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Desiccation and Survival in Plants

Drying Without Dying Desiccation prels 19/3/02 1:42 pm Page ii Desiccation prels 19/3/02 1:42 pm Page iii

Desiccation and Survival in Plants

Drying Without Dying

Edited by

M. Black

King’s College University of London UK

and

H.W. Pritchard

Royal Botanic Gardens, Kew Wakehurst Place UK

CABI Publishing Desiccation prels 4/4/02 2:16 pm Page iv

CABI Publishing is a division of CAB International CABI Publishing CABI Publishing CAB International 10 E 40th Street Wallingford Suite 3203 Oxon OX10 8DE New York, NY 10016 UK USA Tel: +44 (0)1491 832111 Tel: +1 212 481 7018 Fax: +44 (0)1491 833508 Fax: +1 212 686 7993 Email: [email protected] Email: [email protected] Web site: www.cabi-publishing.org

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Library of Congress Cataloging-in-Publication Data Desiccation and survival in plants : drying without dying / edited by M. Black and H.W. Pritchard. p. cm. Includes bibliographical references (p. ). ISBN 0-85199-534-9 (alk. paper) 1. Plants--Drying. 2. Plant-water relationships. 3. Plants--Adaptation. I. Black, Michael. II. Pritchard, H. W. QK870 .D57 2002 581.4--dc21

2001043835 ISBN 0 85199 534 9

Typeset in Melior by Columns Design Ltd, Reading Printed and bound in the UK by Biddles Ltd, Guildford and King’s Lynn Desiccation prels 19/3/02 1:42 pm Page v

Contents

Contributors vii Preface ix

PART I. INTRODUCTION 1 1 Drying Without Dying 3 Peter Alpert and Melvin J. Oliver

PART II. METHODOLOGY 45 2 Methods for the Study of Water Relations Under Desiccation Stress 47 Wendell Q. Sun 3 Experimental Aspects of Drying and Recovery 93 Norman W. Pammenter, Patricia Berjak, James Wesley-Smith and Clare Vander Willigen 4 Biochemical and Biophysical Methods for Quantifying Desiccation Phenomena in Seeds and Vegetative Tissues 111 Olivier Leprince and Elena A. Golovina

PART III. BIOLOGY OF DEHYDRATION 147 5 Desiccation Sensitivity in Orthodox and Recalcitrant Seeds in Relation to Development 149 Allison R. Kermode and Bill E. Finch-Savage 6 Pollen and Spores: Desiccation Tolerance in Pollen and the Spores of Lower Plants and Fungi 185 Folkert A. Hoekstra 7 Vegetative Tissues: Bryophytes, Vascular Resurrection Plants and Vegetative Propagules 207 Michael C.F. Proctor and Valerie C. Pence 8 Systematic and Evolutionary Aspects of Desiccation Tolerance in Seeds 239 John B. Dickie and Hugh W. Pritchard

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vi Contents

PART IV. MECHANISMS OF DAMAGE AND TOLERANCE 261 9 Desiccation Stress and Damage 263 Christina Walters, Jill M. Farrant, Norman W. Pammenter and Patricia Berjak 10 Biochemistry and Biophysics of Tolerance Systems 293 Julia Buitink, Folkert A. Hoekstra and Olivier Leprince 11 Molecular Genetics of Desiccation and Tolerant Systems 319 Jonathan R. Phillips, Melvin J. Oliver and Dorothea Bartels 12 Rehydration of Dried Systems: Membranes and the Nuclear Genome 343 Daphne J. Osborne, Ivan Boubriak and Olivier Leprince

PART V. RETROSPECT AND PROSPECT 365 13 Damage and Tolerance in Retrospect and Prospect 367 Michael Black, Ralph L. Obendorf and Hugh W. Pritchard Glossary 373 Taxonomic Index 383 Subject Index 401 Desiccation prels 19/3/02 1:42 pm Page vii

Contributors

Peter Alpert, Biology Department, University of Massachusetts, Amherst, Massachusetts 01003-5810, USA. [email protected] Dorothea Bartels, Institute of Botany, University of Bonn, Kirschallee 1, D-53115 Bonn, Germany. [email protected] Patricia Berjak, School of Life and Environmental Sciences, University of Natal, Durban 4041, South Africa. [email protected] Michael Black, Division of Life Sciences, King’s College, Franklin Wilkins Building, 150 Stamford Street, London SE1 6NN, UK. [email protected] Ivan Boubriak, The Oxford Research Unit, Open University, Foxcombe Hall, Boars Hill OX1 5HR, UK. [email protected] Julia Buitink, UMR Physiologie Moléculaire des Semences, Institut National d’Horticulture, 16 Bd Lavoisier, F49045 Angers, France. [email protected] John B. Dickie, Seed Conservation Department, Royal Botanic Gardens Kew, Wakehurst Place, Ardingly, West Sussex RH17 6TN, UK. [email protected] Jill M. Farrant, Department of Molecular and Cellular Biology, University of Cape Town, 7700, South Africa. [email protected] Bill E. Finch-Savage, Horticulture Research International, Wellesbourne, Warwick CV35 9EF, UK. bill.ﬁ[email protected] Elena A. Golovina, Laboratory of Plant Physiology, Department of Plant Sciences, University of Wageningen, Arboretumlaan 4, 6703 BD Wageningen, The Netherlands and Timiryazev Institute of Plant Physiology, Botanicheskaya 35, Moscow 127276, Russia. [email protected] Folkert A. Hoekstra, Laboratory of Plant Physiology, Department of Plant Sciences, University of Wageningen, Arboretumlaan 4, 6703 BD Wageningen, The Netherlands. Folkert. [email protected] Allison R. Kermode, Department of Biological Sciences, Simon Fraser University, Burnaby, BC, V5A 1S6, Canada. [email protected] Olivier Leprince, UMR Physiologie Moléculaire des Semences, Institut National d’Horticulture, 16 Bd Lavoisier, F49045 Angers, France. [email protected] Ralph L. Obendorf, Seed Biology, Department of Crop and Soil Sciences, Cornell University, Ithaca, New York, USA. [email protected] Melvin J. Oliver, USDA-ARS Plant Stress and Germplasm Development Unit, 3810 4th Street, Lubbock, Texas 79415, USA. [email protected]

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viii Contributors

Daphne J. Osborne, The Oxford Research Unit, Open University, Foxcombe Hall, Boars Hill OX1 5HR, UK. [email protected] Norman W. Pammenter, School of Life and Environmental Sciences, University of Natal, Durban 4041, South Africa. [email protected] Valerie C. Pence, CREW, Cincinnati Zoo and Botanical Garden, 3400 Vine Street, Cincinnati, OH 45220, USA. [email protected] Jonathan R. Phillips, Max-Planck-Institute for Plant Breeding Research, Carl-von-Linné- Weg 10, D-550829 Köln, Germany. [email protected] Hugh W. Pritchard, Seed Conservation Department, Royal Botanic Gardens Kew, Wakehurst Place, Ardingly, West Sussex RH17 6TN, UK. [email protected] Michael C.F. Proctor, School of Biological Sciences, University of Exeter, Washington Singer Laboratories, Perry Road, Exeter EX4 4QG, UK. [email protected] Wendell Q. Sun, Department of Biological Sciences, National University of Singapore, Kent Ridge Crescent, Singapore 119260. [email protected] Clare Vander Willigen, Department of Botany, University of Capetown, Private Bag, Rondebosch 7701, South Africa. [email protected] Christina Walters, USDA-ARS National Seed Storage Laboratory, 1111 South Mason Street, Fort Collins, CO 80521, USA. [email protected] James Wesley-Smith, School of Life and Environmental Sciences, University of Natal, Durban 4041, South Africa. [email protected] Desiccation prels 19/3/02 1:42 pm Page ix

Preface

Plant survival of desiccation as sporophytic and gametophytic tissues was last reviewed in detail in two books published in 1980. The first, by J. Levitt, on Responses of Plants to Environmental Stresses, Volume II, Water, Radiation, Salt and Other Stresses is a classic. The topic of plant water stress consumes about 200 pages and is set mainly at the introductory level but is still of sufficient detail to stimulate post-graduate researchers. Interestingly, there is no mention in Levitt’s book of desiccation sensitivity in seeds. However, this latter topic was specifically covered in another book by H.F. Chin and E.H. Roberts (Recalcitrant Seeds). At that time recalcitrant seed behaviour was something of a novelty and the book deals mainly with descriptions of germination, a listing of species with such seeds and an indication of how best to store the material in the short term. Aspects of plant desiccation have been considered in other publications dealing with the biology and biophysics of dehydration and in contributions to general works on seeds but a comprehensive treatment of desiccation and plant survival is not yet available. Since 1980 there has been a revolution in plant science as new methods of cell and molecular biology and biophysics have been applied to environmental stress, particularly in relation to desiccation tolerance. The basic level of understanding of how plant cells cope with extreme water stress has increased tremendously and considerable effort has been made in the last 10 years to develop diagnostic markers for desiccation tolerance. At the physiological level, studies have often focused on seed material and on the responses of resurrection plants. At a more mechanistic level, model membrane systems have been used extensively, and exploration of the molecular genetics of desiccation tolerance has begun on developmental mutants, especially of seeds of crop species. These progressive but fundamental changes in approach to investigating the basis of survival of plant tissues under desiccation since the 1980s have meant that our perceptions of this subject have altered significantly. It seems particular appropriate now to take stock of these recent developments, to assess critically the importance of the experimental systems available for investigation and to

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x Preface

consider possible foci for future research work. This book sets out to address these issues. The Introduction surveys the topic of desiccation, and the remainder of the book is divided into four parts, dealing with: (i) the technical background to desiccation tolerance studies; (ii) the frequency and levels of dehydration stress tolerance in biological systems; (iii) mechanisms of damage and tolerance; and (iv) a brief retrospect and prospect. It will not attempt to address in detail plant drought stress (i.e. at relatively high water potentials). This subject has been covered in detail in the last 10 years, for example in Environmental Stress in Plants – Biochemical and Physiological Mechanisms (Cherry. J.H. (ed.), Springer Verlag, 1989) and Plants Under Stress (Jones, H.G., Flowers, T.J. and Jones, M.B. (eds), SEB Seminar Series, Cambridge, 1989). However, drought stress will be referred to in several places within this text. In dealing with the different aspects of desiccation it is inevitable that certain topics will receive consideration in more than one chapter. But the authors and editors have attempted, as far as is possible, to avoid repetition of detail. Extensive cross-referencing has been used, to aid the reader in identifying where, within the special viewpoints of the treatments, similar subjects are considered. This comprehensive presentation on desiccation and survival in plants will be of value to all researchers in the ﬁeld, both beginners and the more experienced, and to those with interests in basic and applied plant sciences – physiology, ecology, conservation biology, agriculture and horticulture.

M. Black H.W. Pritchard 01 Desiccation -Chap 1 18/3/02 1:53 pm Page 1

Part I

Introduction 01 Desiccation -Chap 1 18/3/02 1:53 pm Page 2 01 Desiccation -Chap 1 18/3/02 1:53 pm Page 3

1 Drying Without Dying

Peter Alpert1 and Melvin J. Oliver2 1Biology Department, University of Massachusetts, Amherst, Massachusetts 01003-5810, USA; 2Plant Stress and Water Conservation Laboratory, Agricultural Research Service, US Department of Agriculture, 3810 4th Street, Lubbock, Texas 79415, USA

1.1. Introduction 4 1.2. Defining and Measuring Desiccation Tolerance 4 1.2.1. Operational and conceptual definitions 4 1.2.2. Measuring tolerance 6 1.3. A Brief History of Research on Desiccation Tolerance 6 1.3.1. Early work (1702–1860) on the question of whether life can stand still 6 1.3.2. The next step: establishing records 7 1.4. The Occurrence of Desiccation Tolerance in Plants: Rarity and Ubiquity 8 1.4.1. Seeds, pollen and spores 8 1.4.2. Vegetative tissues 9 1.5. The Ecology of Desiccation Tolerance in Plants: a Diversity of Cycles in Marginal Habitats 13 1.5.1. Habitats 17 1.5.2. Cycles 17 1.5.3. Hypotheses 19 1.6. Mechanisms of Desiccation Tolerance 20 1.6.1. Damage 21 1.6.1.1. Damage during desiccation 21 1.6.1.2. Damage during rehydration 22 1.6.1.3. Poikilochlorophylly 23 1.6.2. Protection 24 1.6.2.1. Proteins 24 1.6.2.2. Sugars 26 1.6.3. Repair 28 1.7. Future Prospects and Agricultural Significance 30 1.8. References 31

4 P. Alpert and M.J. Oliver

1.1. Introduction Though some desiccation-tolerant plants can survive droughts more intense and pro- Water is a universal requirement for life as longed than any that occur almost any- we know it. Water is the most abundant where on earth, tolerant plants are in the compound in all active cells, it is essential minority. Desiccation-sensitive plants dom- for metabolism and all organisms must take inate the world’s vegetation. The rarity of in water to survive. Living things therefore the apparently excellent ability to tolerate face a major problem whenever they desiccation raises a second, cautionary emerge above ground on land: the air is question about desiccation tolerance: How almost always drier than they are and takes does surviving desiccation affect plant sur- water from them. This is a life and death vival? These two questions, one largely problem for most organisms in most habi- genetic and biochemical and the other tats, because the air is at least sometimes mainly physiological and ecological, frame deadly dry. For example, when the relative the topic of desiccation and plant survival. humidity is about 50% and the tempera- The purpose of this introductory chap- ture 28°C, a plant cell that dries to equilib- ter is to summarize some of the current rium will drop to a water potential of about answers to these questions and lead into 100 MPa (Gaff, 1997). This kills over 99% the more detailed reviews of questions and of flowering plants. answers about desiccation and plant sur- Terrestrial plants appear to have vival in the chapters that follow. We begin evolved two solutions to the problem of with some terms and techniques that pro- maintaining an aqueous self in a withering vide concepts and methods for research on world. The majority solution, at least at the desiccation tolerance in plants, and a brief present evolutionary time, is never to dry summary of the surprisingly lively history out – to maintain a chronic disequilibrium of research on desiccation tolerance. We between wet cells and dry air. Some of the then give an overview of the range and most universal features of plant form, such ecology of desiccation tolerance in plants, as waxy coatings on shoots, and pores that subjects that bear on how surviving desic- can open and close on leaves, seem largely cation affects plant survival. Last, we dis- designed to conserve water. cuss mechanisms of desiccation tolerance The minority solution is to dry up but in plants, the keys to understanding how not die – to desiccate during drought and plants survive desiccation, and consider rehydrate and resume growth when the potential for breeding crops that can drought ends. About 300 species of flower- dry without dying. We will sometimes ing plants, or perhaps 0.1% of those named, abbreviate desiccation tolerance to ‘toler- are known to tolerate desiccation ance’, and we will call plants that cannot (Porembski and Barthlott, 2000). Some of tolerate desiccation ‘desiccation-sensitive’ these species can lose all of the free water or ‘sensitive’. We will consider desiccation in their cells or remain dry for up to 5 years tolerance in plants and in some organisms and still recover (Gaff, 1977). These prodi- that are not in the plant kingdom, mainly gious abilities raise the first and fundamen- cyanobacteria, algae and fungi. tal question about desiccation tolerance: How do plants survive desiccation? Most recent research on desiccation tolerance has 1.2. Defining and Measuring focused on discovering the mechanisms of Desiccation Tolerance desiccation tolerance, partly in the hopes of some day engineering tolerance in econom- 1.2.1. Operational and conceptual definitions ically important species and banishing the spectre of famine from drought. Desiccation tolerance can be operationally However, the ability to survive desicca- defined as the ability to dry to equilibrium tion may not always increase the ability of with moderately dry air and then resume plants to survive in natural systems. normal function when rehydrated, where 01 Desiccation -Chap 1 18/3/02 1:53 pm Page 5

Drying Without Dying 5

‘moderately dry’ means 50–70% relative 2000). Some tolerant species are more dam- humidity at 20–30°C . This definition is aged by being held at intermediate water workable because there seems to be a wide contents than at full hydration or complete gap between the maximum tolerance of desiccation (Gaff, 1997), and one advantage sensitive plants and the minimum toler- of rapid desiccation may be to minimize ance of tolerant ones (Gaff, 1997). Almost time spent at intermediate levels of hydra- all species known to recover from complete tion (Kappen and Valladares, 1999; Proctor, drying at 80% relative humidity also 2000; Chapters 3 and 5). Second, cells must recover from drying at 50% (but see preserve enough cellular organization and Bochicchio et al., 1998). functional enzymes so that metabolism can The reason why a gap exists between the resume after rewetting. Preserving a skele- ranges of tolerance of drying in desiccation- tal machinery for metabolism must involve sensitive and desiccation-tolerant plants both protection and repair (Section 1.6). may be that desiccation tolerance depends Enzymes and membranes must be pro- on the ability to reversibly cease metab- tected from loss of configuration and orga- olism as cells dry out. There may not be nization, and the damage that accumulates very many marginally desiccation-tolerant from degradative non-metabolic reactions plants because, once metabolism has while plants are inactive must be repaired. stopped, it cannot be stopped further. Clegg Differences in effectiveness of protec- (1973) argued on biochemical grounds that tion may explain much of why desiccation- metabolism, defined as ‘systematically con- tolerant plants do differ in the intensity trolled pathways of enzymatic reactions’ (minimum water content or water poten- (Clegg, 2001), cannot take place at a cell tial) of desiccation that they can stand. For 1 water content of less than 0.1 g H2O g instance, tolerant angiosperms tend to sur- dry mass because not enough water vive equilibration with lower relative remains to hydrate intracellular proteins. humidities than do tolerant pteridophytes Organisms this dry do show chemical in South Africa (Gaff, 1977). Species with a activity. For instance, dried pollen can greater degree of protection of molecular incorporate water vapour into organic com- configuration and cellular organization pounds (Wilson et al., 1979). However, may survive with smaller fractions of chemical reactions, even some characteris- water. Differences in effectiveness of repair tic of living things such as oxygen uptake, may help explain why species also differ in do not necessarily require metabolism: iron the duration of desiccation (length of time rusts (Clegg, 1986). We propose that desic- in the dried state) that they can stand (e.g. cation tolerance can be conceptually Sagot and Rochefort, 1996). Those with defined as reversible cessation of metab- more effective repair mechanisms may be olism in response to water loss. better able to undo non-metabolic degrada- This suggests that the mechanisms of tion suffered while dry. desiccation tolerance must involve at least There has been some confusion about two key elements (Section 1.6). First, there the difference between ‘desiccation toler- must be an orderly shutdown of metabo- ance’ and ‘drought tolerance’. We would lism during desiccation. Different meta- like to propose that desiccation tolerance is bolic pathways must slow at compatible one form of drought tolerance. Drought rates to avoid fatal accumulations of inter- may be defined as any level of water avail- mediates and generation of free radicals. ability that is low enough to reduce plant Oxidation is a major hazard of desiccation performance. ‘Drought tolerance’ is most (e.g. Smirnoff, 1993), and the advantages of often used to refer to tolerance of water minimizing photo-oxidation may explain availabilities that are suboptimal but not why some desiccation-tolerant plants cease low enough to cause complete drying to photosynthesis at relatively high water equilibrium with the air, i.e. desiccation. contents during drying (e.g. Sherwin and Mechanisms of drought tolerance include Farrant, 1998; Tuba et al., 1998; Farrant, ways of maintaining cell water content, 01 Desiccation -Chap 1 18/3/02 1:53 pm Page 6

