States, it is wide- Keller et al., 2003a). An additional resistance spread throughout the world, except in regions mechanism may be vitrification of penetrating with very low spring and summer rainfall, mycelium by the localized accumulation of sili- such as central California, eastern Washington, cates in the cell walls (Blaich and Wind, 1989). northern Chile, or Western Australia. Because Grape berries, even those of V. vinifera culti- the little rain that falls in the Mendoza region vars, become increasingly resistant to E. necator in Argentina mostly comes in the summer, over the course of their development, which downy mildew is a major problem even in this contrasts with the berries’ increasing vulnera- otherwise arid region. Along with other North bility to B. cinerea. At least in V. vinifera this is American natives (the fungi E. necator, which likely to be a side effect of the accumulation was introduced earlier, and G. bidwellii), of PR proteins for other reasons (e.g., sugar P. viticola wreaked havoc in the European wine accumulation) because this species has not industry when it spread from infested grape had time to evolve specific resistance mechan- material imported in the 1870s from the United isms against this comparatively novel patho- States for use as rootstocks (see Chapter 1.2 and gen. Moreover, the increase in PR protein above) resistant to the previously introduced content following an infection may also raise insect phylloxera to which the susceptible the amount of protein in the juice and wine V. vinifera varieties were succumbing. During 7.5. LIVING WITH OTHER ORGANISMS: DEFENSE AND DAMAGE 305 the event, the discovery of the so-called necrotic and are abscised, whereas the highly Bordeaux mixture (copper sulfate mixed with susceptible young grape berries become lime suspended in water) saved the industry. covered with a grayish “felt.” Incidentally, however, the widespread appear- Having coevolved with the pathogen, most ance of inferior wines made by desperate of the North American Vitis species are partly producers who lacked sufficient V. vinifera resistant (e.g., V. rupestris, V. berlandieri, and grapes and instead used fruit from hybrids V. aestivalis) or fully resistant (e.g., M. rotundifo- resulting from resistance-breeding programs lia, V. riparia, and V. cinerea) to downy mildew. with V. vinifera and American Vitis species dur- Some Asian species (e.g., V. amurensis) also ing the phylloxera-and-mildew crisis indirectly have partial resistance, whereas V. vinifera is spurred the advent of the rigid legislation that highly susceptible. Nonetheless, even among has since become a hallmark of the European the European grape cultivars there are various wine industry. degrees of susceptibility. For instance, Riesling, Like the fungi discussed previously, the Pinot family, and especially Cabernet Sau- P. viticola can infest all green plant parts but vignon are among the less susceptible cultivars, ordinarily colonizes young leaves or young whereas Tempranillo or Albarin˜o are among berries by penetrating through the stomata the most susceptible (Boso and Kassemeyer, (Gindro et al., 2003; Langcake and Lovell, 2008). Some of the resistant species defend 1980; Pearson and Goheen, 1998). Its mycelium themselves against infection of their leaves by then develops an intercellular network in the rapidly secreting callose that plugs their sto- leaf mesophyll and feeds off these cells by mata and coats the pathogen spores (Gindro means of cell wall-penetrating haustoria to et al., 2003; Langcake and Lovell, 1980). This cause downy mildew (also called peronospora). covering probably kills the germinating spores Initial disease symptoms appear on the adaxial and stops mycelial growth while also reducing side of leaves as yellow or, in some cultivars, water loss from the leaves. In addition, the red oily spots (see Figure 7.15), which spread leaves produce stilbenes (resveratrol, pterostil- and later become angular necrotic patches bene, and viniferins) that kill the cells sur- restricted by the leaf veins. The typical whitish rounding infected stomata (Dai et al., 1995; “downy” mildew symptoms arise from sporu- Langcake, 1981; Langcake et al., 1979; Pezet lation of the pathogen through the stomata on et al., 2004a). High plant N status seems to com- the abaxial leaf surface. Even before any dis- promise the leaves’ ability to produce stilbenes ease symptoms appear, however, the invading and leads to higher vulnerability to infection pathogen prevents the stomata from closing at (Bavaresco and Eibach, 1987). night or in response to water deficit so that Young grape berries, even those of suscepti- the unrestrained transpiration may lead to ble species and cultivars, also rapidly accumu- water loss and wilting of infected leaves late stilbenes around sites of infection (Alle`gre et al., 2007). Although in contrast to (Schmidlin et al., 2008), but the berries appear E. necator, P. viticola does not stimulate sugar to lose this ability around veraison. Nonethe- accumulation in infected leaves, it also leads less, the berries become increasingly resistant to repression of photosynthesis and shedding to infection during development (Kennelly of severely damaged leaves. This can adversely et al., 2005). The appearance of necrotic cells impact yield formation, fruit ripening, replen- near invasion sites is termed hypersensitive ishment of storage reserves, and cold hardi- response and is an example of localized pro- ness. Moreover, infected shoot tips, petioles, grammed cell death that stops the pathogen, tendrils, and inflorescences often become which as a biotroph depends on live host cells, 306 7. ENVIRONMENTAL CONSTRAINTS AND STRESS PHYSIOLOGY from spreading to healthy tissues (Busam et al., plants in general have evolved powerful resis- 1997; Kortekamp, 2006; Langcake and Lovell, tance strategies (Bais et al., 2006). One of the 1980). Again, Vitis species differ in their ability few perpetrators is Rhizobium vitis (aka Agrobac- to accumulate stilbenes in response to P. viticola terium vitis, formerly known as Agrobacterium infection (Dai et al., 1995; Pezet et al., 2004a). For tumefaciens biotype 3), the causal agent of instance, stilbenes appear earlier and reach crown gall, which is mainly confined to and much higher concentrations in leaves of the spreads via xylem vessels—both up and down resistant V. riparia than in the susceptible the trunk and roots (Tarbah and Goodman, V. vinifera, whereas V. rupestris seems to be 1987; Young et al., 2001). It may live for years intermediate in both resistance and stilbene within the vascular system of infected vines production. However, the resistant V. cinerea without any outward expression of disease, and V. champinii are also poor stilbene produ- which typically only develops at sites of physi- cers, which indicates that these phytoalexins cal injury. Although commonly regarded as a are not the only actors on the stage of downy soil-borne pathogen, the bacterium survives mildew resistance. Indeed, in addition to the only in Vitis tissues and is usually introduced constitutive chitinase in their leaves, resistant into vineyards with contaminated planting species also activate glucanase upon challenge material (which can be symptomless) that then by P. viticola (Kortekamp, 2006). Greater gluca- serves as a source of inoculum (Burr and Otten, nase activity, and perhaps peroxidase activity, 1999; Burr et al., 1998). It enters the plant pre- in old than in young leaves also may partly dominantly through wounds caused by cold account for the decrease in vulnerability to the injury but also at sites of other injuries such as pathogen as leaves age (Reuveni, 1998). those caused by machinery or grafting/top- As in the case of E. necator, the American working. Therefore, the disease thrives in cli- P. viticola and the European V. vinifera met only mates that experience frequent freeze injury, recently by evolutionary timescales, so it is prob- and damage caused by galls often surpasses able that the ability to produce stilbenes evolved that of the initial injury. The bacteria, it seems, in V. vinifera as a defense against intruders induce the tumors called crown gall when other than P. viticola. The formation of necrotic mechanical wounds rupture the vessels and spots on the leaves of at least partly resistant release the bacteria to the adjacent parenchyma Vitis species and of oilspots on susceptible spe- cells, where they insert some of their own DNA cies is, of course, a result of the way the different into the genome of the infected plant (Burr and species respond to the attacking pathogen. Such Otten, 1999; Tarbah and Goodman, 1987). variation demonstrates the principle that when a These bacterial DNA pieces are called tumor- pathogen “discovers” and invades a new host inducing plasmids, or Ti plasmids. It is this species, the symptoms it provokes are often unique ability to transfer genetic material to different and usually more severe than those plants that also makes Agrobacterium species that occur with the original species, which has the organism of choice for plant genetic engi- “honed” its skills at fending off the invader neering. The bacterial genes induce the host through the long arms race of coevolution. cells to degrade tartrate (Burr and Otten, 1999), to divert auxin away from its normal 7.5.4. Bacteria basipetal movement, and to locally enhance both auxin and cytokinin production to stimu- Although the majority of all bacteria live in late cell proliferation that leads to the typical the soil, extremely few have succeeded at trunk tumors associated with crown gall becoming plant pathogens, suggesting that (Sakakibara, 2006). 7.5. LIVING WITH OTHER ORGANISMS: DEFENSE AND DAMAGE 307

Although no Vitis species have thus far been approximately 1 m above the soil surface, even found to be immune to crown gall, the species on canes. Training up suckers to form new vary in their vulnerability to R. vitis. For trunks is a technique that can be used to instance, V. vinifera cultivars are highly suscep- replace injured trunks, provided the suckers tible, whereas V. labrusca cultivars are some- arise from the asymptomatic base of the trunk what resistant, the rootstocks of V. riparia and and not from a rootstock. On vines grafted to V. rupestris parentage are relatively resistant, resistant rootstocks, galls usually form at or and V. amurensis is quite resistant (Burr and above the graft union (Burr and Otten, 1999). Otten, 1999; Burr et al., 1998; Stover et al., Although infected roots do not usually form 1997). On susceptible species, galls normally galls, they may develop necrotic lesions. develop close to the base of the trunk, from In addition to inducing gall formation, auxin slightly above to slightly below the soil surface and cytokinin also trigger the differentiation (Figure 7.16), but they can also form up to of new vascular bundles, which are then

FIGURE 7.16 Grapevine with typical symptoms of crown gall (left) and tumor formation above the soil surface (right). Photos by M. Keller. 308 7. ENVIRONMENTAL CONSTRAINTS AND STRESS PHYSIOLOGY connected to the vascular system of the vine to been deposited there by xylem-sucking insects, provide water and nutrients to the growing such as sharpshooters (Chatelet et al., 2006). tumor at the expense of the shoots (Aloni, The pathogen may facilitate its own migration 2001; Efetova et al., 2007; Veselov et al., 2003). through the xylem by releasing cell wall The lack of an epidermis around the tumor degrading enzymes (e.g., polygalacturonase can lead to water loss through its large surface and cellulase) that digest some of the pit mem- area. In response to this threat, the galls appear branes that get in its way (Roper et al., 2007). to emit ethylene that then triggers the produc- Bacteria populations build up over time, and tion of abscisic acid in the host plant and its severe infection can sometimes lead to loca- translocation in the xylem and/or phloem to lized plugging of vessels, although the majority the tumor, where it induces production of of the plugging seems to result from the pro- osmoprotectants (e.g., proline; see Chapter 7.1) duction of tyloses and gums by the infected and of suberin for incorporation in the exposed vine. Such xylem occlusion could be a response cell walls as a sealing agent (Efetova et al., 2007; to the release of ethylene by the injured vine Veselov et al., 2003). (Sun et al., 2007). The disruption of water flow, As developing galls expand, they increas- especially in the petioles where the bacteria can ingly restrict movement of water and nutrients induce cavitation, may lead to leaf dehydration with drastic consequences for plant vigor, lon- (McElrone et al., 2008). Water stress may or may gevity, and yield (Schroth et al., 1988). Because not be associated with the symptoms character- of the girdling effect of the tumors that inhibits istic of Pierce’s disease: marginal leaf necrosis phloem transport to the roots, the leaves may that resembles salinity injury, abscission of turn bright red due to the conversion of accu- leaves but not the petiole (leaving so-called mulating sugars into anthocyanins. In severe “matchsticks” on the shoot), and green islands cases, the portion of the vine above the gall on otherwise maturing (i.e., browning) shoots may die, which is a particular problem during (Stevenson et al., 2005; Thorne et al., 2006). The vineyard establishment (see Figure 7.16). In berries of severely infected plants sometimes addition to the striking disease symptoms asso- shrivel up and dry, and vines, too, ultimately ciated with gall formation, R. vitis also causes die from infection. Species that have coevolved root lesions (Rodriguez-Palenzuela et al., with the bacterium, such as M. rotundifolia and 1991), thereby decreasing plant vigor even in V. arizonica, are resistant to the bacterium, but the absence of galls. Upon removal of a vine- the resistance varies according to the home yard, the bacteria can persist in root debris for range of local populations of wild vines several years, which may provide a source of (Fritschi et al., 2007; Ruel and Walker, 2006). inoculum for newly planted vines. In contrast, the bacteria seem to be unable to move to the 7.5.5. Viruses tip of growing shoots. This can be exploited to eliminate R. vitis from propagation material In addition to bacteria, grapevines are also through the process of shoot-tip culturing (Burr vulnerable to infection by a variety of graft- and Otten, 1999; Burr et al., 1998). transmissible viral diseases. Grapevine viruses The bacterium X. fastidiosa, the causal agent only became widely spread in the 20th century of Pierce’s disease, contrasts with R. vitis in that after, in response to the spread