6 P. Alpert and M.J. Oliver

such as osmotic regulation and stomatal undergo cycles of drying and wetting. closure, whereas desiccation tolerance con- Chapter 4 reviews the rapidly expanding sists of ways of surviving the nearly com- range of non-invasive techniques available plete loss of water. Some papers on to study the diffusion of water, the configu- intertidal algae maintain the confusion by rations and interactions of macromole- using ‘desiccation’ to refer to any amount of cules, metabolism, thermal events water loss (e.g. Leuschner et al., 1998; Bjork associated with membrane phase transi- et al., 1999). They are probably wrong, tions, ultrastructure, oxidative stress, fer- since the Oxford English Dictionary (1989) mentation and the physical properties of defines ‘to desiccate’ as ‘to make quite dry; membranes, cytoplasm and protein com- to deprive thoroughly of moisture’. plexes during desiccation and rehydration. Chapter 7 summarizes some of the technical developments in infrared gas analysis 1.2.2. Measuring tolerance and fluoroscopy that have improved our capacity to quantify responses to desicca- Techniques for quantifying the degree of tion on the physiological level. desiccation tolerance in different species are reviewed in Chapters 2–4 and 7. Chapter 2 discusses the advantages and 1.3. A Brief History of Research on limitations of different measures of water Desiccation Tolerance content and techniques for distinguishing water properties in plant cells. Chapter 3 The 300-year history of the science of desic- notes how the survival and recovery rates cation tolerance began with a lengthy of seeds and vegetative tissues vary with period of discovery and doubt. In the rate of drying, light conditions during dry- course of discovering desiccation tolerance, ing, storage conditions and length of time scientists confronted the nature of life. The in the dehydrated state. In general, highly next step was to enumerate the organisms desiccation-tolerant bryophytes can sur- that tolerate desiccation and test the limits vive rapid drying but tolerant angiosperms of their tolerance. In the 1960s, researchers cannot; this seems to be related to differ- started to investigate the physiological ecol- ences in their mechanisms of tolerance ogy of desiccation tolerance in plants, espe- (Section 1.6). A few species appear insensi- cially the cycles of wetting and drying and tive to rate of drying, but most probably their effects on carbon uptake in bryophytes have an optimal rate or optimal range of and lichens. Since the 1980s, emphasis has drying rates. For instance, desiccation in shifted to the biochemistry and molecular less than 6 hours or over more than 7 days biology of desiccation tolerance. We now kills the otherwise highly tolerant pterido- know more about how plants survive desic- phyte Selaginella lepidophylla (Eickmeier, cation than about how tolerating desicca- 1983). Rates and final levels of recovery tion affects plant survival. can decrease with increasing intensity or duration of desiccation (e.g. Gaff, 1977; Alpert and Oechel, 1987; Davey, 1997). 1.3.1. Early work (1702–1860) and the Quantifying desiccation tolerance therefore question of whether life can stand still also requires techniques for imposing known rates, intensities and durations of It took scientists one and a half centuries to desiccation, and for measuring rates and establish that desiccation tolerance exists final levels of recovery (Chapter 3). Rate of (Keilin, 1959). At the end of an often ran- drying is particularly hard to standardize corous debate, the nature of life had been across species. called into question: Can life stop, be con- Investigating the mechanisms of desic- tained in a static array of molecules and cation requires techniques for measuring restart? Anthony von Leeuwenhoek was processes and states in cells as they apparently the first to glimpse desiccation 01 Desiccation -Chap 1 18/3/02 1:53 pm Page 7

Drying Without Dying 7

tolerance, soon after he invented the micro- can be cooled to within 0.5°C of absolute scope. In 1702, he wrote to a friend (see zero, absolute zero being the temperature at Schierbeek, 1959): which all molecular motion is believed to stop, and then revive when rewarmed and The following day the sky was very hot and rehydrated (Becquerel, 1951). The discov- dry and, about nine in the morning, I took some of the sediment which has been in the ery of desiccation tolerance has shown us leaden gutter … and poured on it a small that living things can come to exist in three quantity of rain-water taken out of my stone states: alive, dead and still (Clegg, 2001). cistern … so that if there were still any living animalcules in it they might issue forth; though I confess I never thought that there 1.3.2. The next step: establishing records could be any living creatures in a substance so dried as this was. The scientific battle over whether living I was, however, mistaken; for scarce an things could dry without dying was fought hour had elapsed, when I saw at least a over animals. Starting in the 1960s, toler- hundred of the animalcules before described. ance was identified in the larvae of at least These animals were rotifers. By the mid- one insect and of some other arthropods 1800s, others had seen desiccation toler- (Hinton, 1968; Crowe et al., 1992) but has ance in two more phyla of animals, never been found in any life stage of any nematodes and tardigrades (Keilin, 1959; vertebrate or in the adults of any animals Alpert, 2000). However, others still flatly except microscopic rotifers, nematodes and denied it was possible to survive drying tardigrades. In contrast, tolerance was out. A French biologist, Felix Pouchet found to be widespread in plants. Tolerant (1859), wrote that: ‘Dry and completely bryophytes were reported by 1886, fern mummified animals cannot be resuscitated gametophytes by 1914, fern sporophytes by by hydration. Rational beliefs, observation, 1931 and angiosperms by 1921 (tables and and experiment unite to demonstrate it.’ citations in Kappen and Valladares, 1999; The Société de Biologie in Paris convened Alpert, 2000; Chapter 7). a special commission and conducted its The intensity and duration of desicca- own tests on rotifers. Its report effectively tion that plants and plant-like organisms settled the matter: ‘[organisms] reaching could survive were shown to be remark- the most complete degree of desiccation able. Like tardigrades (Doyère, 1842), cer- that can be realized … may yet retain the tain lichens, bryophytes, pteridophytes and ability to revive in water’ (Broca, 1860). angiosperms survived equilibration with However, the commission was silent on air of nearly 0% relative humidity, in

the deeper question of whether this means closed volumes over concentrated H2SO4 that life can stop and restart. As phrased by or P2O5 (e.g. Lange, 1953; Hosokawa and Pouchet’s main scientiﬁc opponent Kubota, 1957; Gaff, 1977). The liverwort (Doyère, 1842), ‘Has there been a mere Riccia macrocarpa produced new apical slowing down of the vital phenomena, … cells after 23 years of air-dryness (Breuil- or truly an absolute destruction that one Sée, 1993); the moss Grimmia laevigata could compare to death itself?’ grew when rehydrated after 10 years in a The modern consensus on this question herbarium (Keever, 1957); lichens survived seems to be that some organisms can slow 10 years of being desiccated and frozen their metabolism at least to the point at (Larson, 1988); and leaves of ferns and which it cannot be detected against the ﬂowering plants took up neutral red dye or background of physical chemical reactions excluded Evans blue dye after 5 years of and then resume normal metabolism air-dryness (Gaff, 1977). Some desiccation- (Keilin, 1959; Hinton, 1968; Clegg, 2001). tolerant plants clearly survive longer and The most convincing evidence may be that more intense drought than ever occurs some desiccated tardigrades, rotifers, where they grow, raising the question of seeds, spores, algae, lichens and mosses what has selected for such tolerance. 01 Desiccation -Chap 1 18/3/02 1:53 pm Page 8

8 P. Alpert and M.J. Oliver

Dessicated plants were also shown to Porembski and co-workers (e.g. Porembski tolerate other extreme stresses. Various taxa and Barthlott, 2000) are probably the clos- survived extreme cold (Becquerel, 1951; est approaches to surveys for whole plants. Pence, 2000), and some mosses survived The overall pattern one sees is taxonomic heating to 100°C (Glime and Carr, 1974; and geographic breadth contrasted with Norr, 1974). Eickmeier (1986) found that ecological narrowness. more desiccation-tolerant populations of Selaginella were also more heat-tolerant. The fungus Schizophyllum commune pro- 1.4. The Occurrence of Desiccation duced hyphae after 34 years in a vacuum of Tolerance in Plants: Rarity and Ubiquity less than 0.01 mm Hg (Bisby, 1945), showing long-term tolerance of both desiccation Part of the puzzle of desiccation tolerance in and lack of oxygen. Takács et al. (1999) cor- plants is that it is both very uncommon and related desiccation and UV-B tolerance in a nearly universal (Alpert, 2000). The relative set of bryophyte species. biomass of desiccation-tolerant plants in all The correlation between tolerance of des- but the most arid or frigid habitats is very iccation and tolerance of cold, heat and low, and fewer than one in a thousand anoxia has suggested that there may be species of ﬂowering plants is known to toler- some basic properties or mechanisms that ate desiccation. At the same time, desicca- confer ‘broad-spectrum’ tolerance. Since tion-tolerant species are found on all freezing often dehydrates cells, cold and continents, in all major plant groups except desiccation stress have an obvious func- gymnosperms, and among species of all tional link. Another parallel between desic- growth forms except trees; and the great cation and cold tolerance is that both can be majority of ﬂowering plants and also gym- ‘softened’ by periods of low stress and nosperms have desiccation-tolerant seeds or ‘hardened’ by ones of moderate stress. pollen or both. Desiccation tolerance appears Plants may lose some of their desiccation to be a universal evolutionary potential of tolerance after prolonged periods of full plant cells that has been little selected for hydration (e.g. Gaff, 1977; Schonbeck and except in resting stages of the life cycle and Bewley, 1981; Kappen and Valladares, in organisms that have not evolved effective 1999). Desiccation tolerance can vary sea- ways of avoiding desiccation. sonally (Dilks and Proctor, 1976; Gaff, 1980) Detailed reviews of the occurrence of and increase in winter (Kappen, 1964). desiccation tolerance in seeds, pollen and However, the correlation between tolerance other spores, and vegetative tissues are of desiccation and other stresses is not given in Chapters 5, 6, 7 and 8. Other recent absolute. Wood and Gaff (1989) saw no cor- reviews of the occurrence of tolerance in relation between desiccation and salinity adult plants and non-plant non-animals tolerance in species of the grass Sporobolus. include those of Kappen and Valladares As records of desiccation tolerance (1999), Alpert (2000), and Porembski and accumulated, pictures emerged of the taxo- Barthlott (2000). In this section, some of the nomic and geographic ranges of desicca- main points in these reviews will be dis- tion tolerance in plants. These pictures cussed, a few of the examples they give will remain somewhat haphazard because there be mentioned and some additional exam- have been few systematic surveys for desic- ples and points will be presented. cation tolerance within taxa or habitats. Relatively extensive lists exist for seeds (Chapter 8). A survey of all the soil algae at 1.4.1. Seeds, pollen and spores one site was published by Evans (1959). The lists of tolerant vascular plants from As in adult plants, the desiccation toler- different regions published by Gaff and co- ance of seeds can vary greatly between workers (e.g. Gaff, 1977, 1986; Gaff and species within genera, between individuals Latz, 1978) and from rock outcrops by within species and between tissues within 01 Desiccation -Chap 1 18/3/02 1:53 pm Page 9

Drying Without Dying 9

individuals; and there is a continuum of 1.4.2. Vegetative tissues degree of tolerance across species (Chapters 5 and 8). However, whereas des- Desiccation tolerance appears common iccation tolerance is rare in adult flowering though not universal in bryophytes (e.g. plants, it is so much the rule in their seeds Richardson, 1981; Proctor, 1990), common that tolerant seeds are traditionally known in lichens (Kappen and Valladares, 1999), as ‘orthodox’ and desiccation-sensitive uncommon in pteridophytes and rare in seeds as ‘recalcitrant’. Desiccation sensitiv- angiosperms (Chapter 7). No gymnosperms ity may be a derived character in seeds, are known to tolerate desiccation (Gaff, evolved through neoteny, and is probably 1980; Chapter 7), even though gym- associated with large seeds and trees nosperms may have desiccation-tolerant (Chapter 8). Another difference between seeds or pollen (Chapters 5 and 6). desiccation tolerance in adult plants and Desiccation tolerance occurs in non-lich- seeds is that tolerance and desiccation are enized fungi, cyanobacteria and algae (Ried, environmentally induced in adults but may 1960; Mazur, 1968; Bertsch, 1970; be developmentally programmed in seeds. Schonbeck and Norton, 1978; Potts, 1994, Seeds become tolerant as part of develop- 1999; Dodds et al., 1995) but little is known ment and dry because the parent withholds about its extent. It must be very common in or withdraws water from them. Once they free-living algae and bacteria that grow on germinate, the seedlings of desiccation-sen- the surface of plants or soil, where they are sitive species with desiccation-tolerant very probably subject to desiccation. seeds lose their tolerance within hours. Different vegetative parts of a plant The obvious ecological advantage of ortho- may have different degrees of tolerance. doxy is that seeds can survive periods of There seem to be two main patterns. First, drought and disperse the offspring of a in some species only the perennating plant more widely in space and time, structures survive desiccation, such as although orthodoxy is not a prerequisite for corms in Limosella grandiflora (Gaff and dormancy (Chapter 5). Two advantages of Giess, 1986) or special dry-season organs being recalcitrant are that seeds need never in the small shrub Satureja gilliesii stop growing and may germinate more (Montenegro et al., 1979). As in plants rapidly – as in whole plants, there may be that are desiccation-sensitive but have a trade-off between desiccation tolerance desiccation-tolerant seeds, tolerance in and productivity in seeds. these species is confined to relatively Desiccation tolerance is probably also inactive plant parts. Second, leaves may the rule rather than the exception in pollen be more desiccation-tolerant when and spores, and tolerance and desiccation younger. Younger leaves are more tolerant are developmentally programmed in spores than older ones in Chamaegigas intre- as in seeds (Chapter 6). However, there are pidus (Gaff and Giess, 1986) and some at least three differences between tolerance species of Borya (Gaff, 1989). In the leaves in seeds and in spores. Tolerant pollen has of some grasses, only the basal meristem- no dormancy, it survives no more than a atic zone tolerates drying (Gaff and few months of dry storage at room tempera- Sutaryono, 1991). This suggests that some ture, and spores of some pteridophytes can tissues may lose tolerance as they differ- survive cycles of drying and wetting. entiate or age; the processes involved Desiccation-sensitive pollen is relatively could conceivably parallel those that common in species of Poaceae, cause loss of tolerance after germination Cucurbitaceae and Araceae (Chapter 6), of seeds. In all these examples of differen- and may be associated with hot, humid tial tolerance in leaves, there are con- habitats. The prevalence of desiccation tol- geners whose leaves remain tolerant as erance in seeds and spores is one reason to they mature, offering inviting systems for believe that the genetic potential to tolerate comparative studies of the ecology and desiccation exists in all plants. mechanisms of desiccation tolerance. 01 Desiccation -Chap 1 18/3/02 1:53 pm Page 10

10 P. Alpert and M.J. Oliver

No one appears to have assessed the rel- and Valladares, 1999; Porembski and ative prevalence of desiccation tolerance in Barthlott, 2000). Desiccation-tolerant mono- different taxa of bacteria, cyanobacteria, cotyledons outnumber tolerant dicotyle- fungi and algae. Acinetobacter radioresis- dons. The monocotyledonous family tans survives 150 days at 31% relative Velloziaceae may have over 200 tolerant humidity, which helps make it a persistent species (Kubitzki, 1998). At least 39 species source of infection in hospitals (Jawad et of Poaceae tolerate desiccation (Gaff, 1997). al., 1998). At least 400 species of algae and One very small family of angiosperms, the cyanobacteria tolerate desiccation (e.g. Myrothamnaceae, is entirely desiccation- Davis, 1972; Potts, 1994, 1999; Trainor and tolerant (Porembski and Barthlott, 2000). At Gladych, 1995). Evans (1959) found that the other extreme, some species, such as many but not all of the freshwater algae in Borya nitida (Liliaceae), contain both toler- pond mud survived desiccation in the ant and sensitive individuals (Gaff, 1981). ﬁeld; at least two species survived 69 days Phylogenetic analysis suggests that desicca- of desiccation in the laboratory without tion tolerance in active phases of the life forming resting stages. Two interesting phe- cycle has evolved at least eight separate nomena that have been reported from some times in vascular plants (Oliver et al., 2000). green algae but apparently not from other Desiccation-tolerant angiosperms are groups are dependence of tolerance on also widely but unevenly geographically nutrient availability (McLean, 1967, cited distributed. They occur on all continents in Chandler and Bartels, 1999) and loss of except Antarctica, but very few species are capacity to reproduce after desiccation known from Europe or North America. The (Hsu and Hsu, 1998). We know of few European species are all in two genera from reports of desiccation tolerance in non- one family (Ramondia and Haberlea in the lichenized fungi (Bisby, 1945; Gesneriaceae) (Muller et al., 1997; Drazic Zimmermann and Butin, 1973), but there is et al., 1999). The North American species an extensive literature on tolerance in include three grasses (Iturriaga et al., 2000). lichens, at least 50 species of which have The greatest concentrations of known desic- been shown to tolerate desiccation cation-tolerant angiosperms are in southern (Kappen and Valledares, 1999). Africa, western Australia and eastern South Desiccation tolerance is broadly but America (Figs 1.1 and 1.2; Gaff, 1977, 1987; unevenly distributed among taxa in plants. Gaff and Latz, 1978; Porembski and Most of the 25,000–30,000 species of Barthlott, 2000). Different taxa predominate bryophytes probably tolerate at least brief in each of these three areas. desiccation of low intensity (Chapter 7); Desiccation-tolerant plants have a wide the proportion of desiccation-tolerant range of morphological and physiological species appears to differ between orders of characteristics (Porembski and Barthlott, mosses and to be higher in mosses than in 2000). There are desiccation-tolerant annuals liverworts. There are also desiccation-toler- and perennials, graminoids and forbs, and ant hornworts (Oliver et al., 2000). herbs, shrubs and arborescent rosette plants. Porembski and Barthlott (2000) estimated Tolerant species may be caespitose, stolonif- that there are 275–325 desiccation-tolerant erous or rhizomatous. Some species are xero- species of vascular plants. At least nine fam- morphic, such as B. nitida (Gaff and ilies of pteridophytes and seven families of Churchill, 1976); others are not, such as Boea angiosperms contain desiccation-tolerant hygroscopica (Gaff, 1981). A few desiccation- sporophytes (Chapter 7). Some fern gameto- tolerant species, like C. intrepidus, have mor- phytes also tolerate desiccation (e.g. Pence, phological features typical of aquatic plants 2000). Groups of ferns and allies that seem (Gaff and Giess, 1986), and at least one to be relatively rich in desiccation-tolerant species is succulent (Barthlott and species include the family Pteridaceae and Porembski, 1996). Desiccation-tolerant the genera Cheilanthes and Selaginella angiosperms can have crassulacean acid (Gaff, 1977; Gaff and Latz, 1978; Kappen metabolism (Barthlott and Porembski, 1996; 01 Desiccation -Chap 1 18/3/02 1:53 pm Page 11

Drying Without Dying 11 Xerophyta , (c) Xerophyta viscosa Xerophyta , (b) Craterostigma wilmsii Craterostigma (d) (b) . Each is shown in its desiccated (left) and hydrated (right) state. (Photos by J. M. Farrant.) (right) state. (Photos by in its desiccated (left) and hydrated is shown . Each Myrothamnus ﬂabellifolius Southern Africa is a centre of diversity for desiccation-tolerant angiosperms, including (a) for desiccation-tolerant Africa is a centre of diversity Southern , and (d) (c) (a) Fig. 1.1. Fig. retinervis 01 Desiccation -Chap 1 18/3/02 1:53 pm Page 12

12 P. Alpert and M.J. Oliver

(a)

(b) (c)

(d) (e)

Fig. 1.2. The large, isolated, granitic or gneissic outcrops known as inselbergs are a major habitat for desiccation- tolerant vascular plants in Australia, Brazil and Africa. (a) An inselberg in the Mata Atlantica of Brazil; (b) a mat of the pteridophyte Selaginella sellowii on a Brazilian inselberg; (c) an arborescent Brazilian monocot (Velloziaceae); (d) a species of Borya (Boryaceae, shown desiccated) in Australia; (e) Afrotrilepis pilosa (Cyperaceae, shown desiccated), a dominant, mat-forming species on inselbergs in West Africa. (Photos by S. Porembski.)

Markovska et al., 1997) and probably C4 pho- The wide distribution of desiccation tol- tosynthesis (Lazarides, 1992). However, no erance in plants has suggested to some plants more than 3 m tall and hence no trees authors that the basic mechanism of toler- are known to tolerate desiccation, possibly ance must be simple (Chandler and Bartels, because they cannot re-establish upward 1999). According to the ‘water replacement movement of water once the xylem cavitates hypothesis’ of Crowe et al. (1998a), the during desiccation (e.g. Sherwin et al., 1998). evolution of desiccation tolerance in all 01 Desiccation -Chap 1 18/3/02 1:53 pm Page 13

Drying Without Dying 13

organisms depends on the selection and habitats where desiccation-sensitive plants synthesis of sufﬁcient concentrations of do not live (Fig. 1.3). In habitats where molecular substitutes for water (Clegg, water availability and temperature are 2001). Under certain circumstances, tre- moderate and sensitive plants are abun- halose may even induce desiccation toler- dant, desiccation-tolerant vascular plants ance in human cells (Guo et al., 2000). grow mostly on outcrops of bare rock However, tolerance in plants also involves (Porembski and Barthlott, 2000). In the other mechanisms (Section 1.6), and the driest and coldest habitats, especially ecology of desiccation-tolerant plants sug- where dew and fog are major water sources, gests that the evolution of tolerance in desiccation-tolerant bryophytes, lichens, plants is constrained by its consequences algae or cyanobacteria may form the only for growth and competition. vegetation (e.g. Thompson and Iltis, 1968; Friedmann and Galun, 1974; Davey, 1997). Despite the ability of some of these species 1.5. The Ecology of Desiccation to tolerate a drought that is longer and Tolerance in Plants: a Diversity of Cycles more intense than occurs in these habitats, in Marginal Habitats the most xeric microsites are often still bare (e.g. Alpert, 1985). On rocks and soil Desiccation-tolerant plants grow mainly in in the desert, small differences in exposure the interstices and on the margins of the to the sun may determine whether a patch world’s vegetation, in microhabitats and of soil or stone is colonized or not.