of phylloxera, it causes damage mainly in areas with mild European growers were forced to change their winters (Mullins et al., 1992). It lives exclusively propagation methods from simple rooting of inside xylem vessels and uses them to spread cuttings to grafting onto phylloxera-tolerant throughout its many host plants after it has rootstocks. Cultivated grapes throughout the 7.5. LIVING WITH OTHER ORGANISMS: DEFENSE AND DAMAGE 309 world now appear to be infected with more viruses is often not clear-cut. Intriguingly, viruses than any other woody species. These many viruses appear to induce the production viruses are typically introduced into vineyards by their hosts of heat shock proteins (which by infected planting material, although some seem to facilitate virus infection and replica- can also be spread by transport agents called tion) in addition to stilbenes and PR proteins vectors, such as phloem-feeding insects (e.g., among other disease-resistance mechanisms leafroll viruses by aphids or mealybugs, often (Whitham et al., 2006), which might make aided by wind, people, or machinery) or root- virus-infected vines less susceptible to attack feeding nematodes (e.g., fanleaf nepovirus by by fungal pathogens. the dagger nematode Xiphinema index) (Bovey Lower temperatures seem to strongly et al., 1980; OEPP/EPPO, 2002). The latter, of increase a plant’s susceptibility to virus infec- course, also cause direct damage through feed- tions and also lead to more pronounced disease ing on the roots in addition to delivering their symptoms, possibly because cool conditions virus load. Moreover, although some grapevine may favor the multiplication of viruses and genotypes, such as certain rootstocks, can toler- slow the degradation of their RNA (Samach ate very high nematode numbers, these nema- and Wigge, 2005; Wang et al., 2008). Many plant todes can still feed on the roots and transmit viruses induce localized or general yellowing viruses (Mullins et al., 1992). of leaves. The virus particles normally spread Viruses can be classed as biotrophic patho- symplastically in infected tissues and organs, gens because they depend on the metabolism using special movement proteins that enable of their hosts for multiplication (replication), them to travel via plasmodesmata from cell to which leads to systemic infections. All known cell and via the phloem to other organs (Lough viruses infecting Vitis species have genomes and Lucas, 2006; Oparka and Santa Cruz, 2000). that consist of RNA (rather than DNA, as in The leafroll virus (actually a genetically diverse their hosts) and recruit host ribosomes to man- bunch of so-called closteroviruses causing sim- ufacture their proteins. Note that this property ilar symptoms; see Karasev, 2000; Martelli et al., enables such viruses to mutate readily because 2002) is arguably the most destructive of all it dodges the host cells’ ordinary “proofread- grape viruses because it renders the phloem ing” machinery designed to detect and repair nonfunctional and curtails root growth and errors on DNA. This leads to rapid evolution shoot vigor. Phloem blockage occurs not by of novel virus genotypes, although one or a the virus itself (although it is mostly confined few stable genotypes typically dominate infec- to the phloem and adjacent vascular tissues) tions in plants (Garcı´a-Arenal et al., 2001; but due to callose deposition, which may be Karasev, 2000). To further their own needs, an attempt by the vine to limit the spread of many viruses also suppress the expression of viruses. Unfortunately, this defense response some of their host’s genes (a phenomenon called also restricts sugar export from the leaves and host gene shutoff) while triggering the expres- leads to sugar accumulation inside the leaves, sion of other genes (Havelda et al., 2008), thereby which in turn induces feedback inhibition of manipulating the host’s metabolism and, ulti- photosynthesis (Espinoza et al., 2007) and, mately, leading to the development of disease often, the characteristic downward rolling symptoms. Once a virus has colonized a grape- and interveinal reddening (in red cultivars; vine, it remains there for good; a cure is not pos- Figure 7.17) or yellowing (in white cultivars). sible. Moreover, infections with a potpourri of In contrast with V. vinifera, the American Vitis different viruses are common, and the associa- species and their hybrids (including Concord) tion of specific symptoms with particular typically do not develop visual symptoms. 310 7. ENVIRONMENTAL CONSTRAINTS AND STRESS PHYSIOLOGY

behind in maturity, with poor sugar and color and high acidity. Because of restricted growth, replenishment of storage reserves in the peren- nial parts of the vine also may be reduced (Ru¨ hl and Clingeleffer, 1993), which weakens the plant and leads to a gradual decline and, finally, death of the plant, especially if multiple infections with different viruses occur. In addi- tion, the leafroll virus also induces the plant to produce volatiles that attract the insect vectors of the disease. This facilitates spreading of the virus from infected to healthy vines and neigh- boring vineyards. In addition, the so-called “graft incompatibility” of certain scion–root- stock combinations is often associated with one or both of the grafting partners being infected with the leafroll virus. However, the viruses infest own-rooted vines just as readily, and symptoms are identical to those develop- ing on grafted vines. FIGURE 7.17 Typical symptoms of leafroll virus infec- Despite this list of adverse consequences of tion in Cabernet Sauvignon: Bright red areas appear on virus infection, it is possible that viruses may the lamina of a mature leaf, whereas the veins remain green. Photo by M. Keller. not be all bad for grapevines. There is evidence that virus infection may improve drought toler- ance and cold hardiness of plants, although this In addition to causing reduced growth and has yet to be tested in grapes (Xu et al., 2008). plant longevity, some viruses, especially the This outcome seems to be a consequence of leafroll-associated viruses, result in significant the accumulation of sugars in the leaves, which losses in both yield and quality of the fruit leads to feedback inhibition of photosynthesis (Bovey et al., 1980; Cabaleiro et al., 1999; resulting in partial closure of the stomata, Goheen and Cook, 1959; Hewitt et al., 1962; which in turn decreases transpiration. The Lider et al., 1975; Woodham et al., 1983, 1984). lower vigor and, hence, leaf area of infected This is because diminished sugar transport to plants further reduces transpiration. Other the fruit clusters interferes with yield forma- metabolites (e.g., proline and polyamines) tion, grape berry development, and ripening. accumulating in the leaves may confer some Because infected vines become highly sensitive protection against oxidative stress that typically to adverse environmental conditions that limit results from environmental stress (see Chapter photosynthesis, clusters often appear very 7.1). Thus, viruses may further their own sur- loose due to poor fruit set, berry size and ripen- vival by “helping” their hosts to survive ing are uneven, and grapes commonly lag adverse environmental conditions. References

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Note: Page numbers followed by “f” indicate figures and “t” indicate tables.

A Anatomy. See Morphology/anatomy burst of, 51f ABA. See Abscisic acid Androecium, 43–44 cell division/auxin production Abscisic acid (ABA), 90–91 Anemic cultivars,13 in, 54–55 reaction of stomata to, 91 Angiospermae (Magnoliophyta), 3 dormant, 34–35 Acids. See also Organic acids Angiosperms, fertilization in, 74 fruitfulness, 172, 173 grape berries composition/quality Anthesis, 71–73, 71f growth stage of, 51f,52f with, 186–190, 187f Anthocyanins, 194t, 201, 202–205 inflorescences per, 68–69 Allocation, photosynthate, 134–140 Apianic cultivars,13 latent, 34–35 assimilate surplus in, 139–140, 140f Apoplast, 100–101 longitudinal section of, 36f assimilation rate determined by Aquaporins, 85–86 morphology/anatomy of, 33–36, source in, 139 Assimilation, 111–117 34f,36f patterns in, 135–138 ATP to ADP, 113–114, 113f prompt, 34–36 swell, 52f phosphates availability in B regulation of, 134–135 temperatures inducing budbreak f source-sink relation with, 135–138, Bacteria, 306–308, 307 in, 55–56 135f Bark, 33 three types of, 34–35 storage in, 134 Basal buds, 35–36 winter, 52f See three categories for, 134 Berries. Grape berries Bunch rot, 298–302, 300f Biologically effective degree transport in, 134 C utilization in, 134 days, 61–62 Alphonse Lavalle´e., 12–13 Biotroph, 296 Cabernet franc, 11–12 f f American group, 6–8, 6t,7f Bloom, 45–47, 51 ,52 Cabernet Sauvignon, 11–12 Vitis aestivalis,6 yield potential with, 174 Calcium, 258–259 f Vitis berlandieri,7 Bonarda, 12–13 Calvin cycle, 112–114, 113 f f Vitis candicans,7 Boron, 267–269, 268 ATP to ADP in, 113–114, 113 Vitis labrusca,6 Botanical classification, 1–9 carbon into starch production Vitis riparia,7,7f class Dicotyledoneae, 3 with, 115 Vitis rupestris,7 division Angiospermae, 3 first stage: carboxylation, 112–113 f Amino acids, 158 domain Eukarya, 2–3 NADPH to NADP in, 113–114, 113 mesophyll cell produced, 131 family Vitaceae, 4 rubisco in, 112–113, 117 root-delivered, 131 genus Muscadinia, 4–5 second stage: reduction, 113–114 f f temperature influencing genus Vitis, 2, 4–5, 5 sucrose in, 113 concentration of, 217–218 kingdom Plantae, 3 Calyptra, 44–45 xylem/phloem sap with, 127t order Rhamnales, 3–4 Canopy-environment Ammonium assimilation, 160–162 order Vitales, 3–4 interactions, 140–158 glutamate dehydrogenase in, 161f, Bracts, 34–35 assimilation rate determines export 162 Buds rate from, 139 structure/reactions of compounds basal, 35–36 climatic conditions for resource involved in, 161f bracts with, 34–35 availability in, 141, 142 Ampelography, 9 budbreak/vegetative cycle of, 55–58 macroclimate, 141

369 370 INDEX

Canopy-environment interactions UV range with, 149–150, 149f Chloroplast, 116–117 (Continued) south-facing slope in, 142 starch production in, 114–115 mesoclimate, 141 temperature in, 150–154 sugar production in, 114, 114f microclimate, 141 clouds alter, 152 Citric acid cycle (Krebs cycle), 117, f grapevines productivity depends CO2 assimilation influenced 119 on, 140–141 by, 151, 153 Climatic conditions humidity in, 155–156 global climate change with humidity in, 155–156 leaf growth with, 155 increase in, 152–153 light in, 143–150 transpiration with, 155 leaf growth with, 153–154 resource availability with, 141, 142 vine water status with, 156 leaf temperature changes, 150, temperature in, 150–154 ideal, 156–158 151f three levels of, 141 canopy height/row width ratio photosynthesis stimulated macroclimate, 141 in, 156 by, 151–152 mesoclimate, 141 canopy surface area in, 156 wind in, 154–155 microclimate, 141 15 shoots per meter of canopy damage from, 154, 154f wind in, 154–155 in, 156 down row v. across row Clonal variation, 15–16 fruit exposure in, 157 turbulence, 155 Clones, 9–19 fruit zone in, 157 leaf’s boundary layer resistance Clusters gaps in, 157 decreased with, 154–155 initiation/differentiation of, 70f lateral shoot growth limited Carbohydrates, construction costs morphology/anatomy of, 41–43 in, 157 of, 121–122, 121t Collection phloem, 130–131 leaf layers in, 156 Carbon metabolism, 158–167 Convection, 92 north/south oriented rows Carbon uptake, 111–117 Copper, 265–266 in, 156 Calvin cycle with, 112–114, 113f Cordon trained grapevines, 27 productivity upper limit ATP to ADP in, 113–114, 113f Cotyledons, 36 with, 156–157 carbon into starch production Cultivars, 9–19 pruning weight in, 157 with, 115 Alphonse Lavalle´e., 12–13 renewal zone in, 157 first stage: carboxylation, 112–113 Anemic cultivars,13 shoots 15 nodes long in, 157 NADPH to NADP in, 113–114, Apianic cultivars,13 vertically trained foliage in, 156 113f Bonarda, 12–13 yield to pruning weight ratio second stage: reduction, 113–114 Cardinal, 12–13 in, 157 sucrose in, 113f Charbono, 12–13 light in, 143–150 chloroplast with, 116–117 chilling of, 55 grapevine adjustment to, 145–146 starch production in, 114–115 classification, 13–15 importance for plant sugar production in, 114, 114f berry color/yield, 13–14 development of, 143 glucose molecules in, 116 ecogeographical groups, 14 f leaf evolution for, 143 H2O loss with, 112 perceived wine quality, 14 leaf hairs in scatter of sucrose production with, 114, 114f time of maturity, 14, 15t UV, 149–150 uptake of CO2 by leaves what grapes used for, 14 leaf layers alter quantity/quality with, 111–112 winemaking of, 147–148, 147f Carboxylation, 112–113 characteristics, 14–15 leaf’s net CO2 assimilation with Cardinal., 12–13 Corbeau, 12–13 zero, 143–144 Casparian bands, 100–101, 100f hybridization of., 11–12 limited, 144 Cell expansion, 85–89 Ko¨nigin der Weinga¨rten, 12–13 photosynthetically active turgid cell wall loosening with, 88 Mu¨ ller-Thurgau, 12–13 radiation, 143, 144f Cell membranes, 101 Nomentanic cultivars,13 polychrome system for plant Charbono, 12–13 number of., 10 monitoring of, 148–149 Chlorophyll, 108, 109–110, 109f pollination of., 73 range for vine vigor of, 144–145 light energy absorbed/translated Proles occidentalis,14 red/far red leaf absorption with, 108 Proles orientalis,14 of, 147–148, 147f photochemical quenching with, 110 Proles pontica,14 red/far red variations for photosystem II grouping of, 109–110 root growth influenced by, 66–67 day’s, 148 structure of, 109f variety v., 9–13 INDEX 371