(a) (b)

Fig. 1.3. (a) Exposed surfaces of granitic boulders in the western foothills of the Cuyamaca Mountains in southern California are colonized mainly by an assemblage of desiccation-tolerant lichens and bryophytes. (b) Two of the most common mosses are Grimmia laevigata (left) and Grimmia apocarpa (right), shown hydrated. Crevices support desiccation-tolerant pteridophytes such as Pentagramma triangularis (gold-back fern), shown (c) desiccated and (d) hydrated. (Photos by P. Alpert.) 01 Desiccation -Chap 1 18/3/02 1:53 pm Page 14

14 P. Alpert and M.J. Oliver

(a) (b)

(c)

Fig. 1.4. Desiccation-tolerant bryophytes are common even in cool, moist climates. The mosses (a) Orthotrichum anomalum, (b) Anomodon viticulosus, and (c) Tortula latifolia all occur in the UK. Each is shown in its desiccated and its rehydrated state. (Photos by M.C.F. Proctor.) These patterns appear tied to the differ- dry out again in hours. Desiccation-tolerant ent sources of water that different desicca- vascular plants are only known to rewet tion-tolerant plants can use to rehydrate, from rain; they recover in hours to days the rates at which they rewet and dry out, and dry out in days to weeks. The cumula- and their ability to recover after desicca- tive effect of repeated cycles of desiccation tion and achieve a cumulative net gain of on net photosynthesis and growth may resources. Lichens and bryophytes may explain why desiccation-tolerant plants fail rewet from dew, recover in minutes and to survive in the most exposed microsites. 01 Desiccation -Chap 1 18/3/02 1:53 pm Page 15

Drying Without Dying 15

(a)

(b)

(c)

Fig. 1.5. The most intensively studied desiccation-tolerant bryophyte is (a) Tortula ruralis, shown in the fully hydrated state (top), after slow drying (lower right), and after rehydration for 2 min (lower left). T. ruralis is common in dry habitats in North America, as shown on rocks at Mesa Verde National Park, USA (b). One of the most studied desiccation-tolerant angiosperms is the grass Sporobolus stapﬁanus (c), shown after drying in a pot for 14 days (left) and after subsequent immersion in water for 24 h (right). (Photos by M.J. Oliver and B. Mishler.) 01 Desiccation -Chap 1 18/3/02 1:53 pm Page 16

16 P. Alpert and M.J. Oliver

(a) (b)

(c)

Fig. 1.6. Effects of desiccation and rehydration on ultrastructure in leaves of the moss Tortula ruralis. The transmission electron micrographs (after Bewley and Pacey, 1978) show papillose cells in (a) the fully hydrated state (note chloroplast (C) with grana stacks (g), starch grains (s), and plastoglobuli (p, labelled in (b)); a vesicle (V) and rough (RER) and smooth endoplasmic reticulum (SER); a mitochondrion (m) with prominent internal membranes or cristae; and electron-dense bodies (E); and (b) after desiccated plants had been rehydrated for 5 min (note that the chloroplasts are swollen but the nucleus (N) is not). The freeze–fracture micrograph (c) (from Platt et al., 1994) shows a portion of a cell from a slowly dried leaf (note the large, tightly appressed grana stacks (G) in the portion of the chloroplast visible and the mitochondrion (M) outside the chloroplast.) 01 Desiccation -Chap 1 18/3/02 1:53 pm Page 17

Drying Without Dying 17

Trade-offs between tolerance and growth pensation point for photosynthesis and competition with sensitive plants may (Schipperges and Rydin, 1998). In warm explain why desiccation-tolerant plants, and cold deserts, tolerant algae and lichens though they can rise again from ‘apparent grow inside or on the underside of translu- death’ (Doyère, 1842), have not dominated cent rocks (Friedmann and Galun, 1974; the earth. Kappen, 1993; Nienow and Friedmann, 1993). Species of Nostoc, Anacystis and other cyanobacteria form desiccation- 1.5.1. Habitats tolerant crusts on bare walls and rocks from the tropics to the boreal zone (Potts, In contrast to the wide taxonomic and 1994; Lüttge, 1997). Algae, lichens and geographical ranges and the broad mor- bryophytes join cyanobacteria to form phological diversity of desiccation-tolerant crusts on desert soils, which are important vascular plants, their ecological range is in nitrogen cycling (e.g. Nash and Moser, narrowly conﬁned to chronically or sea- 1982; Lange et al., 1994, 1997). Since sonally dry habitats or microhabitats lichens and bryophytes dry so rapidly, they where desiccation-sensitive plants are may be active mostly when conditions are sparse or absent. Porembski and Barthlott effectively mesic and function as ‘shade (2000) estimated that 90% of desiccation- plants’, even in exposed, xeric habitats tolerant vascular plants are associated (Green and Lange, 1994; Proctor, 2000). with rock outcrops, mainly in tropical to Degree of desiccation tolerance seems to lower temperate latitudes. Some species explain some of the relative ability of toler- grow on exposed rock surfaces, while oth- ant species to occupy xeric microsites or ers are associated with crevices (Nobel, habitats (e.g. Hernandez-Garcia et al., 1999; 1978; Gildner and Larson, 1992). Franks and Bergstrom, 2000). For instance, Ephemeral pools on rock outcrops in ability to tolerate desiccation (Mitchell et Africa harbour a set of aquatic, desicca- al., 1999), to maintain photosynthesis dur- tion-tolerant vascular plants (e.g. Volk, ing desiccation (Robinson et al., 2000) and 1984; Gaff and Giess, 1986). Tolerant to recover photosynthesis after repeated angiosperms and pteridophytes also grow cycles of desiccation (Davey, 1997) are in semiarid or desert grasslands, especially associated with occurrence of bryophytes (Eickmeier, 1983; Gaff, 1987; Kappen and in relatively dry sites in Antarctica. The Valladares, 1999) though not invariably ability to tolerate prolonged desiccation (Gaff and Sutaryono, 1991), on shallow and to recover quickly upon rehydration soils. There are exceptions to this narrow- appeared necessary but not sufﬁcient to ness of ecological range. A few tolerant allow mosses to colonize highly insolated vascular species, such as Boeah hygro- surfaces on boulders in chaparral in scopica (Gaff, 1981) and Pentagramma California (Alpert, 1985; Alpert and triangularis (P. Alpert, personal obser- Oechel, 1987). A species of Selaginella vation), occur in forest understoreys. from dry habitats recovered net photosyn- Desiccation-tolerant bryophytes, lichens thesis faster than one from moister habitats and algae occupy a much wider ecological (Eickmeier, 1980). Shirazi et al. (1996) range than do tolerant vascular plants, reported differences in desiccation toler- including both less and more arid sites ance between populations of lichens from (Fig. 1.4). For example, tolerant bryophytes different habitats. and lichens are common on rocks, trunks and soil in moderately moist forests. They may be common in tundra, although the 1.5.2. Cycles Sphagnum species characteristic of tundra do not necessarily recover net photosyn- Desiccation-tolerant plants vary greatly in thesis after losing more than about 10% the rates at which they dry out, rehydrate of the water content they hold at the com- and recover upon rehydration and there- 01 Desiccation -Chap 1 18/3/02 1:53 pm Page 18

18 P. Alpert and M.J. Oliver

fore in the rhythms of desiccation and Xerophyta scabrida began to respire within growth they experience in nature (Tuba et 20 min after rehydration, reached full rates al., 1998; Kappen and Valladares, 1999; of respiration within 6 h, began to synthe- Chapter 7). In general, bryophytes and size chlorophyll after 12 h and did not lichens dry out in hours in the sun, complete synthesis until 36 h (Tuba et al., whereas ferns and angiosperms take a day 1994). Poikilochlorophylly appears to be a or more. Minimum times to net photosyn- programmed rather than a pathological thesis after rehydration range from minutes response to desiccation. For instance, it is a in lichens and mosses rewetted with liquid necessary component of tolerance in the water, to hours in lichens and mosses rehy- leaves of some species: when these leaves drated with water vapour and in some vas- are detached before drying, they stay green cular plants rewetted with liquid, to days as they dry but they die (Gaff, 1981). in other vascular plants (Fig. 1.5; Lange, Natural cycles of wetting and drying 1969; Lange and Kilian, 1985; Gaff and have been followed for a number of Giess, 1986; Reynolds and Bewley, 1993a; mosses and lichens (e.g. Kappen et al., Scott and Oliver, 1994; Scheidegger et al., 1979; Lange et al., 1994; Sancho et al., 1997; Tuba et al., 1998). 1997) but very few desiccation-tolerant Desiccated lichens can resume net pho- vascular plants (Nobel, 1978; Gaff and tosynthesis by taking up water vapour Giess, 1986). During a year in the Negev (Hahn et al., 1993; Schroeter et al., 1994), Desert, thalli of the lichen Ramalina maci- but only if the phycobiont is a green alga formis underwent a cycle of wetting and rather than a cyanobacterium (Kappen and drying almost daily, mostly from dew Valladares, 1999). A few mosses can (Kappen et al., 1979). Some bryophytes in recover at least very slow rates of net pho- semiarid grasslands can likewise experi- tosynthesis by taking up water vapour after ence diurnal desiccation cycles driven by desiccation (Lange, 1969; Rundel and dew during dry seasons (Csintalan et al., Lange, 1980). Both bryophytes and lichens 2000). At the other extreme, some can rehydrate with dew (Lange et al., 1994; angiosperms may undergo a single period Csintalan et al., 2000). Despite the lack of a of desiccation per year, with a cycle of cuticle, differences in thallus, leaf and activity almost like that of an annual plant shoot morphology and packing produce (e.g. Gratani et al., 1998). several-fold differences in drying rates Three factors that determine how cycles between different species of bryophytes of desiccation translate into growth are and lichens (e.g. Gimingham and Smith, light damage, nutrient relations and carbon 1971; Proctor, 1982; Scott, 1982; balance. Photodamage can occur as plants Valladares, 1994); differences in morpho- dry or while they are desiccated, due at logical control of water loss may help least in part to light absorption without explain differences in ability to colonize energy transfer to photosynthesis (e.g. Seel xeric microhabitats. et al., 1992; Gauslaa and Solhaug, 1996). Desiccation-tolerant angiosperms are Desiccation-tolerant plants show a variety known to rehydrate in nature only after of mechanisms likely to reduce photodam- rain. Woody angiosperms may take longer age, including leaf curling, accumulation of to desiccate and rehydrate than herbaceous anthocyanin and carotenoids, and xantho- ones (Sherwin and Farrant, 1996; Farrant et phyll metabolism (e.g. Muslin and al., 1999). The slowest to recover from des- Homann, 1992; Eickmeier et al., 1993; iccation are the poikilochlorophyllous des- Lebkeucher and Eickmeier, 1993; iccation-tolerant plants, monocots that Calatayud et al., 1997; Deltoro et al., 1998; dismantle their photosynthetic machinery Beckett et al., 2000; Farrant, 2000). when they dry and reassemble it again Antarctic mosses, which could be subject when they rehydrate (Sherwin and Farrant, to photodamage during freezing, show 1996; Tuba et al., 1998). In one desiccation reversible photoinhibition and zeaxanthin study, the poikilochlorophyllous species activity (Lovelock et al., 1995). 01 Desiccation -Chap 1 18/3/02 1:53 pm Page 19

Drying Without Dying 19

Little is known about the interaction These factors are most important in des- between desiccation tolerance and nutrient iccation-tolerant plants that tend to have relations. Uptake and metabolism of min- short periods of hydration or frequent eral nutrients must be interrupted from cycles of desiccation, such as lichens and some point during drying to some point mosses in arid habitats, and are probably during rehydration and recovery. In one reason why these species grow so mosses, leakage of solutes during rehydra- slowly (Stark, 1997; Kappen and tion could also reduce net nutrient uptake. Valladares, 1999; Badacsonyi et al., 2000). Increased frequency of desiccation cycles Since brief periods of hydration can result decreases potassium content but not cumu- in net carbon loss (Lange et al., 1994; lative phosphorus uptake in Tortula ruralis Csintalan et al., 2000), it is possible that (Badacsonyi et al., 2000). Activity of nitrate some desiccation-tolerant plants may have reductase decreases rapidly during desicca- been selected for traits that help prevent tion in T. ruralis (Mahan et al., 1998; rewetting by small amounts of water. Water Badacsonyi et al., 2000); activity can repellence in epilithic lichens (Bertsch, recover in less than 8 h after rehydration if 1966) and hair points on some epilithic the moss has dried slowly, but may take mosses (P. Alpert, unpublished data) could 24 h after rapid drying (Mahan et al., be examples. 1998), and may decrease during the ﬁrst hour of rehydration (Marschall, 1998). However, Bates (1997) found that weekly, 1.5.3. Hypotheses 24 h desiccation did not decrease uptake of N, P or K in two mosses compared to The rarity of desiccation-tolerant vascular uptake during continuous hydration, and plants in habitats where other vascular Badacsonyi et al. (2000) saw no difference plants are abundant suggests that surviving between the effect of low water potential desiccation may have negative as well as on nitrate reductase activity in desiccation- positive effects on survival overall. One tolerant and sensitive mosses. hypothesis is that there is a trade-off Cycles of desiccation tend to reduce net between tolerance and growth, and that tol- carbon gain by favouring respiration over erant plants are out-competed by sensitive photosynthesis and by decreasing the ones where the latter can survive, because amount of time that plants are active. the latter grow faster and larger. This Desiccation increases the ratio of respira- should cause selection against tolerance in tion to photosynthesis because: (i) photo- habitats where plants can acquire and con- synthesis ceases before respiration during serve enough water to avoid desiccation. drying and resumes after respiration during An alternative possibility is that tolerance rehydration; (ii) respiration in some species is merely lost in plants that are not increases above normal levels during recov- exposed to desiccation, due to lack of ery from desiccation; and (iii) plants tend to selection pressure to maintain tolerance. stay hydrated at night when they cannot Does desiccation tolerance entail a photosynthesize but do respire, and to des- reduction in growth rate or maximum iccate most rapidly when light levels are size? There are a number of reasons to high (e.g. Alpert, 1979; Proctor, 1982; Lange suppose this, but little direct evidence. et al., 1994; Tuba et al., 1998). Tuba et al. Kappen and Valladares (1999) proposed (1999) examined the hypothesis that an that some morphological features that pro-

increase in atmospheric CO2 might improve mote tolerance also conﬂict with produc- carbon balance during cycles of desicca- tivity. In angiosperms, hairs or scales that

tion. Elevated CO2 does prolong photosyn- reduce water loss and can thus prolong thesis during drying in X. scabrida, but the periods of net photosynthesis also inhibit authors concluded that this aspect of global rehydration (Kappen and Valladares, change was unlikely to favour desiccation- 1999). In lichens and bryophytes, having tolerant over sensitive species. high maximum water content tends to 01 Desiccation -Chap 1 18/3/02 1:53 pm Page 20

20 P. Alpert and M.J. Oliver

prolong hydration but inhibits photosyn- tats? This hypothesis has been partially thesis, since much of the water is typi- tested in bryophytes and lichens (e.g. cally held externally or in upper layers of Alpert, 1990; Pintado et al., 1997; Williams the lichen thallus and so slows gas diffu- and Flanagan, 1998; Kappen and sion (Green and Lange, 1994; Valladares, Valladares, 1999). For example, during a 1994; Lange et al., 1996; Tuba et al., morning after nocturnal rain or dew, 1996a; but see Sojo et al., 1997). bryophytes and lichens growing on sloped Populations of Ramalina capitata in dry, surfaces in north temperate latitudes tend bright sites tend to have greater capacity to dry more rapidly if they are on surfaces to store water and slower gas diffusion that face south or east than if they are on than populations in more shaded sites surfaces that face north or west (Kappen et (Pintado et al., 1997), suggesting that al., 1980; Alpert and Oechel, 1985). Those selection favours water storage more when on north- and west-facing surfaces are light is less limiting. Proctor (2000) pro- more likely to recoup the respiratory losses posed that bryophytes have been selected incurred during the night before desicca- for rapid desiccation to minimize time tion arrests photosynthesis in the morning. spent at intermediate water contents, This probably at least partly explains why which most dispose plants to damage. mosses are ‘more common on the north side Rapid desiccation would also reduce time of the tree’. There appear to be no studies available for photosynthesis and growth. on the effect of microsite on carbon balance Cellular mechanisms of tolerance such as in desiccation-tolerant vascular plants. sugar and protein synthesis (Section 1.6) Oliver et al. (2000) hypothesized that seem likely to impose metabolic costs and desiccation tolerance was once the major- thus reduce growth. If cavitation during ity solution to the problem of living in dry desiccation precludes desiccation-tolerant air. They suggested that tolerance is a prim- plants from exceeding 3 m in height itive characteristic in green plants that (Sherwin and Farrant, 1998), then they allowed them to colonize the land. Once will be overtopped wherever trees can plants evolved vascular tissues and effi- grow. Some comparative studies on cient internal water transport, they lost mosses (Bates, 1997; Arscott et al., 2000) their tolerance of desiccation except in and anecdotal reports on grasses (Gaff, parts that had to be cut off from water 1989) have found that more productive transport – their spores, seeds and pollen. species are less desiccation-tolerant. The Tolerance in adult plants then re-evolved long-standing hypothesis (Grime, 1979) several times in different lineages. that stress tolerance conflicts with pro- Porembski and Barthlott (2000) proposed ductivity is intuitively appealing but that this re-evolution occurred as mechanically elusive. Further compara- angiosperms colonized the bare rock out- tive studies on desiccation-tolerant plants crops where their desiccation-tolerant could help reveal mechanisms that dictate species are now most diverse. If this sce- trade-offs between tolerance and growth. nario is correct, then desiccation tolerance The absence of desiccation-tolerant in plants has evolved not just as a way of plants in some highly xeric habitats where surviving in marginal habitats, but as a way no other plants occur suggests that surviv- of colonizing frontiers, first from water on ing desiccation may not assure survival, to land and then from soil on to stone. even where competition is not a factor. One hypothesis to explain why tolerant plants are not more abundant in barren habitats is 1.6. Mechanisms of Desiccation that the plants cannot maintain a cumula- Tolerance tive positive carbon balance under certain regimes of water availability (Ried, 1960). Until the mid-1970s, it was generally Does carbon balance limit the survival of believed that the mechanisms of desicca- desiccation-tolerant plants in xeric habi- tion tolerance in plants were mechanical 01 Desiccation -Chap 1 18/3/02 1:53 pm Page 21

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(see reviews by Bewley, 1979; Oliver and 1.6.1. Damage Bewley, 1984). Structural features such as flexible cell walls, small vacuoles and Before discussing what is known of the lack of plasmodesmata were suggested as cellular protection and repair mecha- key elements in tolerance (Gaff, 1980; nisms of desiccation tolerance, it is worth Bewley and Krochko, 1982; Oliver and reviewing the effects of desiccation and Bewley, 1984). In a landmark paper, rehydration on cellular integrity in desic- Bewley (1979) articulated the alternative cation-tolerant plants. The critical ques- view that desiccation tolerance is primar- tion, when deciding what type of ily protoplasmic in nature. This theory mechanisms desiccation-tolerant plants argues that certain plants and plant tis- employ, is when might damage occur? Is sues achieve desiccation tolerance as a it during the drying process or upon rehy- result of the inherent properties of their dration? For instance, if damage does not cellular contents (protoplasm). Most evi- actually occur during desiccation, then dence now supports this view, though there is good reason to believe that pro- structural features are clearly important tective mechanisms are in place. If dam- in desiccation tolerance in some cases age occurs upon rehydration, and the cell (Sherwin and Farrant, 1996; Farrant et subsequently recovers, repair mecha- al., 1999). nisms are probably operative. In addition, Bewley (1979) further defined three the amount of damage and the rate at critical features of desiccation tolerance which cells return to a normal status based on the observation that many desic- measure the effectiveness of protective cation-tolerant plants exhibit cellular and repair processes and the overall level changes, some of which can be described of desiccation tolerance. as extensive damage, during and following desiccation. The plant or tissue must: 1.6.1.1. Damage during desiccation (i) limit damage to a repairable level; (ii) maintain its physiological integrity in the The timing of damage is still controversial, dried state (perhaps for extended periods but a consensus is building that little dam- of time); and (iii) mobilize mechanisms age occurs during drying in desiccation- upon rehydration that repair damage suf- tolerant tissues. Much of the work in this fered during desiccation and rehydration. area has focused on the plasma membrane. These criteria laid the experimental foun- All desiccation-tolerant tissues leak dation for the field from the 1980s solutes during rehydration (Simon, 1978; onwards and continue to influence the Bewley, 1979; Bewley and Krochko, 1982), way we think about how plants survive indicating that the cell membrane has been desiccation. In particular, it is now compromised. Early electron microscopy widely accepted that the cells of desicca- of seeds (Webster and Leopold, 1977; tion-tolerant plants employ mechanisms Morrison-Baird et al., 1979) and bryophyte that protect them from the rigours of tissues (reviewed by Oliver and Bewley, extensive water loss and also mecha- 1984) suggested that membranes in dried nisms, at least in the case of vegetative plant cells were completely disorganized. cells, that repair damage suffered during With the advent of more sophisticated desiccation or rehydration (Bewley and technologies, these observations were Oliver, 1992). This introductory overview determined to be artefacts of sample of the mechanisms of desiccation toler- preparation and chemical fixation (Bewley, ance will therefore concentrate on cellu- 1979; Thompson, 1979; Bewley and lar features (the so-called ‘inherent Krochko, 1982; Oliver and Bewley, 1984). properties’ of desiccation-tolerant cells) The use of non-aqueous fixatives elimi- that have been suggested to play a major nated some of these artefacts but the heavy role in protection and repair. Details are use of chemical treatments still made covered in subsequent chapters. interpretation difficult (Thompson, 1979; 01 Desiccation -Chap 1 18/3/02 1:53 pm Page 22