Vinum francicum,14 fruit yield with active leaf area American group, 6–8, 6t,7f Vinum hunicum,14 for, 174–175 Vitis aestivalis,6 Cytokinins, 58 inflorescence necrosis with, 175, Vitis berlandieri,7 production of pollen stimulated 175f Vitis candicans,7 by, 72–73 size of grape berries with, 176 Vitis labrusca,6 Cytosol, sucrose production in, 114, temperature in fruit development Vitis riparia,7,7f 114f delay with, 176–177 Vitis rupestris,7 Dicotyledoneae (Magnoliopsida), Eurasian group, 6t, 8–9 D 3 Vitis amurensis,9 Day length, 49–53 Dormant buds, 34–35 Vitis coignetiae,9 DELLA proteins, 58 Downy mildew, 304–306 Vitis sylvestris,8 Developmental physiology, 169–226 Vitis vinifera,8 grape berry composition/ E Global climate change quality, 178–225 Electromagnetic spectrum, 107–108, photorespiration with, 117–118 f f acids in, 186–190, 187 108 temperature CO2 increase crop load in, 223–225 Electron transport chain, 117–118 with, 152–153 light influencing, 212–214 Endocarp, 47 Glutamate dehydrogenase lipids in, 205–209 Endosperm nucleus, 76 (GDH), 161f, 162 maturity of, 211 Energy capture, 107–111. ability to oxidize glutamate mineral nutrients in, 190–193 See also Photosynthesis of, 165–166 nitrogenous compounds in, Eukarya domain, 2–3 Glycolysis, 119–120, 119f 190–193 Eurasian group, 6t, 8–9 Grafting, 17 nutrient status in, 220–223 Vitis amurensis,9 Grape berries phenolics in, 193–205, 194t, Vitis coignetiae,9 bloom of, 45–47 196f, 201f Vitis sylvestris,8 composition/quality of, 178–225 sources of composition variation Vitis vinifera,8 acids in, 186–190, 187f in, 209–210 Exocarp, 45–47 crop load in, 223–225 sugars in, 183–186, 185f F light influencing, 212–214 temperature influencing, 214–218, lipids in, 205–209 216f Fertilization, 73, 74 maturity of, 211 volatiles in, 205–209 in angiosperms, 74 mineral nutrients in, 190–193 water in, 181–183 cell division following, 78–79 nitrogenous compounds f water status in, 218–220 flower, 135 in, 190–193 t yield formation in, 169–178 Flavonols, 194 , 203, 204–205 nutrient status in, 220–223 components of grapevine Flowers phenolics in, 193–205, 194t, yield, 169–170 androecium of, 43–44 196f, 201f fruit production depresses vine calyptra of, 44–45 sources of composition variation f capacity for, 171 development, 135 in, 209–210 f genotype/environment fertilization, 135 sugars in, 183–186, 185f f f interactions for, 170 growth stage of, 51 ,52 temperature influencing, 214–218, pruning depresses vine capacity gynoecium of, 43–44 216f for, 171 morphology/anatomy of, 43–47 volatiles in, 205–209 f shoot vigor for, 171 ovule of, 44 ,45 water in, 181–183, 218–220 f vine capacity for, 171 pistil of, 43–45, 44 diagrammatic representation of vine growth maintenance proportion developing into berries size/color of, 179f for, 170–171 for, 77 endocarp of, 47 f vine self-regulation for, 171 stamens of, 43–44, 44 exocarp of, 45–47 vineyard yield, 170 vascular bundles of, 44–45 growth stages of, 51f,52f, 79–82, Vitis vinifera yield potential in, 172–178 , 43–44 80f, 135f bloom occurrence with, 174 G stage I: rapid increase, 79 bud fruitfulness for, 172, 173 stage II: lag phase, 80 See cell enlargement with, 176–177 GDH. Glutamate dehydrogenase stage III: ripening period, 80 defined, 172 Geographical distribution, 1–9 idioblast of, 45–47 372 INDEX

Grape berries (Continued) transpiration in leaf of, 90, 104–105 carbon investment initially required mesocarp of, 47 trellis system for support of, 27 for, 126 morphology/anatomy of, 43–47 trunk of, 27–33 CO2 assimilation in darkness pericarp of, 45, 46f,47 water status of, 156 of, 143–144 f Proportion of flowers developing Growing degree days, calculation CO2 uptake with H2O loss in, 112 into, 77 of, 61 cotyledons, 36 seedless, 82 Growth cycle, 49–84, 51f,52f cross section of, 37f size of, 176 day length and, 49–53 emergence of, 52f skin of, 45–47 reproductive cycle of, 68–83 formation of, 36 solute unloading in, 132–133 seasons, 49–53 fruit yield with active leaf area structure of, 46f vegetative cycle of, 53–68 of, 174–175 vascular bundles of, 45 Gynoecium, 43–44 growth stage of, 51f,52f Grape-growing regions, 142 humidity with, 155 Grapes H hairs in scatter of UV light, brandy, 14 Heat dissipation, 91–92 149–150 history of, 9–10 convection for, 92 ideal canopy with layers of, 156 hybridization of., 11–12 radiation for, 92 lamina, 36–37, 38–39 juice, 14 transpiration for, 92 layers alter quantity/quality of mutations in genome of, 10–11 Heliothermal index, 61–62 light, 147–148, 147f raisin, 14 Hydroxybenzoic acids, 194t mesophyll of, 40, 40f table, 14 I morphology/anatomy of, 36–41, uses for, 14 37f wine, 14 Idioblast, 45–47 palisade parenchyma of, 40, 40f f Grapevines Inflorescence necrosis, 175, 175 petiole of, 36 adjustment to light by, 145–146 Inflorescences photosynthetic machinery of, 40 bleeding, 53–54, 53f beginning of bloom with, 71–72, plastochron of, 36 f components of yield in, 169–170 72 reddening, 59–60, 60f cordon trained, 27 buds with, 69 red/far red light absorption growth cycle for, 49–84, 51f,52f flower differentiation with, 69–70, by, 147–148, 147f f day length and, 49–53 70 senescence, 59–61, 60f seasons, 49–53 number of flower per, 71 serrated margins of, 37–38, 38f heat dissipation of, 91–92 Ion channels, 101–102 stomata of, 39–40 f convection for, 92 Iron, 262–264, 263 temperature changes in, 150, 151f radiation for, 92 J temperature response to growth transpiration for, 92 of, 153–154 light capture adapted wild, Jumping genes, 10–11 transpiration in, 90 146 K unfolding of, 52f morphology/anatomy of, 20–47 uptake of CO2 by, 111–112 Ko¨nigin der Weinga¨rten., 12–13 buds, 33–36, 34f Light, 143–150 Krebs cycle. See Citric acid cycle clusters, 41–43 grape berries composition/quality flowers, 43–47 L influenced by, 212–214 grape berries, 43–47 grapevine adjustment to, 145–146 Lamina, 36–37, 38–39 leaves, 36–41, 37f importance for plant development cell division in growth of, 38–39 nodes, 33–36, 34f of, 143 function of, 36–37 tendrils, 41–43 leaf evolution for, 143 Latent buds, 34–35 nitrates stored/distributed in, leaf hairs in scatter of UV, 149–150 Latitude-temperature index, 61–62 160 leaf layers alter quantity/quality Leaves photoinhibition in, 146–147 of, 147–148, 147f anatomical adaptation to light range productivity of, 140–141 leaf’s net CO assimilation with by, 143 2 pruning, 27 zero, 143–144 assimilate surplus with premature root of, 21–27, 23f,26f limited, 144 senescence of, 139–140, 140f shade tolerant quality of, 146 photosynthetically active boundary layer resistance decreased shoots of, 27–33, 28f,29f,31f radiation, 143, 144f with wind, 154–155 INDEX 373

polychrome system for plant growth stage of, 51f,52f epidermis of, 30–31 monitoring of, 148–149 gynoecium of, 43–44 growth stage of, 51f, 55–58, 57f, range for vine vigor of, 144–145 ovule of, 44f,45 59–60, 61, 62–63, 68 red/far red leaf absorption of, pistil of, 43–45, 44f leaf-opposed tendril production 147–148, 147f stamens of, 43–44, 44f with, 30 red/far red variations for day’s, 148 vascular bundles of, 44–45 longitudinal section of, 28f UV range with, 149–150, 149f grape berries, 43–47 parenchyma cells of, 32–33 Light absorption, 107–111. bloom of, 45–47 pearls of, 30–31, 31f See also Photosynthesis endocarp of, 47 phyllotaxy of, 27–28 chlorophyll for, 108, 109–110, 109f exocarp of, 45–47 procambium of, 31–32 electromagnetic spectrum growth stage of, 51f,52f three-node pattern of Syrah, with, 107–108, 108f idioblast of, 45–47 29f photochemical quenching with, 110 mesocarp of, 47 vascular cambium of, 31–32 translation of, 108 pericarp of, 45, 46f,47 Vitis vinifera,32f visible light with, 107–108 skin of, 45–47 tendrils, 41–43 Lipids structure of, 46f intercalary growth of, 41–43 construction costs of, 121–122, 121t vascular bundles of, 45 trunk, 27–33 grape berries composition/quality leaves, 36–41, 37f bark of, 33 with, 205–209 cotyledons, 36 cortex of, 31 Lizard’s tail strategy, 95 cross section of, 37f pith cells of, 29–30 formation of, 36 Mu¨ller-Thurgau., 12–13 M growth stage of, 51f,52f Muscadinia genus, 4–5 Macroclimate, 141 lamina, 36–37, 38–39 Muscadinia munsoniana,5 Magnesium, 259–262, 261f mesophyll of, 40, 40f Muscadinia popenoei,5 Magnoliophyta. See Angiospermae palisade parenchyma of, 40, 40f Muscadinia rotundifolia,4 See Magnoliopsida. Dicotyledoneae petiole of, 36 N Malate, 189–190 photosynthetic machinery of, 40 NADPH to NADP, 113–114, 113f Manganese, 266–267 plastochron of, 36 Necrotroph, 295–296 Matrix potential, 87 serrated margins of, 37–38, 38f Nickel, 269 Mesocarp, 47 stomata of, 39–40 Nitrogen, 249–253, 250f, 252f Mesoclimate, 141 nodes, 33–36, 34f Nitrogen assimilation, 158–167 Mesophyll, 40, 40f shoots distinguished from, ammonium assimilation Mesophyll cell, amino acids produced 33–34 with, 160–162 in, 131 root, 21–27, 23f,26f glutamate dehydrogenase in, Microclimate, 141 apex of, 22 162 Mineral nutrients, grape berries cell differentiation in formation structure/reactions of composition/quality with, of, 22, 23f compounds involved in, 161f 190–193 cell division in, 22 cells to plants with, 162–167 Molybdenum, 267 elongation zone of, 22–24, 23f energy from photosynthetic Morphology/anatomy, 20–47 endodermis of, 22–24 electron transport for, 163 buds, 33–36, 34f,36f growth in vegetative cycle of, leaves in assimilation for, basal, 35–36 64–68, 67f 163–164 bracts with, 34–35 hypocotyl development of, 21–22 nitrogen as nucleic acid dormant, 34–35 meristem cells of, 22–24 component for, 162 growth stage of, 51f,52f phloem of, 24–25 latent, 34–35 surface area of, 22 nitrogen distribution in soil longitudinal section of, 36f tip, 22 for, 162–163 roots/shoots nitrate assimilation prompt, 34–36 vascular cambium of, 25 three types of, 34–35 Vitis vinifera,26f for, 163 clusters, 41–43 xylem of, 24–26 sufficient photosynthate energy flowers, 43–47 shoots, 27–33, 28f for, 164–165 necessity of, 158 androecium of, 43–44 apical meristem of, 28–29 calyptra of, 44–45 cortex of, 31 nitrate uptake/reduction with, 159–160, 159f 374 INDEX

Nitrogen assimilation (Continued) O release, 130–131 grapevine store/distribute, 160 Organic acids three sectors of, 130–131 nitrate reductase forms for, 160 construction costs of, 121–122, 121t transport, 130–132, 134 protein ferredoxin in, 160 xylem/phloem sap with, 127t Phloem sap root cells in, 159–160 Osmosis, 85–89 composition of, 127–128, 127t Nitrogenous compounds, grape aquaporins with, 85–86 flow velocities of, 128, 129–130, 129f berries composition/quality diffusion with, 86f movement of, 127t, 128 with, 190–193 osmotic potential with, 86 Phosphorus, 253–255 Nodes osmotic pressure with, 86 Photochemical quenching, 110 ideal canopy with shoots of 15, 157 osmotic solutes with, 86, 87–88 Photoinhibition, 146–147 morphology/anatomy of, 33–36, turgor pressure with, 86 Photorespiration, 117–118 34f Ovule, 44f,45 global climate change with, 117–118 shoots distinguished from, 33–34 photosynthesis efficiency reduced shoots with pattern of three, 29f P with, 117 Nomentanic cultivars,13 Palisade parenchyma, 40, 40f Photosynthate, 114 Nutrient assimilation, photosynthesis Panicles, 43 allocation/partitioning of, 134–140 with, 125–126 PAR. See Photosynthetically active assimilate surplus in, 139–140, Nutrient uptake, 85–106, 92–105 radiation 140f active transport with, 102 Parthenocarpic fruit assimilation rate determined by Casparian bands with, 100–101, 100f development, 76–77 source in, 139 cell membranes with, 101 Partitioning, 134–140 patterns in, 135–138 ion channels with, 101–102 communication with, 137 phosphates availability in movement through root of, 100–101, competition with, 137 regulation of, 134–135 100f connection with, 136 source-sink relation with, tissue growth rates with, 105 defined, 135–138 135–138, 135f transpiration stream with, development with, 137 storage in, 134 103–104 interference with, 136 three categories for, 134 water flow dependant, 99 patterns in, 135–138 transport in, 134 Nutrients. See also Mineral nutrients proximity with, 136 utilization in, 134 deficiency or excess stress source-sink relation with, 135–138, sufficient energy for, nitrogen with, 243–274, 244f 135f assimilation