22 P. Alpert and M.J. Oliver

Öpik 1980, 1985; Tiwari et al., 1990; 1.6.1.2. Damage during rehydration Smith, 1991). Freeze–fracture electron microscopy, however, has yielded the most As noted above, all plant tissues leak reliable data. Dried tissues are eminently solutes when rehydrated following a dry- suited for freeze–fracture preparation ing event. In desiccation-tolerant tissues, because their low water content virtually however, this is a transient event (Simon, eliminates the formation of ice crystals, 1978; Bewley, 1979; Bewley and Krochko, which make high-quality replicas difficult 1982). Several hypotheses have been to obtain. Freeze–fracture studies clearly offered to explain imbibitional (or rehydra- demonstrated that the membranes of seeds tive) leakage (Simon, 1974; Senaratna and (Thompson and Platt-Aloia, 1982; Bliss et McKersie, 1983a,b; Crowe et al., 1989, al., 1984) and pollen (Platt-Aloia et al., 1992; Hoekstra et al., 1992). The prevailing 1986) could retain normal bilayer organi- hypothesis is that imbibitional leakage is zation and dispersal patterns of intramem- the result of lipid-phase transitions occur- branous particles at water contents as low ring in the plasma membrane as a result of 1 dehydration and rehydration (Crowe et al., as 0.08 g H2O g dry mass. The plasma and organelle membranes of vegetative 1992). During drying, membranes pass cells of the desiccation-tolerant pterido- from the liquid crystalline to the gel phase, phyte Selaginella lepidophylla and the and they return to the liquid crystalline moss Tortula ruralis also retain normal phase during rehydration. In artificial organization and dispersal patterns in the membranes, this transition can lead to a dried state (Platt et al., 1994; Fig. 1.6). transient leakage event (Hammoudah et al., The effects of desiccation on cellular 1981), and, since phase transitions have components that cannot be observed by been demonstrated in drying and rehydrat- freeze–fracture microscopy are more diffi- ing desiccation-tolerant cells (Crowe et al., cult to evaluate, largely due to the likeli- 1989; Hoekstra et al., 1992), it has been hood of partial rehydration and the generally accepted that phase transition is production of artefacts during chemical the basis of imbibitional leakage in most fixation. In seeds, the uncertainty is com- desiccation-tolerant tissues. In seeds, how- pounded by the fact that the tissues ever, it is thought that membrane-phase are part of a developing system. changes do not occur because of the pres- Nevertheless, observations tend to sug- ence of a seed coat, which impedes the gest that desiccation of tolerant plants passage of water to the dried cells. generates an ordered ‘collapse’ of the Hoekstra et al. (1999) suggested that the cellular milieu that results in little ultra- slow rate of penetration of water may set structural damage (Oliver and Bewley, up a ‘pre-hydration’ state where the mem- 1984; Gaff, 1989; Goldsworthy and branes are in a liquid crystalline state Drennan, 1991; Sherwin and Farrant, before liquid water surrounds the rehydrat- 1996; Farrant et al., 1999). If desiccation- ing cells. Since leakage does occur during tolerant plants successfully avoid damage the rehydration of these tissues (Hoekstra during the dehydration process, as it et al., 1992; Tetteroo et al., 1996), it has appears they do, is there any consequence been concluded that leakage must occur at all of desiccation in these plants? The through an intact lipid bilayer, as suggested answer appears to be yes. All desiccation- by Senaratna and McKersie (1983b). tolerant plants and plant tissues show Recently, a new hypothesis has emerged signs of cellular damage when the dried from some exciting new studies on dehy- tissue is rehydrated. It is, however, debat- drating and rehydrating pollen (Hoekstra et able whether or not the damage occurs al., 1997, 1999; Golovina et al., 1998; during the drying process (but is not Buitink et al., 2000; Chapter 10). This body of observable at an ultrastructural level) or work using amphiphilic spin probes demon- as the result of the inrush of water into strates that during dehydration endogenous the cells during rehydration. amphiphilic substances partition from the 01 Desiccation -Chap 1 18/3/02 1:53 pm Page 23

Drying Without Dying 23

aqueous cytoplasm into pollen membranes. Within minutes after rehydration, the Using data obtained from liposome-based ex- chloroplasts of the green gametophytic tis- periments, Golovina et al. (1998) suggested sues of desiccation-tolerant bryophytes that it is the presence of these amphiphiles appear swollen and globular. Their outer in the membrane that causes imbibitional membranes are folded and separated from leakage and that as the pollen rehydrates the the thylakoids, which are no longer com- amphiphilic substances move out of the pacted (Oliver and Bewley, 1984). The membranes and leakage stops. This hypo- extent of thylakoid disruption increases thesis could explain how transient leakage with the rate of prior desiccation. The can occur through an intact membrane. In a chloroplasts of desiccation-tolerant more recent study, Buitink et al. (2000) angiosperms tend to be more resistant to demonstrated that the movement of disruption than those of bryophytes, amphiphilic compounds into membranes although vesicularization within the also occurs in imbibing radicles of peas and chloroplast internal membranes is common cucumbers. This study, using electron para- (Gaff and Hallam, 1974; Gaff et al., 1976; magnetic resonance (EPR) spectroscopy and Sherwin and Farrant, 1996). In all desicca- inserted nitroxide spin probes, demon- tion-tolerant plants, mitochondria swell strated a difference in partitioning behav- and exhibit disruption of the cristae iour between desiccation-tolerant and (reviewed by Bewley and Krochko, 1982). sensitive tissues. Spin probes partitioned Swelling and disruption of mitochondria into the membranes at higher water content are not affected by rate of desiccation. In in desiccation-sensitive tissues than in toler- all cases, organelles regain normal struc- ant tissues. These authors suggest, from in ture within 24 h. vitro portioning experiments, that it is the microviscosity of the cytoplasm that con- 1.6.1.3. Poikilochlorophylly trols portioning of amphiphilic compounds into the plasma membrane. What remains to At least eight genera of desiccation-tolerant be determined is the role of the native monocots are ‘poikilochlorophyllous’, i.e. amphiphilic compounds in membrane dam- they reversibly lose their chlorophyll and age and, if they are important in desiccation dismantle their chloroplasts during desic- tolerance, the role they play in the long-term cation (Gaff, 1989; Tuba et al., 1998). The stability of membranes in the dried state. thylakoid system within desiccated chloro- Golovina et al. (1998) speculated that plasts is completely replaced by small amphiphiles may have antioxidant proper- groups of plastoglobuli and by osmophilic, ties that protect membranes from damage stretched lipid material, which appears to by free radicals generated during desicca- occupy the positions previously occupied tion and rehydration. If so, imbibitional by the thylakoids (Hallam and Luff, 1980; leakage may be a necessary trade-off for Tuba et al., 1993a,b; Sherwin and Farrant, protection. Much work will be required 1996). After 10–12 h rehydration, when before the importance of native full turgor and maximum leaf water con- amphiphilic compounds in desiccation tol- tent are reached, synthesis of chlorophylls erance can be determined, but amphiphiles and carotenoids and the reassembly of thy- are an intriguing new development in our lakoids begin. Early in reassembly, sets of understanding of desiccation tolerance. two primary thylakoids stack to form Rehydration-induced damage other than grana. Within 72 h the chloroplasts appear leakage is difﬁcult to distinguish from nor- normal and full photosynthetic capacity is mal development in seeds and pollen but restored (Tuba et al., 1993b, 1994). From is clearly evident in the tissues of most these studies and later physiological in- desiccation-tolerant vegetative tissues, vestigations (Tuba et al., 1997), it appears especially in organelles (reviewed by Bewley that these changes can be classiﬁed as and Krochko, 1982; Oliver and Bewley, genetically programmed responses to des- 1984; Gaff, 1989; Oliver and Wood, 1997). iccation rather than damage. 01 Desiccation -Chap 1 18/3/02 1:53 pm Page 24

24 P. Alpert and M.J. Oliver

1.6.2. Protection reach a maximum about three days before the seed begins to desiccate (Galau and Much of what we know of the cellular pro- Hughes, 1987; Galau et al., 1987). The tection mechanisms involved in desiccation other class contains 12 transcripts, which tolerance in plants comes from studies of appear late in maturation and achieve max- orthodox seeds (Bewley and Black, 1994; imum expression just before and during Chapter 5) and, to a slightly lesser extent, desiccation. LEA proteins make up 30% of pollen (Crowe et al., 1992; Hoekstra et al., the non-storage protein and 2% of the total 1992). The ability of seeds to withstand soluble protein in the mature cotton desiccation is acquired during their devel- embryos and are uniformly localized opment. This acquisition is usually sub- throughout the cytoplasm (Roberts et al., stantially earlier than the culmination of 1993). LEA proteins and the acquisition of the drying event itself, which is the termi- desiccation tolerance during seed matura- nal event in orthodox seed maturation. tion have been linked in other dicots (e.g. Seeds of some species can withstand pre- soybean: Blackman et al., 1995) and in mature desiccation well before the mid- monocots (e.g. maize: Mao et al., 1995; point of their development (Bewley and Wolkers et al., 1998). Black, 1994; Chapter 5). Among the meta- A set of LEA proteins arises in develop- bolic changes that take place just prior to or ing barley and maize embryos at the time during drying is the synthesis of proteins that tolerance of desiccation is acquired. A and sugars, which have long been postu- small subset of these proteins is induced lated to form the basis of a series of overlap- when barley embryos at the intolerant stage ping protective mechanisms that limit are cultured in abscisic acid (ABA) (Bartels damage to cellular constituents (Bewley, et al., 1988; Bochicchio et al., 1991), and a 1979; Leprince et al., 1993; Oliver and causal relationship between ABA and lea Bewley, 1997). These two components have gene expression has been suggested. since been widely implicated as being criti- Evidence for, and against, this relationship cal for desiccation tolerance in all plant exists in the literature. In cotton embryos, cells including vegetative cells (Ingram and high expression of the first class of lea Bartels, 1996; Oliver and Bewley, 1997; genes occurs as ABA content increases. Scott, 2000). Over the years it has also High expression of the second set of lea become clear that the synthesis of antioxi- genes, however, occurs at the start of, and dants and enzymes involved in oxidative during, maturation drying, when the metabolism also play a critical role in cellu- endogenous ABA content is low. There are lar protection and desiccation tolerance explanations for this lack of correlation, (Chapter 10). However, this aspect of pro- e.g. there is an early-regulated, ABA-con- tection will not be addressed here. trolled mechanism, which operates only later when drying commences. On the other hand, an ABA-independent pathway 1.6.2.1. Proteins may be involved in the synthesis of the Only one subset of proteins that accumu- second group of LEA proteins. late at the time of the acquisition of desic- LEA proteins have been identified in the cation tolerance has been extensively vegetative tissues of all desiccation-tolerant investigated, the late embryogenesis abun- plants studied so far (Ingram and Bartels, dant (LEA) proteins, first described in cot- 1996; Oliver and Bewley, 1997; Blomstedt ton (Galau and Hughes, 1987; Galau et al., et al., 1998) and proteins related to some of 1987, 1991; Chapter 5). The genes that the LEA proteins, e.g. dehydrins (see encode LEA proteins in developing cotton- below), have been associated with the seeds are comprised of two distinct classes response of non-tolerant plants to water whose regulation is coordinated. One class stress (Skriver and Mundy, 1990; Bray, contains six different lea transcripts, which 1997). In nearly all instances, the induction appear relatively early in development and of LEA protein synthesis in vegetative tis- 01 Desiccation -Chap 1 18/3/02 1:53 pm Page 25

Drying Without Dying 25

sues can be elicited by exogenous ABA LEA protein synthesis is also highly application (Ingram and Bartels, 1996; induced in the vegetative tissues of desic- Campalans et al., 1999). cation-tolerant angiosperms during drying LEA proteins fall into five groups by (Bartels et al., 1993; Blomstedt et al., 1998; virtue of sequence similarities (Dure et al., Bartels, 1999). Callus derived from vegeta- 1989; Ingram and Bartels, 1996; Cuming, tive tissue of the desiccation-tolerant plant 1999). All are highly hydrophilic and all are Craterostigma plantagineum is not inher- very stable, as evidenced by their resistance ently tolerant but can be made so by the to the denaturing effects of boiling (with the application of ABA (Bartels et al., 1990). exception of Group 5 LEA proteins). Group The application of ABA to this tissue 1 LEA proteins are characterized by a 20- results in the synthesis of novel proteins, amino acid motif and are represented by the some of which are LEA proteins including wheat Em protein, the first LEA protein the Group 2 LEA proteins, the dehydrins identified (Cuming and Lane, 1979). Group (Bartels et al., 1993). The desiccation-toler- 2 LEA proteins are characterized by a 15- ant moss T. ruralis utilizes a more primi- amino acid motif, the K-segment, a stretch tive mechanism of desiccation tolerance of serine residues and a conserved motif (Oliver et al., 2000), which involves a con- near the N-terminus of the protein (Close, stitutive cellular protection strategy, and in 1997). This group of proteins is also called this plant, unlike others, dehydrins are not the dehydrins and these are the most wide- induced by dehydration or by ABA but are spread and most studied of the LEA pro- constitutively expressed (Bewley et al., teins. Group 3 LEA proteins share a 1993). Dessication-sensitive species characteristic 11-amino acid repeat motif exposed to sub-lethal dehydration stress (Dure et al., 1989), which is predicted to also respond by synthesizing LEA proteins form an amphipathic -helix. These amphi- and LEA-like proteins, in particular dehy- pathic helices are postulated to form intra- drins (Close, 1997). These examples and and intermolecular interactions that may many more all point to the importance of have important consequences for their func- LEA proteins in dehydration responses and tion (Baker et al., 1988; Dure, 1993a). The desiccation tolerance. least studied of the LEA proteins are those in The most convincing pieces of evidence Groups 4 and 5, which are somewhat to suggest that LEA proteins have an atypical (Dure, 1993b; Galau et al., 1993). important role in cellular protection come Group 5 LEA proteins are more hydrophobic from transgenic studies using a barley than other LEA proteins and are not resistant Group 3 lea gene, HVA1. This gene, when to high temperature. Most of the LEA protein expressed in a constitutive fashion in groups have been identified in many differ- transgenic rice, increased its tolerance to ent plants. All groups are thought to play a water and salt stress (Xu et al., 1996). role in desiccation tolerance, and the evi- HVA1 overexpression in wheat, driven by a dence for this viewpoint is growing. maize ubiquitin promoter, resulted in The evidence for the involvement of transgenic lines that performed in a supe- LEA proteins in desiccation tolerance is rior fashion under soil-water deficits circumstantial but compelling. LEA protein (Sivamani et al., 2000). synthesis in seeds, as mentioned above, is There are a variety of suggested mecha- associated with both the acquisition of des- nisms by which LEA proteins might pro- iccation tolerance and the final stage of tect cellular components. Many LEA seed maturation just prior to desiccation. proteins have extensive regions of random In addition, ABA-deficient (aba) and ABA- coiling, which has been postulated to pro- insensitive (abi3) double-mutants of mote the binding of water, helping to main- Arabidopsis seeds do not dry on the parent tain a minimum water requirement (Ingram plant, do not tolerate desiccation and lack and Bartels, 1996). For instance, the Em several LEA proteins (Koorneef et al., 1989; protein of wheat is considerably more Meurs et al., 1992). hydrated than most common proteins, and 01 Desiccation -Chap 1 18/3/02 1:53 pm Page 26

26 P. Alpert and M.J. Oliver

over 70% of the Em protein is configured in accumulation during desiccation. as random coils (McCubbin et al., 1985). Constitutive expression of HSPs is unusual Baker et al. (1988) suggested that the ran- in vegetative tissues and resembles the dom coil nature of some LEA proteins may expression pattern of these proteins in allow them to conform to the shape of cel- seeds. In addition, exogenous ABA lular constituents and thus, by virtue of induced both the expression of HSPs and their hydroxyl groups, help to maintain the acquisition of desiccation tolerance in their solvation state when water is C. plantagineum callus tissues (Alamillo et removed. These authors also suggested that al., 1995). Finally, a LEA-like HSP, HSP-12, the Group 2 LEA proteins (dehydrins), by from yeast was shown to be capable of pro- virtue of their amphipathic helical repeats, tecting liposomal membranes from the provide surfaces when bundled together damaging effects of desiccation in a way that would sequester ions. This may be similar to that seen with the sugar tre- crucial as the increasing ionic strength dur- halose (Sales et al., 2000). Thus it appears ing drying could cause irreversible damage that small HSPs may also play a role in cel- to cellular proteins and structural compo- lular protection during desiccation: per- nents. Recently, Velten and Oliver (2001) haps this capability is related to their described an LEA-like protein from T. chaperonin-like activities, which may help ruralis that contains 15 15-amino-acid maintain protein structure under denatur- repeats predicted to form amphipathic ing conditions. Other proteins whose tran- helices. This protein appears to be synthe- scripts accumulate during the dehydration sized during the rehydration event and phases of vegetative desiccation-tolerant may serve to trap valuable ions that would angiosperms have been identified but little otherwise be lost. Studies using individual has been done to confirm their roles in des- LEA proteins in in vitro assays also add to iccation tolerance (Kuang et al., 1995; the possible mechanisms by which these Ingram and Bartels, 1996; Blomstedt et al., proteins exert protection of cellular compo- 1998; Bockel et al., 1998; Neale et al., nents. Wolkers (1998) suggested from data 2000). See Chapters 5 and 11 for further obtained from the study of a pollen Group discussion of all these proteins. 3 LEA protein and its effect on sucrose glass formation that LEA proteins may act 1.6.2.2. Sugars as anchors in a structural network that stabilizes cytoplasmic components during The accumulation of soluble sugars is also drying and in the dried state. strongly correlated to the acquisition of At this point it seems likely that each desiccation tolerance in plants and other individual group of LEA proteins may have organisms (for reviews see Crowe et al., different, complementary effects. Most des- 1992; Leprince et al., 1993; Vertucci and iccation-tolerant tissues contain a represen- Farrant, 1995; Chapters 5 and 10). Soluble tative of most, if not all, of the different sugars, especially sucrose, accumulate in groups of LEA proteins, and it is also likely seeds (Leprince et al., 1993), pollen that all are needed to achieve the highest (Hoekstra et al., 1992) and in desiccation- degree of desiccation tolerance. tolerant vegetative tissues (Bewley and There is mounting evidence that another Krochko, 1982; Ingram and Bartels, 1996; class of proteins, the small heat-shock pro- Oliver and Bewley, 1997). In Craterostigma teins (HSPs), may play a role in cellular plantagineum, 2-octulose stored in the protection during desiccation. Small HSPs hydrated leaves is converted to sucrose accumulate in maturing seeds of many during drying to such an extent that in the plant species (Vierling, 1991; Wehmeyer et dried state it comprises about 40% of the al., 1996) prior to desiccation. Alamillo et dry weight (Bianchi et al., 1991). al. (1995) reported that small HSPs are Sucrose is the only free sugar available expressed constitutively in the vegetative for cellular protection in desiccation-toler- tissues of C. plantagineum and increased ant mosses, including Tortula ruraliformis 01 Desiccation -Chap 1 18/3/02 1:53 pm Page 27

Drying Without Dying 27

and T. ruralis (Bewley et al., 1978; Cytoplasmic glass formation has also Smirnoff, 1992). The amount of this sugar been postulated to maintain the structural in gametophytic cells of T. ruralis is and functional integrity of macromolecules approximately 10% of dry mass, which is (Sun and Leopold, 1997; Crowe et al., sufficient to offer membrane protection 1998b), which has been well demonstrated during drying, at least in vitro (Strauss and with in vitro models (Roos, 1995). Hauser, 1986). Moreover, neither drying Intracellular glasses, by virtue of their high nor rehydration in the dark or light results viscosity, drastically reduce molecular in a change in sucrose concentration, sug- movement and impede diffusion of reac- gesting that it is important for cells to tive compounds in the cell. It is by this maintain sufficient amounts of this sugar property that glasses are thought to prolong (Bewley et al., 1978). The lack of an the longevity of desiccated tissues by slow- increase in soluble sugars during drying ing down degradative processes during appears to be a common feature of desicca- storage. Buitink et al. (1998) recently tion-tolerant mosses (Smirnoff, 1992). demonstrated a strong relationship It is thought that sugars protect the cells between molecular mobility and storage during desiccation by two mechanisms. longevity in both pollen and pea seeds. First, the hydroxyl groups of sugars may Thus, although glass formation may not be substitute for water to maintain important in the initial acquisition of des- hydrophilic interactions in membranes and iccation tolerance, it may be crucial for sur- proteins during dehydration (Crowe et al., vival of the dried state (as suggested by 1992). This has so far only been demon- Buitink, 2000; Chapter 10). strated in vivo, using liposomes and iso- Other carbohydrates besides sucrose lated proteins (Crowe et al., 1992). accumulate in desiccation-tolerant tissues, Secondly, sugars are a major contributing the principal ones being the oligosaccha- factor to vitrification, the formation of a rides stachyose and raffinose (Horbowicz biological glass, of the cytoplasm of dry and Obendorf, 1994), and have been postu- cells (Leopold et al., 1994; Chapter 10). lated to play a part in desiccation toler- This mechanism has been the subject of ance. The presence of these compounds intense research over the last 15 years. has also been correlated with seed Vertucci and Leopold (1986) suggested longevity (Hoekstra et al., 1994; that desiccation tolerance in seeds had to Horbowicz and Obendorf, 1994), which be associated with some feature or solute has linked them to a possible role in the combination that would avoid crystalliza- stabilization of intracellular glasses tion of the cytoplasm as dehydration pro- (Leopold et al., 1994; Bernal-Lugo and gressed. Burke (1986) proposed that high Leopold, 1995; Sun, 1997). However, concentrations of sugars lead to vitrification Buitink et al. (2000) demonstrated that the of the cytoplasm during desiccation and reduction in oligosaccharides in primed

thus prevent crystallization. Glass forma- seeds did not alter Tg (the glass-to-liquid tion has since been demonstrated in seeds transition temperature) or viscosity and (Williams and Leopold, 1989; Leopold et thus they contended that oligosaccharides al., 1994; Leprince and Walters-Vertucci, do not affect the stability of intracellular 1995), pollen (Buitink et al., 1996) and in glasses. These results support the earlier leaf tissues of C. plantagineum (Wolkers et studies of Black et al. (1999), which had al., 1998). Walters (1998) went as far as to shown a lack of a temporal correlation say that glass formation is an intrinsic prop- between the induction of desiccation toler- erty of any complex system that can survive ance by a mild dehydration treatment and desiccation. However, glass formation may the appearance of rafﬁnose in wheat not be sufﬁcient to confer desiccation toler- embryos. These studies cast doubt on the ance since desiccation-sensitive tissues are role of oligosaccharides in the acquisition capable of forming cytoplasmic glasses of tolerance and the maintenance of viabil- (Sun et al., 1994; Buitink et al., 1996). ity in the dried state. 01 Desiccation -Chap 1 18/3/02 1:53 pm Page 28

28 P. Alpert and M.J. Oliver

1.6.3. Repair In the desiccation-tolerant fern Polypodium virginianum rehydrating tis- The repair processes associated with des- sues do accumulate novel transcripts but iccation tolerance have been difficult to their identities have not been investigated detail and characterize. In seeds, repair (Reynolds and Bewley, 1993b). Recent mechanisms are difficult to separate from work with the desiccation-tolerant grass events that are associated with germina- Sporobolus stapfianus has identified two tion and early seedling growth, but evi- transcripts that accumulate during the dence for repair does exist. In vegetative early phases of dehydration but also accu- angiosperms, the major emphasis appears mulate during rehydration. The first of to be on effective cellular protection and these is a transcript coding for a plant much of the research in angiosperms has Rab2, a small GTP-binding protein that in focused on this component. The most other systems is an important protein in promising models for investigating a direct the targeting of membrane vesicles in role for cellular repair in desiccation toler- vesicular trafficking pathways and a path- ance appear to be the highly desiccation- way directly involved in membrane con- tolerant bryophytes. struction (O’Mahony and Oliver, 1999a). In seeds, most of the evidence for cellu- The second transcript encoded a polyubi- lar repair derives from investigations into quitin, a protein involved in protein the causal relationship between cellular turnover (O’Mahony and Oliver, 1999b). In damage and loss of viability during storage C. plantagineum very few transcripts were (Bewley and Black, 1994). Consequently, identified as being specific for the rehydra- one has to keep in mind that the repair tion process. Those that were appeared to processes that have been identified may be involved in the metabolism of sugars not play a major role in desiccation toler- that is required to re-establish the pools of ance per se but rather in the ability to sur- octulose required for the generation of vive long term in the dried state. There are sucrose during dehydration (Bernacchia et two reports, however, that indicate the al., 1996). repair of cellular components, proteins and Cellular repair, as a component of desic- DNA, which may directly affect desicca- cation tolerance mechanisms, is more easily tion tolerance as well as storage longevity. defined in desiccation-tolerant bryophytes. Mudgett et al. (1997) demonstrated that Desiccation-tolerant bryophytes are thought proteins containing abnormal L-isoaspartyl to employ a mechanism for desiccation tol- residues could be repaired in aged barley erance that represents the most primitive seeds by the activity of the enzyme L-isoas- form expressed in land plants (Oliver et al., partyl methyltransferase. These authors 2000). Unlike the acquisition of desiccation argue that this type of repair is particularly tolerance in seeds, which may be develop- important during dehydration where pro- mentally programmed, and in desiccation- tein turnover rates are slow. Boubriak et al. tolerant angiosperms, which is (1997) demonstrated that one of the earliest environmentally induced by drying, desic- activities seen in imbibing cereal grains is cation tolerance in most bryophytes the repair of damage to genomic DNA appears to be constitutive (Oliver and incurred whilst the seeds were dry and in Bewley, 1997). The difference in mecha- storage. If the repair processes were nisms of tolerance in these systems is blocked during imbibition then DNA reflected in their biology. Desiccation-toler- degradation became severe. If universal, ant angiosperms have morphological and DNA repair would certainly qualify as a physiological adaptations in place that can key process in the mechanism of desicca- retard the loss of water. The mechanism of tion tolerance in seeds (Chapter 12). desiccation tolerance that has evolved in The identification of repair processes in these plants takes advantage of these adap- vegetative tissues of desiccation-tolerant tations by being inducible. As the rate of tracheophytes has received little attention. water loss is relatively slow, there is time 01 Desiccation -Chap 1 18/3/02 1:53 pm Page 29

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to establish the protective measures bolism when rehydrated. During the first 2 required, and the plant can thus survive a h following rehydration of dried T. ruralis, drying event. If water loss is too rapid, the synthesis of 25 proteins (termed these plants succumb to the damaging hydrins) is terminated or substantially effect of water loss and die (Gaff, 1989; decreased, and the synthesis of 74 proteins Bewley and Oliver, 1992; Oliver and (termed rehydrins) is initiated or substan- Bewley, 1997). Bryophytes, on the other tially increased (Oliver, 1991). Controls hand, have little in the way of adaptations over changes in synthesis of these two to retain water within the plant and, as a groups of proteins are not mechanistically result, the internal water content of these linked. It takes a certain amount of prior plants rapidly equilibrates to the water water loss to fully activate the synthesis of potential of the environment (Proctor et al., rehydrins upon rehydration. Perhaps this 1998). A consequence of this is that many is a strategy that has evolved to link the bryophytes experience drying rates that are amount of energy expended in repair to extreme and therefore have insufficient the amount of damage potentiated by dif- time to induce and set in place protective fering degrees of drying. measures. Thus, it appears that bryophytes In T. ruralis, there also appears the have evolved a constitutive mechanism for capability of preparing the cell for a rapid desiccation tolerance, one that has protec- recovery if drying rates are sufficiently tive measures that are always in place. This slow (4–6 h). Using cDNA clones corre- conclusion is supported by the observa- sponding to T. ruralis transcripts that are tions that both sucrose and LEA proteins preferentially translated during rehydra- are maintained at constant levels in desic- tion (Scott and Oliver, 1994), it was deter- cation-tolerant bryophytes during drying mined that several ‘recovery’ transcripts and rehydration (see above). The constitu- accumulate during slow drying (Oliver tive protection mechanism appears to be and Wood, 1997; Wood and Oliver, 1999). particularly effective in preventing damage Recent studies clearly demonstrate that to the photosynthetic apparatus, as evi- these transcripts are sequestered in the denced by the very rapid recovery of pho- dried gametophytes in messenger ribo- tosystem II activity (Tuba et al., 1996b; nucleoprotein (mRNP) particles (Wood Csintalan et al., 1999; Proctor and and Oliver, 1999). Of 18 rehydrin cDNAs Smirnoff, 2001). isolated (Scott and Oliver, 1994) and How does this relate to cellular repair sequenced (Oliver et al., 1997; Wood et and the uniqueness of bryophytes for al., 1999) in T. ruralis, only three exhibit studying this aspect of desiccation toler- significant sequence homology to known ance? It appears that the level of protec- genes in the Genbank databases. Tr155 has tion that bryophytes are capable of a strong sequence similarity to an alkyl maintaining is not sufficient to completely hydroperoxidase linked to seed dormancy prevent damage, especially to membranes, in barley (Aalen et al., 1994) and during rehydration. To achieve desicca- Arabidopsis embryos (Haslekas et al., tion tolerance, bryophytes thus rely heav- 1998), and in rehydrated but dormant ily on repair mechanisms induced during Bromus secalinas seeds (Goldmark et al., the initial phases of hydration following 1992). Tr213 exhibits a high degree of sim- rewetting (Oliver and Bewley, 1997; ilarity to polyubiquitins from several Oliver et al., 1998). plant sources, suggesting that protein Most work on repair in mosses has cen- turnover may be an important part of tred on the proteins whose synthesis is repair in mosses as well as in induced immediately upon rehydration of angiosperms. Tr288 encodes an LEA-like desiccated gametophytic tissue. Early protein (see above), which suggests that work (see Bewley, 1979, for review) estab- LEA proteins may have a protective role lished the ability of T. ruralis and other during rehydration and a role in cellular mosses to rapidly recover synthetic meta- repair (Velten and Oliver, 2001). 01 Desiccation -Chap 1 18/3/02 1:53 pm Page 30

30 P. Alpert and M.J. Oliver

1.7. Future Prospects and Agricultural tolerance and thus the genetic information Significance necessary for expanding their drought tolerance may not be exploitable or indeed Our present agricultural system is almost present. In contrast, more may be gained by totally dependent upon the ability of ortho- understanding how stress-tolerant plants or dox seeds to tolerate desiccation. The iden- plant structures accomplish tolerance, and tification and functional analysis of genes from such sources genes that contribute involved in the developmentally pro- directly to tolerance can be identified. As grammed desiccation tolerance stage of pointed out by Bartels and Nelson (1994), seeds is a vital step in understanding this the limiting factor for the improvement of complex trait. The knowledge gained from abiotic stress tolerance in crops is ‘the such studies will impact on many diverse availability of structural genes and regula- areas of agricultural concerns such as tory elements which positively contribute germplasm preservation, seed production to stress tolerance improvement’. If our and seedling establishment. Apart from efforts to utilize our understanding of the genetic considerations, the ongoing studies underlying mechanisms of desiccation tol- on the role of biological glasses in desicca- erance are to bear fruit, we must discover tion tolerance and the determination of and identify the genes that are central to which proteins and sugars are important in this trait. their stability will affect our ability to pre- Cushman and Bohnert (2000) describe a serve viable germplasm for longer periods strategy for cataloguing genes central to of time, preserving genetic diversity for particular traits in their discussion on future breeding needs. This goal will also genomic approaches to plant stress. The be benefited by our continued progress in initial phase of gene discovery is the large- understanding of how desiccation-tolerant scale sequencing of randomly selected tissues deal with oxidative stress. cDNA clones, termed Expressed Sequence Over 35% of the world’s land surface is Tags (ESTs), which are synthesized from considered to be arid or semiarid, experi- mRNA pools representing a specific devel- encing precipitation that is inadequate for opmental stage or response state. EST col- most agricultural uses. Ramanathan (1988) lections that will enhance gene discovery has argued, based on predictions of global in desiccation tolerance have been started, environmental changes, that developing the most extensive of which is that for crops that are more tolerant to water developing seeds of Arabidopsis thaliana deficits while maintaining productivity (Girke et al., 2000). Smaller EST collec- will become a critical requirement in the tions have been made for C. plantagineum early part of this century. Understanding leaves that had been dried for an hour or how plant cells tolerate water loss is a vital fully dried (Bockel et al., 1998) and S. stap- prerequisite for developing strategies that fianus leaves during dehydration can influence agricultural and horticultural (Blomstedt et al., 1998; Neale et al., 2000). crop productivity and survival under these Wood et al. (1999) have established a lim- conditions of decreasing water availability. ited sample of ESTs (152) from a cDNA Much has been accomplished in the dis- library developed from the mRNP fraction covery and characterization of those genes of slowly-dried T. ruralis gametophytes. In that are expressed during the response of the EST collections from vegetative desic- crop and model plants to water deficit or cation-tolerant plant tissues, many of the salt stress. From this work, our knowledge sequenced clones were of unknown iden- of stress tolerance has improved tity (71% for Tortula) and/or not previ- immensely and some success has been ously associated with water stress. achieved, but these traits are very complex Cushman and Bohnert (2000) suggested and breeding progress has been slow. This that this may indicate, as suggested above, approach is also restricted in that most that these plants may possess ‘unique gene crops have a limited capacity for drought complements or regulatory processes that 01 Desiccation -Chap 1 18/3/02 1:53 pm Page 31

Drying Without Dying 31

contribute to desiccation stress’ and, by The next step in the search for insight inference, novel genes that may prove use- into gene function and regulatory controls ful in breeding for drought stress tolerance. in desiccation tolerance will come from the The cataloguing of gene products that are use of expression profiling with cDNA expressed during the acquisition and estab- microarrays. Such work is under way but lishment of desiccation tolerance is only the little has been reported as yet. The first first step. The ultimate goal is to determine information is likely to come from the which genes are central to desiccation toler- analysis of Arabidopsis EST microarrays in ance and what functions they perform. The extensions of the studies reported by Girke major approach used to gain a functional et al. (2000), which may pinpoint tran- understanding of individual gene products scripts to the exact time of the acquisition has been the overexpression of genes in of desiccation tolerance in developing transgenic plants. We have already men- seeds. Direct approaches to elucidate func- tioned the studies of Xu et al. (1996) and tionality of individual genes or gene fami- Sivamani et al. (2000) concerning the over- lies will be slower to develop. The vast expression of HVA1, a Group 3 lea gene, and array of genetic tools, such as transgenic their success in improving drought toler- capability, mutant generation and screen- ance in rice and wheat. Two other groups ing tools, T-DNA and transposon-tagged have attempted to modify sugar metabolism knockouts, and map-based cloning tech- to improve tolerance by engineering tre- nologies, will make Arabidopsis and seed halose 6-phosphate synthetase genes from desiccation tolerance the initial foci of non-plant sources, from yeast (Holmström et functional studies. Nevertheless, tools are al., 1996) and from bacteria (Pilon-Smits et becoming available for vegetative desicca- al., 1998) into tobacco. The aim was to pro- tion-tolerant model plants and these will mote trehalose accumulation in leaf cells, play an important role in evaluating target and both groups were successful and genes. An example of this has been the achieved greater tolerance to water deficits exciting use of activation tagging, by trans- in tobacco. The exciting conclusion from genic random insertion of a highly active these studies, and those with the Group 3 foreign promoter to ‘activate’ native genes, LEA protein, is that the engineering of a sin- by Furini et al. (1997) to isolate a gene gle gene can achieve results that affect such (cDT-1) involved in regulation of the a complex trait as drought tolerance. It will response of C. plantagineum callus to des- be interesting to see how these results will iccation. Advances in the fields of molecu- translate into an advancement in our under- lar biology, genetics, genomics and standing of how these gene products func- biophysics have put us on the threshold of tion to achieve greater tolerance and if these a new era in our quest to understand one of plants will have an impact on drought- the most complex and important traits in tolerance breeding efforts. plant biology: desiccation tolerance.

1.8. References

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Part II

Methodology 02 Dessication - Chap 2 18/3/02 1:53 pm Page 46 02 Dessication - Chap 2 18/3/02 1:53 pm Page 47

2 Methods for the Study of Water Relations Under Desiccation Stress

Wendell Q. Sun Department of Biological Sciences, National University of Singapore, Kent Ridge Crescent, Singapore 119260

2.1. Introduction 48 2.2. Expression of Water Status 48 2.2.1. Mass-based measures for tissue hydration 48 2.2.1.1. Water content 48 2.2.1.2. Relative water content 49 2.2.2. Thermodynamic measures for tissue hydration 50 2.2.2.1. Water activity 50 2.2.2.2. Chemical potential of water and water potential 51 2.3. Measurement of Tissue Water Potential 53 2.3.1. Psychrometric and hydrometric methods 53 2.3.2. Osmometric or cryoscopic method 54 2.3.3. Isothermal equilibrium method 55 2.4. Water Relations – the Thermodynamic Approach 55 2.4.1. The Höﬂer diagram and pressure–volume curve 55 2.4.1.1. Change of cell turgor pressure during desiccation 55 2.4.1.2. Change of osmotic potential during desiccation 57 2.4.1.3. The volume of water in symplast, apoplast and intercellular spaces 57 2.4.1.4. Volumetric elasticity of the cell wall 59 2.4.2. Analysis of water sorption isotherms 60 2.4.2.1. Theoretical models 60 2.4.2.2. Temperature dependency of water sorption 62 2.4.2.3. Monolayer hydration and water-clustering function 65 2.4.2.4. Occupancy of water-binding sites 66 2.5. Measurement of Drying Rate and Desiccation Stress 68 2.5.1. Driving force for water loss and expression of drying rate 68 2.5.2. Quantiﬁcation of desiccation stress 68 2.6. Water Relations – the Kinetic and Functional Approach 70 2.6.1. General considerations 72

48 W.Q. Sun

2.6.1.1. Time scale 72 2.6.1.2. Structural complexity and dynamics of molecular ordering 72 2.6.1.3. The model-dependent interpretation: the pitfalls 73 2.6.2. Biophysical techniques 74 2.6.2.1. Differential scanning calorimetry 74 2.6.2.2. Thermally stimulated current (TSC) method 75 2.6.2.3. Nuclear magnetic resonance (NMR) 76 2.6.2.4. Electron spin resonance 77 2.7. Concluding Remarks 78 2.8. References 79 Appendix 84