with, 164–165 grape berries composition/quality Path phloem. See Transport phloem translocation/distribution with, 220–223 Pericarp, 45, 46f,47 of, 125–140 macronutrients, 249–262 Petiole, 36 amino acids in, 127t, 131 calcium, 258–259 Phenolics flexibility with, 127 magnesium, 259–262, 261f anthocyanins, 194t, 201, 202–205 nutrient assimilation nitrogen, 249–253, 250f, 252f biosynthetic pathways for with, 125–126 phosphorus, 253–255 production of, 196f phloem sap in, 127–128, 127t, 129f potassium, 255–257, 256f compound classes of, 194t sink with, 125–127, 132 sulfur, 257–258 construction costs of, 121–122, 121t solute unloading in, 132–133 shoot/root growth influenced by, flavonols, 194t, 203, 204–205 sucrose in, 127–128, 127t, 133–134, 68 grape berries composition/quality 133f transition metal/ with, 193–205, 196f, 201f xylem sap in, 127–128, 127t micronutrients, 262–270 hydroxybenzoic acids, 194t Photosynthesis, 107–124 boron, 267–269, 268f stilbenes, 194t, 197–198 carbon uptake/assimilation copper, 265–266 tannins, 194t, 198–199, 200, 204–205 with, 111–117 iron, 262–264, 263f xydroxycinnamic acids, 194t Calvin cycle with, 112–114, 113f manganese, 266–267 Phenology, 49–84, 49. See also Growth glucose molecules in, 116 f molybdenum, 267 cycle H2O loss with, 112 nickel, 269 Phloem uptake of CO2 by leaves salinity, 270–274, 273f collection, 130–131 with, 111–112 silicon, 269–270 pressure-driven mass flow cells to plants with, 121–123 zinc, 264–265 in, 129–130 chlorophyll for, 108, 109–110, 109f INDEX 375

chloroplast with, 116–117 R cell division in, 22 starch production in, 114–115 Radiation, 92 elongation zone of, 22–24, 23f sugar production in, 114, 114f Red/far red light, 147–148, 147f endodermis of, 22–24 construction costs of compounds Release phloem, 130–131 flow of water with pressure of, 96 with, 121–122, 121t Reproductive cycle, 68–83 gravity sensors with, 66 electromagnetic spectrum anthesis in, 71–73, 71f growth in vegetative cycle of, 64–68, with, 107–108, 108f beginning of bloom with, 71–72, 72f 67f energy overload with, 111 berry growth stages in, 79–82, 80f auxin stimulating, 64–65 energy transfer with, 110–111 stage I: rapid increase, 79 lagging behind shoot growth importance of, 107 stage II: lag phase, 80 of, 65–66 light absorption/energy capture stage III: ripening period, 80 moist soil with, 67–68 with, 107–111 cell division following fertilization nutrient availability influence light energy absorbed/translated in, 78–79 on, 68 with, 108 chromosome mixing in, 75 species, rootstock, and cultivar nutrient assimilation with, 125–126 cluster initiation/differentiation influencing, 66–67 organic product of, 114 in, 70f hypocotyl development of, 21–22 photochemical quenching with, 110 endosperm nucleus division in, 76 meristem cells of, 22–24 photon energy exploited with, 108 evolution and, 82–83 morphology/anatomy of, 21–27 photorespiration, reduced efficiency fertilization in, 73, 74 nutrient and water uptake/ from, 117 flower initiation in, 69–71 transport through, 100–101, rete of sucrose export with, fruit set variations in, 78 100f 115–116 gamete production in, 75 phloem of, 24–25 sugar production in chloroplast inflorescences and flower pressure, 54 for, 114, 114f differentiation in, 69–70, 70f surface area of, 22 temperature stimulates, 151–152 inflorescences per bud with, 69 tip, 22 visible light with, 107–108 meiosis in, 75 vascular cambium of, 25 Photosynthetically active radiation ovary abortion in, 77–78 Vitis vinifera,26f (PAR), 143, 144f perennial woody species, 69 water uptake proportional to Photosystem II (PSII), 109–111 pollination in, 73, 76 surface area of, 99 Phyllotaxy, 27–28 production of pollen, 72–73 xylem of, 24–26 Physiology. See Developmental seedless grape berries in, 82 Rootstocks, 9–19 physiology; Stress physiology vegetative phase of seed grapes agronomic characteristics of, 18t Phytoalexins, 296–297 in, 68–69 commonly used, 17 Pinot blanc, 11–12, 12f zygote division in, 76 grafting of, 17 Pinot gris, 11–12, 12f Respiration, 107–124, 118–121 root growth influenced by, 66–67 Pinot Meunier, 11–12 cells to plants with, 121–123 Rubisco Pinot noir, 11–12, 12f citric acid cycle with, 117, 119f Calvin cycle with, 112–113, 117 Pistil, 43–45, 44f construction costs of compounds evolution of, 117–118 Plantae kingdom, 3 with, 121–122, 121t global climate change and, 117–118 Plastochron, 36 electron transport chain S Pollen, production of, 72–73 with, 117–118 f Pollination, 73, 76 glycolysis with, 119–120, 119f Salinity, 270–274, 273 ovary development triggered intermediate carbon compounds Seasons, 49–53 by, 76 generated by, 118 source-sink relations changing f Potassium, 255–257, 256f photosynthetically fixed carbon with, 135 Powdery mildew, 302–304 consumed with, 122 Shoots Proles occidentalis,14 three processes of, 118–119 apical meristem of, 28–29 Proles orientalis,14 Rhamnales, 3–4 cortex of, 31 Proles pontica,14 Root, 21–27, 23f,26f epidermis of, 30–31 f Prompt buds, 34–36 amino acids delivered by, 131 green tip, 52 f f Pruning, 27 apex of, 22 growth stage of, 51 , 55–60, 57 ,61 vine capacity depressed with, 171 cell differentiation in formation nutrient availability influence PSII. See Photosystem II of, 22, 23f on, 68 376 INDEX