2.1. Introduction of water relations that are directly related to desiccation tolerance of plant tissues will be In hydrated plant cells, water is the main introduced. The strengths and limitations of constituent. The organization of cellular various methods or techniques of measure- structures (both supramolecular assemblies ment of water relations during desiccation and micromolecular structures) and the over- will be discussed. An effort will be made to all biochemistry (the thermodynamics of bio- give a basic understanding of terms and logical processes and their rate parameters) of concepts concerning cellular water status an organism depend on water. Water is a sol- and the expression of dehydration stress. vent and a medium in which diffusion of solutes and biochemical reactions take place in plant cells. It is often a participant and/or 2.2. Expression of Water Status a product of various biochemical reactions. In low-moisture systems such as naturally The most important quantity that has to be dried pollen grains and plant seeds, cellular measured in all studies of desiccation toler- water also plays an important role as a plasti- ance is the degree of dehydration stress. So cizer, influencing the translational or rota- far, there is no agreed parameter of dehydra- tional motions of entire molecules, or tion stress measurement. The change in segments of macromolecules and intramolec- water content of plant tissues and organs is ular motions. Water is involved in virtually often used as an indicator of dehydration. every dynamic process in a living cell. However, insufficient attention has been The loss of water from plant cells is an paid to problems commonly associated with important environmental stress. Changes in the use of water content as an indicator of the aqueous environment influence the dehydration stress. For example, different complex thermodynamics and kinetics of concepts and approaches are currently used structural stability and all aspects of biologi- by research groups working on biological cal functions. The accurate measurement of systems, ranging from bacteria and fungal the status of cellular water is essential for spores to microscopic animals, pollen the study of both desiccation stress in plants grains, large seeds and resurrection plants. and the mechanisms of plant desiccation tolerance. The method of quantification and 2.2.1. Mass-based measures for tissue interpretation must be applicable not just to hydration the narrowly defined desiccation conditions, but also to all other types of physio- 2.2.1.1. Water content logical stresses with a dehydration component, such as freezing and salinity. In Water content on a wet-weight basis (WC, % this chapter, several fundamental principles w.b.) is widely used in the literature of desic- 02 Dessication - Chap 2 18/3/02 1:53 pm Page 49

Methods for Studying Water Relations Under Stress 49

cation studies, and is adopted by the WC (g g1 dw) = WC (% w.b.)/[100 – WC International Seed Testing Association (% w.b.)] (3) (ISTA, 1993) for the expression of seed water content. WC (% w.b.) is the percentage mass 2.2.1.2. Relative water content fraction of water of the total tissue mass: Relative water content (RWC) is another WC (% w.b.) = (fresh weight dry mass-based parameter. RWC is widely used weight)/fresh weight 100 (1) in the pressure–volume analysis of plant WC (% w.b.) is not a linear expression of tissue water stress. RWC is a simple and water content in tissues, because fresh useful measure of the extent to which a tis- weight appears in both the numerator term sue is in water deﬁcit. It is related to tissue

and the denominator term in Equation (1). water content at full turgor (WCF). During a When WC (% w.b.) is used to monitor the dehydration experiment, RWC is calcu- loss of water during desiccation, the lated by dividing water content at a given decrease of WC (% w.b.) does not necessar- time by water content at full turgor, and ily reflect the exact extent of dehydration expressed as a fraction value or as a per- stress. The change of WC (% w.b.) during centage. If water content in the tissue is drying is, in fact, related to the change of determined as WC (g g1 dw), the calcula- the reciprocal of tissue fresh weight. For tion of RWC is straightforward, being example, when the tissue of 80% WC (% WC/WCF. But, if water content is deter- w.b.) is dried to 70% and 60% water con- mined as WC (% w.b.), RWC is calculated tent, the tissue actually loses 41.7% and by the equation: 62.5% of its initial water quantity, respec- RWC = [WC (100 – WC )]/[WC tively, not just 12.5% and 25% reduction as F F (100 – WC)] 100 (4) implied by the values of WC (% w.b.). The quantity of water lost during dehydration RWC is a linear expression of moisture from 80% to 70% WC (% w.b.) is twice as condition. The change in RWC over time much as water loss during dehydration serves as a good indicator for the rate of from 70% to 60% WC (% w.b.). dehydration. Physiological responses of Water content on a dry-weight basis mea- plant water deficit are highly correlated sures the mass ratio between water and the with RWC (Sinclair and Ludlow, 1985). The dry mass in tissues, and is often expressed use of RWC is particularly advantageous for by g water per g dry weight (i.e. g g1 dw): comparative studies, in which initial water content at full turgor or full hydration WC (g g1 dw) = (fresh weight dry varies considerably among different weight)/dry weight (2) species, different tissues of the same WC (g g1 dw) is a linear expression of species or the same tissue at different water content, and the change of WC (g g1 developmental stages, such as seeds. In cer- dw) during drying is proportional to the tain cases, it may be even preferred over loss of water in a tissue. On the mass basis, water potential, because RWC also accounts a tissue with a WC of 0.20 g g1 dw is for the effect of osmotic adjustment in hydrated exactly twice as much as the tis- affecting plant water status. For example, sue with a WC of 0.10 g g1 dw, and four- two plants with the same leaf water poten- fold as much as the sample with a WC of tial can have different RWCs if they differ 0.05 g g1 dw. For this reason, some in their ability for osmotic adjustment. researchers have argued that WC (g g1 dw) In many desiccation studies on higher is a more sensible expression than WC (% plants, water content of a tissue at full tur- w.b.) for the measurement of dehydration. gor was not specifically determined, and At WC < 15%, the difference between WC instead water content after full hydration in (% w.b.) and WC (g g1 dw) is fairly small. water was used. Typically, leaf samples (e.g. WC (% w.b.) is converted to WC (g g1 dw) discs or sections) of higher plant species using the following equation: are taken and weighed immediately. The 02 Dessication - Chap 2 18/3/02 1:53 pm Page 50

50 W.Q. Sun

samples are then hydrated in distilled based parameters for the expression of water for 4–6 h, after which they are dehydration stress has been shown by a weighed again and their water contents are number of studies. The critical onset water determined by drying the samples in an potential of Quercus rubra (Pritchard, 1991) oven. In higher plants, the amount of inter- and Quercus robur (Pritchard and Manger, cellular water is small or non-existent 1998) is about –3 MPa, but the correspond- (Oertli, 1989). However, it should be noted ing mass water contents vary substantially that some plant tissues do contain intercel- due to different seed oil content. Sun and lular water, which is held in spaces Gouk (1999) studied the water relation between cells of a tissue at relatively high responses of three recalcitrant (desiccation- water potential (near zero). Therefore, water sensitive) seeds (Aesculus hippocastanum, content at full turgor has different physio- Andira inermis, Q. rubra) during controlled logical meaning from water content of the dehydration. The critical water potentials tissue at full hydration when the tissue confor seeds are quite similar for all three tains intercellular water. If intercellular species (7 to 8 MPa), but their corre- water is present, water content of the tissue sponding critical water contents are 0.45, at full turgor has to be estimated from the 1.10 and 0.35 g g1 dw for A. hippocas- plot of water potential on water content (g tanum, A. inermis and Q. rubra, respec- g1 dw). To determine the water content of tively. If the critical water content were the tissue at full turgor, two linear regres- used to express the relative desiccation tol- sion lines can be ﬁtted, respectively, with erance, one would conclude incorrectly data points where water potential remains that seeds of A. hippocastanum and Q. almost unchanged during initial water loss rubra are much more desiccation-tolerant and the next few points where water poten- than seeds of A. inermis. Therefore, a mass- tial starts to fall. The intercept of these two based parameter of water loss may not be a regression lines gives the water content at reliable indicator for the degree of desicca- full turgor. Beckett (1997) reported that the tion stress in plant tissues. amount of intercellular water varied greatly among species of bryophytes. If intercellular water exists in a tissue, correction needs 2.2.2. Thermodynamic measures for tissue to be made to the raw RWC readings, which hydration are calculated according to the water content at full hydration. The method used to The response of plant tissues to desiccation correct the raw RWC readings was is related to the thermodynamic and described in detail by Beckett (1997). kinetic status of tissue water, rather than to There are shortcomings in using mass- actual water content. The water status of based parameters for the expression of plant tissues can be expressed in terms of water content. Plant tissues are heteroge- energy status of water molecules, i.e. the neous, complex biological systems, in partial molar Gibbs’ free energy or water which carbohydrates, proteins and lipids potential. This thermodynamic approach is and other components have different hydra- preferred to the mass-based expression of tion properties. As a consequence, when tissue hydration, because thermodynamic plant tissues of various species are equili- parameters (i.e. energy status) are related brated under given conditions of tempera- directly to the numerous biophysical and ture and relative humidity, equilibrium physiological events that contribute to des- water content varies considerably among iccation stresses and desiccation tolerance species. For example, seeds with large lipid of plant tissues. reserves equilibrate to lower water contents than starchy seeds, even though the chemi- 2.2.2.1. Water activity cal potential of water molecules is the same

for all tissues when equilibrium is Water activity (aw) is used to describe achieved. The disadvantage of using mass- water status in the studies of desiccation 02 Dessication - Chap 2 18/3/02 1:53 pm Page 51

Methods for Studying Water Relations Under Stress 51

tolerance and storage survival for spores, In such cases, the measured vapour pres- pollen, seeds and resurrection plants (Ellis sure of water may not be the equilibrium et al., 1990, 1991; Berjak and Pammenter, vapour pressure, but the vapour pressure of 1994; Vertucci et al., 1994, 1995; Walters, a ‘stationary’ state that is time-dependent. 1998a). Water activity is measured as the However, studies in food sciences have ratio of the vapour pressure of water in a suggested that water activities measured system to the vapour pressure of pure are likely to be close to equilibrium and the water at the same temperature. It is related differences should be within the uncer- to the equilibrium relative humidity (RH) tainty associated with the experimental of the air surrounding the system (i.e. RH = determination (Chirife and Buera, 1996). aw 100). Water activity can be viewed as The usefulness of the water activity con- the ‘effective’ water content, which is ther- cept in seed storage stability has been dis- modynamically available to various physi- cussed by Walters (1998b). ological processes in cells. For the survival of organisms under water stress, the ‘effec- 2.2.2.2. Chemical potential of water and tive’ water is more important than the total water potential amount of water present in the tissue. Water activity of fresh plant tissues may The quantity of free energy of a component

vary only between 0.980 and 0.996. Within (µj) in a system is measured by its chemical this narrow range, it is not useful for the potential. The chemical potential of water expression of dehydration stress or tissue (µw) in a system is deﬁned by: water status. However, for the studies of – µ = µ* + RT ln a + V P + z FE + m gh (5) severe water stress and extreme desicca- w w w w w w

tion, water activity has several advantages where µ*w is the chemical potential of pure over water content, including its concep- water at ideal reference conditions. The

tual simplicity, measurability, easy experi- second term RT ln aw is for water activity. R mental manipulation, and its applicability is the gas constant (8.314 103 kJ mol1 to both simple and complex systems. A K1), T is the absolute temperature (K, in number of physiological processes that are kelvin), and a is water activity (RH/100). – w relevant to desiccation tolerance or damage Vw is the partial molal volume of water (i.e. have been shown to occur at speciﬁc water the differential increase or decrease in vol- activities, and some of those are presented ume when a differential amount of water is – in Fig. 2.1. added or removed). Vw is inﬂuenced by the Water activity in plant tissues can be presence of solutes and is also tempera- determined using the hygrometric method ture-dependent (1.805 105 m3 mol1 at and the isothermal equilibrium sorption 20°C). P is the hydrostatic pressure on method. The hygrometric instrument water in excess of atmospheric pressure 3 3 – method directly measures the equilibrium (MPa, 1 MPa = 10 kJ m ). The term VwP RH of plant tissues in a closed chamber. represents the effect of pressure on the With the equilibrium sorption method, chemical potential of water and is samples of plant tissues are equilibrated to expressed in energy per mole (kJ mol1).

a series of known water activities at a spec- zwFE is the electrochemical potential of iﬁed temperature. The relationship water, which equals zero because water is

between water content and water activity uncharged (zw = 0). The last term mwgh is upon equilibrium (i.e. the sorption the gravitational term, representing the isotherm curve) is then used to calculate work needed to move 1 mole of water to a

water activity of plant tissues at different given height. Practically, mwgh will remain water contents. Water activity is deﬁned at constant in most circumstances of desicca- equilibrium. However, plant tissues at low tion studies. and intermediate moisture levels may not The water potential is proportional to be in a true state of equilibrium at all, but the chemical potential of water (µw µ*w) in in an amorphous metastable state instead. a system as described in Equation (5). 02 Dessication - Chap 2 18/3/02 1:53 pm Page 52

52 W.Q. Sun

Therefore, water potential is actually the osmotic potential and h is the gravita- potential energy of water per unit mass. tional potential. The total water potential is While water content tells how much water the sum of hydrostatic, osmotic and gravita- is in a sample, water potential tells you tional components. The gravitational term how available that water is. By convention, ( h) depends on the position of water in a water potential is defined as follows: gravitational field and is not relevant to most desiccation studies. Osmotic potential = + π + (6) P h depends on the concentration of dissolved where p = P and is hydrostatic pressure substances in water. Osmotic potential is on water as defined in Equation (5), π is related to water activity by the equation:

0 species w

–50 in situ Coffea w solution –100 2 Lysozyme activity stops Lysozyme Cell respiration starts to cease

–150 Mean of minimum value for bacterial growth Minimum required for photosynthesis Nucleic acids and proteins fully hydrated

Water potential of water vapour (MPa) Water –200 DNA disordered and damagedDNA at lower a Typical exposure of NostocTypical colonies Desiccation tolerance of embryos Water vapour above saturated NaCl solution Water

–250 Orthodox seeds typically survive at lower a Water vapour aboveWater saturated CaCl

20 40 60 80 100

Relative humidity (%)

Fig. 2.1. Water activities (relative humidities) that limit physiological activities and cell growth. Physical parameters and physiological processes are drawn with data from Wolfe and Leopold (1986), Potts (1994) and Sun and Gouk (1999). The relationship between relative humidity and water potential is calculated according to Equation (7) at 25°C. A similar diagram that is speciﬁc to a plant tissue can be established. Such a diagram would serve as a valuable reference for experimental design and data interpretation, since it gives a clear concept about the possible sequence of potential physiological and biochemical events and their interactions as the tissue loses water. 02 Dessication - Chap 2 18/3/02 1:53 pm Page 53

Methods for Studying Water Relations Under Stress 53

– Vw π = RT ln aw (7) face. This technique, however, is unsuitable for many desiccation-tolerant plant tissues, During dehydration, the water content in e.g. lichens, bryophytes, spores, pollen grain seed tissues is reduced, resulting in an and seeds. This is because the pressure increase in the concentration of solutes and chamber method measures the xylem ten- thus a decrease in water activity and sion, which is broadly equal to the leaf osmotic potential (i.e. becomes more π water potential. Water potential of plant negative). A similar situation occurs during parts that do not have vascular systems can- freezing. The formation of ice leads to the not be measured with the pressure chamber dehydration of the protoplast and the con- method. However, water potential of plant centration of solutes. tissues can be measured by a number of The interactions of water with biological other techniques. These techniques use surfaces and interfaces are of great impor- either the relationship of the sample water tance to desiccation tolerance of plant tis- potential to the equilibrium vapour pressure sues, especially at low moisture levels. The immediately around the sample or the prin- inﬂuence of such interactions on water ciple of the freezing-point depression in the potential in a tissue is commonly called liquid solution. ‘matric’ potential. Rapid water uptake by dry seeds during the early stage of germination is mainly attributed to large matric 2.3.1. Psychrometric and hydrometric potentials. Another example is the reduced methods rate of water loss as the tissue is dried to lower water content. Matric potential Both methods are widely used for the mea- depends on the adsorptive forces that bind surement of tissue water potential. The mea- water to a matrix. The amount of matrix- surement of water potential by a bound water in recalcitrant Q. robur psychrometer and a hydrometer is called embryonic axes is as high as 0.25–0.30 g the wet-bulb depression method and the g1 dw (Pritchard and Manger, 1998). dew-point depression method, respectively. However, the forces of such water–matrix A psychrometer measures water potential of interactions are adequately represented by samples (placed in closed chambers) their contributions to hydrostatic pressure through its ability to determine the RH of (P) and osmotic potential ( ). For exam- π the closed environment. The instrument ple, the presence of aqueous interfaces in uses high-sensitivity thermometers to mea- cells lowers water activity through interfa- sure temperature reduction resulting from cial attractions and binding of water near the heat of vaporization of water in a sample their surfaces, which has already been relative to pure water. It can measure water included in the osmotic component in potential of solid tissue materials and Equation (7). Therefore, matric potential droplets of solutions. The sample is ﬁrst does not represent additional new forces. sealed in a small chamber containing a thermocouple. After an equilibration period, a cooling current is applied to the thermocou- 2.3. Measurement of Tissue Water ple in order to condense water on the ther- Potential mocouple junction. The amount of condensed water is proportional to the A pressure chamber (pressure bomb) is com- water potential of the tissue. The water is monly used to measure directly leaf water allowed to evaporate, causing a change in potential of higher plants. The detached leaf the thermocouple output, and the output is is sealed in a steel chamber with the cut calibrated for water potential, using stan- petiole protruding out. Pressure that is dard salt solutions. On the other hand, a applied to the chamber is taken as the hydrometer maintains the dew-point xylem (leaf water) potential when the sap depression temperature during the measure- meniscus appears at the petiole xylem sur- ment using a thermocouple. The dew-point 02 Dessication - Chap 2 4/4/02 2:18 pm Page 54

54 W.Q. Sun

depression temperature is the temperature osmolal aqueous solution, the osmotic to which the air in the sealed chamber must potential at 0°C is ideally 2.271 MPa, and be reduced so that the air becomes saturated the freezing-point depression is 1.86°C. with water vapour. Psychrometric and (An osmole is the mass of a substance that hydrometric methods can be used to mea- when dissolved in 1 kg water generates an sure both water potential and osmotic osmotic pressure equivalent to that pro- potential of plant tissues. To measure duced by 1 mole of an ideal solute dis- osmotic potential, a sample has to undergo solved in 1 kg water. After dissolving, an the freeze–thaw cycles to disrupt the cellu- ideal solute gives 6.023 1023 osmotically lar structures before the measurement, active particles.) Theoretically, the osmotic whereas the water potential is measured potential of an unknown sample can be using undisrupted tissues. estimated from the depression of its freez- A psychrometer is very sensitive to tem- ing point by the following relationship: perature change because it measures very −2. 271 MPa small temperature differences. A change in Ψ∆∆π = TT=−1. 221 (8) ° water potential of 1.0 MPa is reﬂected by a 186. C change in wet-bulb temperature depression where T is the depression of the freezing of only approximately 0.085°C. A hydro- point. The effect of osmotic potential on meter is less affected by the changes in freezing-point depression also holds for ambient temperature during the measure- non-ideal solutions such as plant saps. ment compared with a psychrometer. However, freezing-point depression is non- Psychrometric and hydrometric methods linear with concentration changes during are suitable only for plant tissues of high dehydration. Water potential (MPa at 0°C) water content. At low water content, the can be derived by the empirical equation equilibrium may take several hours to (Crafts et al., 1949): achieve. The nominal range of the Peltier Ψ = 1.206T + 0.0021T 2 (9) thermocouple measurement is limited from 0 to 6.0 MPa for these two methods. Yet With the osmometric method, a sample many desiccation-tolerant plant tissues can is usually supercooled a few degrees below survive far below 6.0 MPa. Even if one its freezing point to induce immediate uses the Richards thermocouple, it extends crystallization. As the heat of fusion is only to –25 MPa and the accuracy released, the sample temperature rises to decreases to –0.1 MPa at –10 MPa its freezing point, and its equilibrium tem- (Decagon Devices Inc., Pullman, perature is measured. Alternatively, the Washington, USA), corresponding to an RH temperature at which ice crystals start of ~84% at 25°C. melting can be measured and taken as the equilibrium freezing temperature (i.e. Ramsay’s method). The applicability of 2.3.2. Osmometric or cryoscopic method Equations (8) and (9) to the measurement of water potential or osmotic potential in A freezing-point osmometer measures the plant tissues was examined by Sun and osmotic concentration of a biological liquid Gouk (1999), using seed tissues that were using the principle of the freezing-point pre-equilibrated with saturated salt solu- depression. The freezing-point depression tions (from 3 to 35 MPa). The freezing- is one of the four colligative properties of a point depression was determined with a solution. The freezing point is the unique differential scanning calorimeter, using the temperature at which the ice phase and the onset temperature for the exotherm on liquid phase can coexist in equilibrium at cooling. Calculated water potentials were standard pressure. When a solute is dis- found to be very close to the pre-freezing solved in the water, the freezing point of water potentials of seed tissues, with the water is lowered in proportion to the Equation (9) ﬁtting the data slightly better osmotic potential of the solution. For a 1.0 than Equation (8). 02 Dessication - Chap 2 18/3/02 1:53 pm Page 55