Shoots (Continued) phytoalexins, 296–297 leaf temperature changes, 150, temperatures inducing in, 55–56, powdery mildew, 302–304 151f 62 viruses, 308–310, 310f photosynthesis stimulated time of rapid, 62–63 responses to stress in, 227–231 by, 151–152 ideal canopy with, 156, 157 temperature: too cold or too warm shoot growth with, 55–56, 62 leaf-opposed tendril production in, 274–289 stomatal action with, 90 with, 30 chilling stress with, 274–276 stress with too cold or too longitudinal section of, 28f cold acclimation/freeze damage warm, 274–289 morphology/anatomy of, 27–33, 28f with, 276–286, 279f, 281f, 284f transpiration with, 90 parenchyma cells of, 32–33 heat acclimation/damage Tendrils pearls of, 30–31, 31f with, 286–289, 288f intercalary growth of, 41–43 phyllotaxy of, 27–28 water: too much or too little morphology/anatomy of, 41–43 procambium of, 31–32 in, 231–243 Transpiration, 89–92 three-node pattern of Syrah, 29f Sucrose defined, 89 vascular cambium of, 31–32 Calvin cycle with, 113f grapevine leaf with, 90, 104–105 vigor of, 61 cytosol in production of, 114, 114f heat dissipation with, 92 vigor with yield formation from, 171 import into sink tissues of, 133–134 humidity with, 155 Vitis vinifera,32f pathways for import of, 133, 133f stomatal conductance with, 90 Silicon, 269–270 photosynthate translocation temperature with, 90 Sink of, 127–128 turnover of water with, 91–92 leaves initially, 126 production of, 114, 114f water flow rate matches, 97–98 nonphotosynthetic plant organ rete of export of, 115–116 water losses from, 93 as, 125–126 xylem/phloem sap with, 127–128, water potential with, 89 relation with source and, 135–138, 135f 127t Transpiration stream, 103–104 relations changing with growing Sugar xylem as 99% length of, 104 seasons, 135f grape berries composition/quality Transport phloem (Path solutes unloaded in, 132 with, 183–186, 185f phloem), 130–132 sucrose imported into tissues pathways for import of, 133, 133f Trellis system, 27, 144–145 of, 133–134 production, 114, 114f Tricarboxylic acid cycle (TCA woody organs as, 126 Sulfur, 257–258 cycle), 117, 119f. See also Citric Stamens, 43–44, 44f Symplast, 100–101 acid cycle Stilbenes, 194t, 197–198 Trunk Stomata, 39–40, 90–91 T bark of, 33 reaction to abscisic acid of, 91 Tannins, 194t, 198–199, 200, 204–205 cortex of, 31 Stomatal action, 89–92 TCA cycle. See Tricarboxylic acid cycle morphology/anatomy of, 27–33 temperature with, 90 Temperature, 150–154 pith cells of, 29–30 transpiration in grapevine leaf amino acids concentration rises Turgor pressure, 86 with, 90 with, 217–218 Stress physiology, 227–310 budbreak with, 55–56 U nutrients: deficiency or excess clouds alter, 152 UV light, 149–150, 149f in, 243–274, 244f CO2 assimilation influenced by, 151, leaf hairs in scatter of, 149–150 f 153 macronutrients, 249–262, 250 , V 252f, 256f, 261f cold acclimation/freeze damage transition metal/ with, 276–286, 279f, 281f, 284f Vegetative cycle, 53–68 micronutrients, 262–270, 263f, freezing in vegetative cycle, 63–64 anthocyanin production in, 268f, 273f fruit development delay 59–60 f organisms: defense and with, 176–177 bleeding grapevines, 53–54, 53 damage, 289–310, 290f global climate change with increase budbreak in, 55–58 bacteria, 306–308, 307f in, 152–153 cell division/auxin production in biotroph, 296 grape berries composition/quality buds, 54–55 bunch rot, 298–302, 300f influenced by, 214–218, 216f chilling of cultivars in, 55 downy mildew, 304–306 heat acclimation/damage cytokinins in, 58 necrotroph, 295–296 with, 286–289, 288f DELLA proteins in, 58 leaf growth with, 153–154 freezing temperatures in, 63–64 INDEX 377

GDD calculation, 61 root of, 26f damage from, 154, 154f initial growth phase in, 58 shoot of, 32f down row v. across row leaf senescence in, 59–61, 60f Volatiles, grape berries composition/ turbulence, 155 reddening leaves in, 59–60, 60f quality with, 205–209 leaf’s boundary layer resistance root growth in, 64–68, 67f decreased with, 154–155 auxin stimulating, 64–65 W Winkler regions, 61 gravity sensors with, 66 Water Winter bud, 52f lagging behind shoot growth grape berries composition/quality of, 65–66 with, 181–183, 218–220 X moist soil with, 67–68 stress from too much or too Xydroxycinnamic acids, 194t nutrient availability influence little, 231–243 Xylem, 104 on, 68 Water potential, 85–89 Xylem conduits, 96–97 species, rootstock, and cultivar equation for, 88–89, 89t Xylem sap, 92–95 influencing, 66–67 transpiration dependant on, 89 composition of, 127t root pressure, 54 water’s availability in aqueous Xylem vessel seed grapes in, 69 system as, 88–89 cavitation of, 95–96, 100f shoot growth in, 51f, 55–58, 57f, Water relations, 85–106 elements of, 127t 59–60, 61 aquaporins with, 85–86 lizard’s tail strategy protecting, 95 cycle, 59–60 cell expansion with, 85–89 water deficit and narrower, 96 matrix potential with, 87 nutrient availability influence Y on, 68 nutrient uptake, 92–105 root growth lagging osmosis, 85–89 Yield formation, 169–178 behind, 65–66 percent of grapevine’s mass as, components of grapevine temperatures inducing in, 55–56, 85–86 yield, 169–170 62 solute/pressure forces in plants/ fruit production depresses vine time of rapid, 62–63 soils with, 87 capacity for, 171 temperatures inducing budbreak stomatal action with, 89–92 genotype/environment interactions in, 55–56 transpiration with, 89–92 for, 170 Winkler regions of, 61 water uptake/transport, 92–105 pruning depresses vine capacity Vigor, 61 Water uptake/transport, 92–105 for, 171 light range for vine, 144–145 Casparian bands with, 100–101, 100f shoot vigor for, 171 yield formation with shoot, 171 cell membranes with, 101 vine capacity for, 171 Vine capacity, 171 dry soil with, 98–99 vine growth maintenance fruit production depresses, 171 flow matches transpiration rate plus for, 170–171 pruning depresses, 171 bound water in, 97–98 vine self-regulation for, 171 Vineyard yield, 170 flow of water from root pressure vineyard yield, 170 Vinum francicum,14 in, 96 Yield potential, 172–178 Vinum hunicum,14 hydraulic architecture in, 96–97 bloom occurrence with, 174 Viruses, 308–310, 310f influx proportional to root system bud fruitfulness for, 172, 173 Vitaceae, 4 surface area for, 99 cell enlargement with, 176–177 Vitales, 3–4 ion channels with, 101–102 defined, 172 Vitis aestivalis,6 movement through root of, 100–101, fruit yield with active leaf area Vitis amurensis,9 100f for, 174–175 Vitis berlandieri,7 resistances in, 97 inflorescence necrosis with, 175, f Vitis candicans,7 transpiration losses from, 93 175 Vitis coignetiae,9 water pressure as driving force size of grape berries with, 176 Vitis genus, 2, 4–5, 5f for, 92–93 temperature in fruit development Vitis labrusca,6 xylem conduits in, 96–97 delay with, 176–177 Vitis riparia f ,7,7 xylem sap in, 92–95 Z Vitis rupestris,7 xylem vessel cavitation with, 95–96, Vitis sylvestris,8 100f Zinc, 264–265 Vitis vinifera,8 xylem vessel narrower with, 96 Zygote, 76 flowers of, 43–44 Wind, 154–155