Methods for Studying Water Relations Under Stress 55

2.3.3. Isothermal equilibration method cessfully in seed desiccation studies by a number of workers (Pritchard, 1991; Poulsen It is more difficult to measure directly and Eriksen, 1992; Vertucci et al., 1994; Sun water potential of low-moisture systems. et al., 1997; Tompsett and Pritchard, 1998). Perhaps the convenient, yet accurate and reliable method is first to establish the empirical relationship between water con- 2.4. Water Relations – the tent and water potential for a particular Thermodynamic Approach plant tissue. Samples of plant tissues are equilibrated over different salt solutions 2.4.1. The Höfler diagram and the that would maintain a series of constant pressure–volume curve water vapour pressures (i.e. RH) in closed containers. Upon equilibrium, the water Water relations parameters of plant tissues contents of tissue samples are determined can be presented by the Höfler diagram and gravimetrically, and their water potential at the pressure–volume curve (PV curve). The equilibrium is then the same as the water Höfler diagram shows the relationship potential of the air in the closed containers, between water potential and relative water which in turn equals the osmotic potential content (Fig. 2.2a). The PV curve is a plot of the salt solutions used. Therefore, water between the reciprocal of water potential potential of tissue samples is calculated by (1/) and RWC or water loss (1 – RWC) the equation: during desiccation (Fig. 2.2b). Both the Höfler diagram and the PV curve are RT %RH = – ln (10) widely used to characterize water relations V 100 w of plant tissues. To construct a Höfler dia- where R is the gas constant, T is the gram or a PV curve, the changes in water – absolute temperature (kelvin), Vw is the par- potential and RWC are monitored as the tial molal volume of water, and %RH is the tissue is dehydrating. Several important percentage relative humidity inside the parameters can be obtained by analysing containers. (Note that Equation (10) is the components of cell water potential, essentially the same as Equation (7).) The including the osmotic potentials at full tur- empirical relationship between water con- gor and at the partially dehydrated state, tent and water potential can be described the apoplastic and symplastic water vol- by exponential and polynomial (Poulsen ume in tissues, a plot of turgor pressure and Eriksen, 1992) or other functions (Sun (i.e. hydrostatic water potential in Equation and Gouk, 1999). The derived mathematical (6)) as a function of RWC, and the tissue expression is then used to calculate water bulk modulus of elasticity. Without know- potential of plant tissues at any water con- ing these biophysical metrics, it would be tent within the limit of experimental range. impossible to identify different kinds of This method is particularly useful in moni- cellular stresses associated with the loss of toring the change of tissue water potential water in the tissue and to examine the sig- during desiccation. Water potential of dehy- nificance of an array of biochemical and drating tissues can be calculated immedi- physiological responses during desicca- ately from the data of water loss. tion. Moreover, valid comparisons of the Constant RH can be achieved using satu- response of cell function to water stress rated or non-saturated salt solutions, poly- among different organisms cannot be made ethylene glycol solutions and glycerol without such knowledge. solutions. Physico-chemical data of various salts and their solutions are presented in the 2.4.1.1. Change of cell turgor pressure during Appendix. This technique does not need spe- desiccation cial instruments to measure water potential, and can avoid the difficulty in measuring RH In fully turgid cells, turgor pressure is equal accurately. This method has been used suc- to the osmotic potential (with opposite 02 Dessication - Chap 2 18/3/02 1:54 pm Page 56

56 W.Q. Sun

(a) 2 p

–2 External water Water potential (MPa) Water –4

–6 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Relative water content (RWC)

(b)

p )

–1 0 p 0.7 1.0 1.3 (MPa

Incipient RWC plasmolysis –1/ ( p = 0) –1/ Reciprocal of water potential

0.0

–0.2 0.0 0.2 0.4 0.6 0.8 1.0 1 – RWC

Fig. 2.2. Cellular water relations. (a) Höﬂer diagram showing the components of cell water potential. Intercellular or external water (RWC > 1.0) in many plant tissues is held at near-zero water potential and, during the initial dehydration, cell water potential ( ) and turgor pressure ( p) do not change signiﬁcantly (the horizontal dashed line). Maximum osmotic potential is found at the point of full turgor (RWC = 1.0), where p = π. As the plant tissue loses water, turgor pressure decreases, and at the turgor-loss point (RWC = ~0.8), = π (the vertical dashed line). At RWC < ~0.8, the relationship between RWC and π follows a rectangular hyperbola (RWC = a + b/π). Osmotic potential at RWC = ~0.8–1.0 is extrapolated from the rectangular hyperbola relationship. Turgor pressure is the difference between the measured water potential and the extrapolated osmotic potential. (b) The pressure–volume curve showing the relationships between , p and π during dehydration. The reciprocal of water potential is plotted against (1 RWC). Beyond the turgor-loss point (incipient plasmolysis), the relationship between (1 RWC) and 1/ (or 1/π) is linear. The extrapolation of this linear relationship toward the y-axis intercept gives osmotic potential (the dashed line) of the tissue when the tissue is still turgid. The difference between the measured water potential and the extrapolated osmotic potential is turgor pressure (inset). 02 Dessication - Chap 2 18/3/02 1:54 pm Page 57

Methods for Studying Water Relations Under Stress 57

signs). During dehydration, the PV curve of cation of negative turgor pressure. It is con- a plant tissue initially displays a concave ceivable that the development of negative region, beyond which the curve is linear turgor pressure may reduce mechanical (Fig. 2.2b). The loss of turgor is marked by damage on cellular structures by preventing the point at which the relation of 1/ to collapse of the cells. (1 RWC) deviates away from linearity. Turgor pressure ( ) is calculated as the p 2.4.1.2. Change of osmotic potential during difference between the extrapolated linear desiccation portion of the PV curve and the water potential actually measured, and is often When cell turgor pressure falls to zero dur- plotted as a function of RWC. The relation- ing desiccation, the water potential of the ship between turgor pressure and RWC can cell is equal to the osmotic potential (see be described sufficiently by a quadratic or Equation (6)). As desiccation continues, cubic function. osmotic potential and cell water potential Certain plant tissues might develop nega- are equal and inversely proportional to the tive turgor pressure before the cells collapse volume of osmotically active water. The rela- and p can become zero under severe water tionship between RWC and the reciprocal of stress. When negative p develops, the PV osmotic potential is a straight line. The curve would fall below the extrapolated lin- osmotic potential at full turgor is calculated ear portion of the graph (Fig. 2.3a). If the from the extrapolation of the linear portion cells are sufficiently strong, do not collapse of the PV curve to the RWC at full turgor (i.e. and the plasma membrane remains firmly RWC = 1.0 in Fig. 2.2b). In the Höfler dia- attached to the cell wall, the formation of an gram, the relationship between osmotic intracellular gas bubble will increasingly potential and RWC is represented by a rec- become possible (cavitation). The develop- tangular hyperbolic function to the data ment of negative turgor pressure and intra- points corresponding to the linear part of the cellular cavitation appear to play some roles PV curve (dashed part of the π in Fig. 2.2a). in desiccation tolerance of certain cells. A good example of a cell surviving large nega- 2.4.1.3. The volume of water in symplast, tive turgor pressure and cavitation is the apoplast and intercellular spaces ascospore of Sordaria (Milburn, 1970). The volume of Sordaria ascospores changes very In hydrated plant tissues, water may exist little, and the protoplast remains in contact in the symplast, in the apoplast (i.e. the with the spore wall at all times. Under porous spaces in the cell wall) and in the water stress (by air-drying or in osmotic intercellular spaces (large voids) as dissolution), these cells might generate nega- cussed before. Intercellular water, also tive p as much as –4 MPa. Beyond this called ‘external’ cell water by some work- negative turgor pressure, a small bubble ers, may account for up to 35% of total appears inside the protoplasm suddenly, water in certain plant tissues, such as which increases slowly in size and lichens, liverworts, mosses and fern fronds approaches the walls quite closely without (Beckett, 1997; Proctor, 1999) and develop- losing its spherical appearance. Honegger ing embryos of higher plants (W.Q. Sun, (1995) and Scheidegger et al. (1995) also unpublished data). During desiccation, showed that ascomycetous lichen myco- water potential and turgor potential do not bionts form large intracellular gas bubbles fall with initial water loss at RWC > 1.0 when desiccated. More recently, the PV (Fig. 2.2a and b inset). The volume of water analysis by Beckett (1997) suggested the that is lost before turgor pressure starts to existence of negative turgor pressure in veg- fall is assumed to be intercellular water. etative cells of several desiccation-tolerant The volume of symplastic water represents (poikilohydric) plants (e.g. Dumortiera hir- the amount of osmotically active water in suta and Myrothamnus flabellifolia). PV the tissue, and is obtained by subtracting curves of most plants do not show any indi- the apoplastic water volume from the water 02 Dessication - Chap 2 18/3/02 1:54 pm Page 58

58 W.Q. Sun

(a)

1 p 2 0 ) –1 1 0.6 0.8 1.0

(MPa RWC –1/ Reciprocal of water potential 2 0.0

–0.2 0.0 0.2 0.4 0.6 0.8 1.0 1 – RWC

(b) 1000

100

Water potential (–MPa) Water 1.0

0.1

0.0 0.2 0.4 0.6 0.8 1.0 Relative water content (RWC)

Fig. 2.3. (a) The pressure–volume curves of plant tissues that develop negative turgor pressure (curve 1) and intracellular cavitation (curve 2) during desiccation. The inset in (a) shows the change of cell turgor pressure ( p) during the early stage of drying. When intracellular cavitation occurs, the p suddenly changes to zero (curve 2, inset), and is equal to π (curve 2). If intracellular cavitation does not occur, the cell wall will collapse or deform when the p develops beyond the threshold to which the cell wall can resist (i.e. (1 RWC) > 0.15). The collapse or deformation of the cell wall will lead to a gradual increase in (curve 1) and p (curve 1, inset). (b) The semi-logarithmic plot between RWC and tissue water potential. The high RWC break point corresponds to the turgor-loss point, whereas the low RWC break point corresponds to the volume of apoplastic water. Drawn with data from Quercus rubra seeds (Sun, 1999).

content at full turgor. Symplastic water Apoplastic or osmotically inactive water generally declines over a range of water is present in very small pores and strong potential from about 0.5 to 10 MPa, in water-binding sites of biological surfaces in line with that of osmotic potential. plant tissues. This fraction of water is held 02 Dessication - Chap 2 18/3/02 1:54 pm Page 59

Methods for Studying Water Relations Under Stress 59

by matric and molecular forces, and lost tion of cellular membrane and molecular only when plant tissues are desiccated to a assemblies. So far, workers have paid little water potential less than 15 MPa attention to the location of water in plant (Meidner and Sheriff, 1976). The loss of tissues. The difference in the relative vol- apoplastic water in some species extends ume of external, symplastic, and apoplastic to approximately 800 MPa. The amount water should be taken into account in the of such matrix-bound water in plant tissues comparative studies on mechanisms of des- can be as high as 0.1–0.2 RWC or up to iccation tolerance among cells, tissues or 0.25–0.35 g g1 dw. This fraction of water plants. A similar analysis of water relations does not generally act as a solvent in cells, was found to be very useful in developing a and is not readily freezable. From the mechanistic understanding of the role of Höfler diagram, the apoplastic volume is dehydration in freezing tolerance in earth- estimated from the fitted hyperbolic func- worms (Holmstrup and Zachariassen, 1996). tion. From the PV curve, the volume of apoplastic water is commonly estimated by extrapolation of the linear relationship 2.4.1.4. Volumetric elasticity of the cell wall between RWC and the reciprocal of The cell wall may undergo elastic expan- osmotic potential to the (1 RWC) axis sion or contraction. Elastic (mechanical) after the loss of turgor pressure. However, properties of cell walls play an important the simple extrapolation from the PV curve role in cell water relations. For example, is not a reliable method of estimating the the negative turgor pressure that can apoplastic volume, and in some cases gives develop in a cell largely depends on the negative values (Proctor et al., 1998). mechanical properties of the cell wall. The The apoplastic volume of water should elasticity of the cell wall is represented by be derived with data from the isothermal the volumetric elasticity module , where sorption study at low water potentials depends on both p (turgor pressure) and (water activity), rather than the extrapola- V (cell volume) and is defined as: tion method, because the linear relationship between RWC and the reciprocal of = p V (11) osmotic potential does not hold for the V apoplastic volume of water (which is where V is volume change caused by a osmotically inactive). Compared to the given pressure change p. Equation (11) removal of osmotically active (symplastic) indicates that a high value of implies a water, the measured osmotic potential rigid cell wall, whereas a low value implies (including the term of matric potential) a more elastic cell wall. The value can be declines much more rapidly when the calculated from the relationship between apoplastic water is removed. Therefore, the p and RWC (Steudle et al., 1977; volume of apoplastic water is marked by Stadelmann, 1984). The change of as a the point at low water content at which the function of RWC is given by the first deriv- relationship of 1/ to (1 – RWC) again ative of the quadratic or cubic function of deviates away from linearity (Fig. 2.3b). turgor pressure on RWC. The value of the The volume of apoplastic water roughly p/RWC derivative curve at RWC = 1.0 is corresponds to the primary hydration in usually taken as the bulk modulus of elas- tissues (including both strong and weak ticity and used for purposes of comparison. water-binding sites). A pressure probe technique can be used One can expect that plant tissues would directly to determine the turgor pressure respond differently to the loss of external, and the for individual plant cells. This symplastic and apoplastic water. The loss of technique is useful for continuous mea- symplastic water can cause osmotic pertur- surement of cell turgor pressure, cell wall bation of physiological and biochemical elasticity and hydraulic conductivity of the processes, whereas the loss of apoplastic cell membrane in single cells (Hüsken et water may disrupt the structure and func- al., 1978). The intracellular hydrostatic 02 Dessication - Chap 2 18/3/02 1:54 pm Page 60

60 W.Q. Sun

pressure is transmitted to the pressure rated salt solutions in closed desiccators transducer via an oil-filled microcapillary until constant weights are achieved, introduced into the cell, which transforms whereas an adsorption isotherm is devel- into a proportional voltage. This technique oped by rehydrating dried tissues over satu- permits volume changes and turgor pres- rated salt solutions. A desorption curve can sure changes to be determined with an also be developed during drying of tissues accuracy of 105–106 µl and 3–5 103 in any atmospheric condition by measur- MPa, respectively. ing, at various points in time, the water At present, very little information is content of the tissue and the equilibrium available on cell wall properties of desicca- RH of its surrounding air in a closed con- tion-tolerant plant tissues. Proctor (1999) tainer. Similarly, the dry tissue can be rehy- found that two highly desiccation-tolerant drate with a given quantity of water to raise liverworts have low values of bulk elastic the water content and equilibrium RH. modulus. He thought that extensible cell Sophisticated instruments such as con- walls might be a part of structural adapta- trolled atmosphere microbalance and tion to rapid changes of cell volume in dynamic vapour sorption systems (Surface their intermittently desiccated habitats. Measurement Systems, London, UK) use Ultrastructural studies on dry mesophyll the latter methods. Desorption and adsorp- cells of desiccation-tolerant Selaginella tion isotherms are used, respectively, to lepidophylla by Thomson and Platt (1997) study the properties of dehydration and showed highly folded cell walls and con- rehydration of plant tissues. Desorption and tinuous apposition of plasmalemma to the adsorption curves are rarely the same: the walls. Vicre et al. (1999) studied the cell desorption curve usually gives a higher wall architecture of leaf tissues of water content than the adsorption isotherm. Craterostigma wilmsii (a resurrection The difference in the equilibrium water plant), and also observed extensive folding content between two curves is called hys- of the cell wall during desiccation. The teresis. Hysteresis is evidence of the irre- folding of the cell wall allows the plasma versibility of the sorption process, and membrane to remain firmly attached to the therefore indicates the limited validity of wall as the cell loses water. Biochemical the equilibrium thermodynamic approach modifications of the cell wall were to investigate the dehydration–rehydration observed during desiccation and rehydra- properties of plant tissues. Hysteresis might tion, leading to the change in its tensile be an important issue when considering strength that may prevent the total collapse critical water activities for desiccation of the walls in the dry tissue and avoid stress during dehydration–rehydration rapid expansion upon rehydration. The cycles and when investigating storage stabil- change in cell wall elasticity during desic- ity after manipulation of moisture content of cation can be determined easily by taking seeds and pollen. the first derivative of the function of turgor pressure on RWC. 2.4.2.1. Theoretical models Plant tissues show a sigmoid sorption 2.4.2. Analysis of water sorption isotherms isotherm (Fig. 2.4a). The inflection point of the isotherm is believed to indicate either a The water sorption isotherm is the depen- change of water-binding capacity and/or dence of water content on water activity of the relative amount of ‘bound’ or ‘free’ the surrounding environment at a given water. Water sorption data are normally temperature. There are two types of sorp- analysed using theoretical models, from tion isotherms: desorption isotherm and which useful biophysical parameters of adsorption isotherm (Fig. 2.4a). water relations are derived. Commonly Conventionally, a desorption isotherm is used models include the Brunauer– developed by drying fresh tissues over satu- Emmett–Teller (BET) model, the 02 Dessication - Chap 2 18/3/02 1:54 pm Page 61

Methods for Studying Water Relations Under Stress 61

Guggenheim–Anderson–de Boer (GAB)  −  aw = C 11+ model and the D’Arcy–Watt model.  aw (12) Ma()1 −  M  MC The BET model (Brunauer et al., 1938) ww m m is derived from statistical and thermody- where aw is the water activity, Mw is equi- namic considerations. The equation can be librium water content in the tissue, Mm is written as: the BET monolayer (water content corre-

(a)

I II III

Desorption Water content Water

Adsorption

Water activity

(b) III II I Enthalpy

Free energy Sorption enthalpy

Entropy

Water content

Fig. 2.4. The analysis of water sorption isotherms. (a) The typical shape of desorption curves and adsorption curves of plant tissues. The difference between these two curves shows hysteresis, which indicates the irreversibility of water sorption in the tissues during dehydration and rehydration. The sigmoid shape of sorption curves is presumably due to the existence of three types of water-binding sites in tissues (strong (I), weak (II) and multilayer molecular sorption sites (III)). (b) Differential enthalpy (H), free energy (G) and entropy (S) of hydration. Desorption curves can be used to calculate H and S of tissue hydration. See text for detailed discussion. 02 Dessication - Chap 2 4/4/02 2:18 pm Page 62

62 W.Q. Sun

sponding to the monolayer hydration) and amount of water in those three regions can C is temperature dependence for sorption be estimated. K is the number of strong excess enthalpy (Brunauer et al., 1938, water-binding sites, multiplied by the mole-

1940). BET equation parameters, Mm and C, cular weight of water and divided by 23 can be calculated by plotting aw/[Mw (1 Avogadro’s number (6.023 10 ); K is the aw)] against aw. The y-axis intercept of the strength of the attraction of the strong water- straight line is equal to 1/(MmC) and the binding sites for water; c is a measure (lin- slope is equal to (C 1)/Mm. The BET is ear approximation) of the afﬁnity and the valid only for aw < 0.5, thus data points number of weak water-binding sites; k within that range are used to estimate the relates to the number of multimolecular

monolayer value (Mm). The BET model is water sorption sites; and k relates to the an effective method for estimating the activity of water (D’Arcy and Watt, 1970). amount of water bound specifically to The number of water-binding sites in tissues polar sites (monolayer), but cannot be used can be calculated from the derived equation to give a complete estimation of specific coefficients. The number of strong, weak hydration parameters. and multimolecular water-binding sites are The GAB model is an extension of the K N/M, cN/(M o), and k N/M, respectively, BET model, taking into consideration the where N is Avogadro’s number, M is the modified properties of the sorbing materi- molecular weight of water and o is the satu- als in the multilayer region and the bulk rated vapour pressure of pure water. liquid properties through the introduction The D’Arcy–Watt model has been used of a third constant, K. The GAB equation is extensively for the analysis of desiccation- written as: tolerant and desiccation-intolerant plant tissues (Vertucci and Leopold, 1986, 1987a, M CKa M = m w (13) b; Sun et al., 1997). Both the GAB and the w (1 Ka )(1 Ka + CKa ) w w w D’Arcy–Watt models are valid over a wider range of water activities for plant tissues. where C and K are temperature-dependent The GAB model has some advantages over coefficients. Constants, Mm, C and K are the D’Arcy–Watt model, which assumes the estimated via the curve fitting of sorption three types of water-binding sites. The GAB data. In the field of food sciences, the GAB model does not have such an assumption. model is the most widely accepted due to For biological systems it is more reasonable its accuracy and its validity over a wide to assume that the number of water-binding range of water activities from 0.05 to 0.9 sites is changing continually along with the (Rahman and Labuza, 1999). binding energies. Moreover, the GAB model The D’Arcy–Watt model was developed can be more easily applied to other thermal for the analysis of sorption isotherms of analyses (e.g. water-clustering function). non-homogeneous materials (D’Arcy and Watt, 1970). This model assumes that there is a fixed number of water-binding sites 2.4.2.2. Temperature dependency of water with different discrete binding energies. The sorption D’Arcy–Watt equation can be written as: Desiccation involves the transfer of liquid water in plant tissues into the vapour KKaw ++kkaw M w =caw (14) phase. Temperature influences evaporation 11+ Ka −κa w w rate through the heat supply as well as where K, K, c, k and k are equation coeffi- through its effect on the partial water cients (adjustable parameters). The equation vapour pressure in air and the energy sta- has three terms, which represent the tus of water in plant tissues. In isothermal amounts of water that are strongly bound, conditions, air acts as an osmotic mem- weakly bound and sorbed in multimolecular brane and equilibrium is often slow and water clusters, respectively. For a tissue that dependent on temperature. An increase in

is in equilibrium with a given aw, the temperature generally results in a decrease 02 Dessication - Chap 2 18/3/02 1:54 pm Page 63

Methods for Studying Water Relations Under Stress 63

in equilibrium water content of plant tis- event. A high negative H value at low sues at a given RH (i.e. water activity) or an water content suggests the strong affinity of increase in equilibrium water activity at adjacent water molecules toward ionic sites constant tissue water content. The shift of and/or other polar sites of the substrate. As water activity at the constant water content water content increases, the H becomes by temperature is mainly due to the change less negative (Fig. 2.4b). The primary hydra- in water binding, dissociation of water, tion process (i.e. strong and weak binding physical state of water or increase in solu- sites) is considered to be completed when bility of solute in water. Tensile strength of the differential enthalpy of hydration (H) water, the pressure holding molecules approaches zero (Luscher-Mattli and Ruegg, together, increases by 81.6 mbars on aver- 1982; Rupley et al., 1983; Bruni and age for a reduction of 1°C. Temperature Leopold, 1991). The change of S reflects dependence of isotherm shift is described the relative degree of order, and the S peak by the Clausius–Clapeyron equation: is presumably associated with the saturation of all primary hydration sites. It should be a q +  11 ln =w2 = w  −  (15) clearly noted that the relationships of a R TT w1 21 H/WC, G/WC and S/WC describe ther- where q is the excess heat of sorption; w is modynamic interactions between water and the latent heat of vaporization for water biomaterials, but not necessarily the func- (44.0 kJ kg1 at 25°C); R is the gas constant; tions of water and biological structures in aw1 and aw2 are water activities for a given physiological processes. A possible associa- equilibrium water content at temperature tion between water sorption behaviours and T1 and T2, respectively. The plot of ln aw desiccation tolerance of plant tissues was against 1/T at any given tissue water con- discussed in a number of studies (Vertucci tent is a straight line and its slope gives (q and Leopold, 1987b; Farrant et al., 1988; w)/R, from which the excess heat of Pritchard, 1991; Sakurai et al., 1995; Eira et sorption, q, can be derived (Fig. 2.5a). al., 1999; Sun, 2000). No consistent differ- In practice, some thermodynamic quan- ence in water sorption characteristics has tities of tissue hydration can be calculated been found between desiccation-sensitive according to isotherms at two different (recalcitrant) and desiccation-tolerant temperatures. The aw1 and aw2 for a given (orthodox) seed tissues (Sun, 2000). equilibrium water content at two tempera- The van’t Hoff relationship provides tures can be taken from water sorption another convenient means to analyse tem- curves or calculated from fitted sorption perature dependence of sorption isotherm. equations (Fig. 2.5b and Fig. 2.7a). The van’t Hoff equation and the Differential enthalpy of hydration (H, Clausius–Clapeyron equation are essen- including q and w), differential free tially the same in theory, but different in energy of hydration (G) and differential their mathematical treatment of experimen- entropy of hydration (S) are given by: tal data. The Clausius–Clapeyron equation RT T a handles two temperature points, whereas H 1 2 ln w1 (16) T T a the van’t Hoff equation can handle a series 2 1 w2 of temperature points at once. The van’t G RT ln (aw) (17) Hoff equation expresses the relationship of the equilibrium water activity (a ) for a S H G (18) w T given water content against the temperature (1/T) (Fig. 2.5b), and is written in its These thermodynamic quantities are the differential mathematical form as: functions of water content in tissues. The H relationships of H/WC, G/WC and ∂ ln a ∂(1/T) (19) w R S/WC provide important information with regard to the hydration properties of tissues where T is absolute temperature in kelvin, (Fig. 2.4b). Water sorption is an exothermic and R is the gas constant. The H is the 02 Dessication - Chap 2 18/3/02 1:54 pm Page 64

64 W.Q. Sun

differential enthalpy of water sorption. It is require the storage of desiccation-sensitive important to note that the relationship seeds and other tissues in a refrigerated

between ln(aw) and (1/T) is not necessarily condition or at liquid nitrogen tempera- a straight line. Within a relatively narrow ture. When the extrapolation is used, the range of temperature, linear approximation non-linear nature of the relationship may be used to calculate H accurately. between ln(aw) and (1/T) needs to be taken However, there is considerable interest in into consideration. The study on tempera- studying water sorption properties of bio- ture dependence of water sorption using logical tissues at a much wider range of the van’t Hoff equation (Fig. 2.5a and b) is temperature. For example, long-term used to establish the theoretic framework preservation of genetic resources may for the optimization of germplasm preser-

(a) 0.0 0.09 g g–1 dw

–1.0

–2.0 0.04 g g–1 dw

or RH/100) or

w a

–3.0 ln ( ln

–4.0 0.02 g g–1 dw

3.3 3.4 3.5 3.6 1/Temperature (K, 10–3)

(b) 0.14

0.12 dw) –1 0.10 50% RH

0.08

0.06 Water content (g g Water 0.04 10% RH

0.02 0 5 10 15 20 25 Temperature (C)

Fig. 2.5. (a) Temperature dependence of water sorption for the same seed material at different water contents (i.e. the van’t Hoff plot). Drawn with data from Eira et al. (1999). (b) Equilibrium water content at speciﬁc water activities as a function of temperature for whole seeds of Coffea arabica cv. Mundo Nova. This relationship is called ‘isopleth’. 02 Dessication - Chap 2 18/3/02 1:54 pm Page 65

Methods for Studying Water Relations Under Stress 65

vation protocols (Vertucci et al., 1994, polar hydration sites. A recent study using 1995; Eira et al., 1999). the D’Arcy–Watt model suggested that water redistribution among different types of hydration sites might be related to the 2.4.2.3. Monolayer hydration and water- rapid loss of seed viability during storage clustering function after osmotic priming and drying back (Sun The monolayer hydration values of plant et al., 1997). tissues, the amount of water bound to spe- Water clustering in binding sites is ciﬁc polar sites, can be easily determined, another important hydration event that is using BET or GAB isotherm models. For of signiﬁcance to desiccation tolerance of most plant tissues and their major chemi- plant tissues and the survival of tissue in cal components, the monolayer value at the dried state. Clustering formation is ambient temperature is estimated to be related to a number of transport phenom- between 0.04 and 0.09 g g1 dw using the ena. For example, clustering reduces the BET or GAB model (Rahman and Labuza, effective mobility of water by increasing 1999). The BET monolayer value of many the size of the diffusing molecular group or orthodox seeds was also found to be in this by increasing the tortuosity of diffusion range (Vertucci and Leopold, 1987a,b; paths (Stannett et al., 1982). The range of Bruni and Leopold, 1991; Vertucci and water activity where the self-association of Roos, 1993; Sun et al., 1997). The mono- water takes place can be examined by the layer value of Typha pollen was much less clustering function (Lugue et al., 1995; than that of orthodox seeds (Buitink et al., Dominguez and Heredia, 1999). The clus- 1998b). The monolayer hydration is gener- tering function is written as: ally complete at a water activity of G /V V [(a /V )/a ] 1 (20) 0.20–0.30 (i.e. 150 to 250 MPa). It is 11 1 2 w 1 w

important to note that the monolayer value where G11/V1 is the clustering function, V1 decreases rapidly as temperature increases, is the volume fraction of water, V2 is the and increases as temperature declines. The volume fraction of biopolymers, and aw is monolayer water is of great importance for water activity (Zimm and Lundberg, 1956).

the survival of many dry organisms (e.g. The subscript ‘11’ in G11/V1 denotes the spores, pollen grains and seeds) during water–water interaction as a function of storage. In food science, the water activity water content (component 1). The cluster- at the monolayer value is defined as the ing function can be applied to an isotherm critical water activity. At a water activity sorption model such as the GAB equation above 0.20–0.30, the rate of chemical reac- with some modifications. The GAB equations begins to increase significantly tion needs to be rewritten in terms of vol- because of the greater solubility and mobil- ume fraction instead of weight fraction. ity of the reactants. At water contents The GAB equation can be rewritten as: below the monolayer value, the rate of MmCKaw lipid oxidation and associated free radical Mw V1p1/V2p2 (21) (1Ka )(1Ka CKa ) damage increases. The presence of mono- w w w layer water inhibits the undesirable inter- or actions between polar groups on adjacent carbohydrate or protein molecules, thereby (1Ka )(1Ka CKa ) a /V w w w (22) preserving their rehydration ability and w 1 M CKp V biological functions (Rahman and Labuza, m 2 2

1999). where p1 and p2 are the density of water There is no deﬁned monolayer parame- and biopolymers. The density of sorbed ter in the D’Arcy–Watt model. However, water is assumed to be equal to 1.0 g cm3.

the ﬁrst term of the D’Arcy–Watt equation Substituting aw/V1 in Equation (20) with may be used as an approximation, as it rep- Equation (22), the clustering function can resents water that is bound strongly to be expressed as: 02 Dessication - Chap 2 18/3/02 1:54 pm Page 66

66 W.Q. Sun

(2KCKM CK)(2CK22K2)a expressed as the percentage of the corre- G /V m w (23) 11 1 sponding maximum value in the fully MmCKp2 hydrated tissues (Fig. 2.7b). Therefore, the According to Equation (23), G /V is pro- 11 1 occupancy relationship indicates the portionally related to a and the reciprocal w degree of hydration for different types of of polymer density (p ) function. G /V 2 11 1 hydration sites during desiccation. Figure can be solved easily by the substitution of 2.7b shows that the occupancy for three M , C and K constants from the GAB equa- m types of hydration sites changes as the tion. Figure 2.6 shows a plot of the waterwater content of Q. rubra seed tissues clustering function of soybean axes. The decreases during desiccation. Desiccation clustering plot is basically a straight line of seed tissues to 0.30 g g 1 dw (the critical against water activity. When G /V is 11 1 water content) removed about 90% of mul- greater than 1, water is expected to clus- tilayer molecular sorption water, but only ter (Zimm and Lundberg, 1956). The auto- about 10% of water molecules attached to association (clustering) of water in a few the weak hydration sites in seed tissues. desiccation-tolerant seeds is observed to The removal of water from weak hydration occur at water activity ranging from 0.55 to sites appears to be related to desiccation 0.60 (W.Q. Sun, unpublished data). damage in Q. rubra seeds (Sun, 1999). The critical water content of Q. robur axes also 2.4.2.4. Occupancy of water-binding sites corresponds to the amount of matrix-bound water (Pritchard and Manger, 1998). The D’Arcy–Watt model can be used to However, the question of whether the examine the occupancy of water-binding water-binding or sorption behaviour in sites as a function of water content accord- seed tissues is related to their desiccation ing to Luscher-Mattli and Ruegg (1982). tolerance remains unresolved. The loss of The occupancy represents the amount of viability in many recalcitrant seeds occurs water attached to certain hydration sites, at a water content that is much higher than

–4 Clustering function

–8

–12

0.0 0.2 0.4 0.6 0.8 1.0 Water activity

Fig. 2.6. Water-clustering function showing the waterwater association in soybean seed axes as a function of equilibrium water activity. Apparent water clusters ﬁrst appear at a water activity of 0.58 (arrow). The water-clustering function Equation (23) was solved through the study on biopolymer volumetric change

during hydration [i.e. P2= f (V1)] by applying water sorption analysis. See text for further explanation. 02 Dessication - Chap 2 18/3/02 1:54 pm Page 67

Methods for Studying Water Relations Under Stress 67

that of ‘bound’ water (Pammenter et al., recently released manual by Bell and 1991; Berjak et al., 1992). Clearly, more Labuza (2000). This book generally dis- comprehensive studies are needed. cusses water activity in food materials, but Readers who wish to know more about the principles are also applicable to plant water sorption analysis may refer to a desiccation tolerance studies. Practical

(a) 0.8

4.974/ 0.0219/ 0.6 WC = + 0.0673/ + 1 + 90.2/ 1 – 0.990/ dw)

–1 25C

1.279/ 0.026/ 0.4 WC = + 0.0373/ + 1 + 27.4/ 1 – 0.986/

Water content (g g Water 0.2

0.0

0.0 0.2 0.4 0.6 0.8 1.0 Water activity

(b) 100

80 5C 25C

60 Strong binding site

40 Weak binding site

Multilayer sorption

Sorption sites occupied (%) 20

0 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 Water content (g g–1 dw)

Fig. 2.7. (a) The interpretation of desorption isotherms of Quercus rubra cotyledonary tissues, using the D’Arcy–Watt model. Equation coefﬁcients are derived though curve-ﬁtting of experimental data ( / o = aw). See text for further explanation. (b) The occupancy for three types of hydration sites in Q. rubra cotyledonary tissues at different water contents. The occupancy is based on the percentage of the corresponding maximum values in the fully hydrated state (i.e. full turgor). The change of occupancy reveals how and when water is removed in different types of hydration sites during dehydration. 02 Dessication - Chap 2 18/3/02 1:54 pm Page 68

68 W.Q. Sun

examples are provided to elucidate how to curves are biphasic. During the first drying solve many equations. phase, the loss of water follows a simple exponential function. During the second phase, water content does not decrease 2.5. Measurement of Drying Rate and much because the tissue is very much Desiccation Stress closer to achieving equilibrium with the air. Because water loss during the first 2.5.1. Driving force for water loss and phase is described by an exponential func- expression of drying rate tion, the rate constant () of water loss can be used as an expression of drying rate. The loss of water from tissues depends on two factors: the gradient in water potential between tissue surface and external air or 2.5.2. Quantification of desiccation stress solution, and the hydraulic conductivity of the tissue. The volume flow of water from The response of plant tissues to desiccation the tissue to air can be described by: is significantly affected by dehydration conditions, such as drying rate (see Chapter 3). V AL ( ) (24) w p o i Under slow-drying conditions, plant tissues

where Vw is the volume ﬂow of water per stay longer at intermediate water contents. unit time (m3 s1), A is the surface area of Fast drying is often reported to improve 2 the tissue (m ) and Lp is the hydraulic con- desiccation tolerance of recalcitrant plant 1 1 ductivity of the tissue (m s Pa ). The o seeds (reviewed by Pammenter and Berjak, and i are water potentials of external air 1999). There is no doubt that the level of and the tissue, respectively. The difference desiccation stress would vary with drying in water potential ( o i) is a measure of rate, and the questions are: (i) how desicca- the driving force for dehydration. i is a tion stress can be quantiﬁed; and (ii) how function of time that describes the decrease drying rate affects the level of desiccation in tissue water potential during drying. The stress. The change in chemical potential of

hydraulic conductivity of the tissue (Lp) is a cellular water is a good measure for the measure of the diffusional resistance of the degree of desiccation stress. When the water transport pathway within the tissue. chemical potential of water is compared, A good measure for the drying rate is *w and mwgh in Equation (5) cancel out. essential for comparative studies on desic- The difference in chemical potential of cel- cation tolerance. According to Equation lular water between the dehydrated state D H (24), the rate of water loss from the tissue is ( w) and the hydrated state ( w) is: – time-dependent. Under constant RH and H D RT(ln aH ln aD )V (PHPD ) (26) temperature, the water content of the tissue w w w w w w w is expected to decrease exponentially over According to Equation (26), the degree of desiccation stress is proportional to time until i reaches o (Fig. 2.8a). The curve of water loss can be described by: changes in osmotic potential and hydrostatic pressure (P) in cells. Therefore, the WC = exp(t) (25) change of water potential, d/dt, can be where is the initial water content, is the used to quantify the level of desiccation rate constant of water loss, and t is time of stress. Figure 2.8b shows the plots of tissue drying. This relationship was ﬁrst used by water potential against drying time. Under Tompsett and Pritchard (1998) to compare the conditions of constant temperature and the dehydration rate of A. hippocastanum relative humidity, such plots are straight seeds. Drying curves of other seed tissues lines down to the fraction of apoplastic have been examined under a wide range of water. Water potential of the tissue desiccation conditions, and they conform decreases faster and deviates away from to Equation (25) (Li and Sun, 1999; Liang the straight line when the apoplastic water and Sun, 2000). Typically, water content is lost (see Fig. 2.3b, the low-RWC break 02 Dessication - Chap 2 18/3/02 1:54 pm Page 69

Methods for Studying Water Relations Under Stress 69

point). The slope of each straight line por- mathematical evaluation of this linear relation (d/dt) represents the degree of direct tionship will not be presented here. physical stress under different desiccation If the plant tissue is viewed as a viscoelas- conditions. The relationship between tic system, the mechanical stress caused by d/dt and the rate constant () of water water loss can be considered as a simple loss (drying rate) is linear (Fig. 2.9). A stress–strain response. The physico-chemical

(a) 3.2

33% RH 88% RH 2.4 dw)

–1 94% RH

1.6

= 0.00502 Water content (g g Water 0.8

= 0.0211 0.0 = 0.103

0 80 160 240 320 400 Drying time (h)

(b) 0 33% RH 88% RH –5 94% RH

–10

d/dt = –0.032

Water potential (MPa) Water –15 d/dt = –0.160

d/dt = –0.689 –20

0 80 160 240 320 400 Drying time (h)

Fig. 2.8. Measurement of drying rate and quantiﬁcation of desiccation stress for Theobroma cacao axes. (a) Drying curves of isolated axes in three constant relative humidities (RHs). The data are ﬁtted with exponential functions (WC = exp(t)), and the rate constants of water loss, , are shown near each curve. (b) Plots of tissue water potential against drying time. The mechanical stress on tissue caused by the water loss can be considered as a simple stress–strain response. The slope of /time plot, d/dt, is directly related to the intensity of desiccation stress. Data from Liang and Sun (2000). 02 Dessication - Chap 2 18/3/02 1:54 pm Page 70

70 W.Q. Sun

1.6

) 1.2 t /d

0.8

0.4 Dehydration rate (–d

0.0

0.00 0.06 0.12 0.18 0.24 0.30 Rate constant of water loss ()

Fig. 2.9. The relationship between the rate constant of water loss () in Equation (25) and dehydration rate (d/dt) for Theobroma cacao (cocoa) axes. Isolated axes were dehydrated at 16°C under constant relative humidities ranging from 6 to 94% to achieve different drying rates and stress conditions. Drawn using data from Liang and Sun (2000).

aspect of desiccation stress can be assessed by treats biological systems as fully reversible integrating the function of tissue water poten- ones and does not give much considera- tial over time (Fig. 2.8b). Figure 2.10a and b tion to the term time, one of the most shows an example of the quantitative analysis important factors in any biological of desiccation stress. The cumulative water response. This limitation is particularly stress during desiccation at three different RHs relevant to the study of desiccation. The was plotted against drying time and water application of thermodynamics is gener- content, respectively. Under the slow-drying ally sufficient in many cases for fully condition (94% RH), the mechanical stress hydrated tissues. However, at intermediate (d/dt) is small (Fig. 2.8b); however, the or low moisture levels, the non-equilib- cumulative physico-chemical stress is remark- rium, kinetic principles play a more ably high because the time to dry to the same important role. During desiccation, the water content increases exponentially as the biological system basically shifts from a drying rate decreases (Fig. 2.10a and b). The thermodynamic state to a non-equilibrium quantitative analysis of mechanical and kinetic state (Leopold et al., 1994; Sun et physico-chemical aspects of desiccation stress al., 1994; Sun, 1997, 1998). The thermo- has led to an understanding of the physiologi- dynamic approach does not sufficiently cal basis of the optimal drying rate to achieve address the kinetics of various reactions the maximum desiccation tolerance of and processes in intermediate- to low- Theobroma cacao axes (Liang and Sun, 2000). moisture systems. The kinetic and functional approach to cellular water relations focuses on how the 2.6. Water Relations – the Kinetic and interactions between water and other cellu- Functional Approach lar components can influence the structures and biological properties of each The thermodynamic approach to water other. In this chapter, principles of the relations has its limitations, because it kinetic approach and the interactions 02 Dessication - Chap 2 18/3/02 1:54 pm Page 71

Methods for Studying Water Relations Under Stress 71

(a) 2000 h) 1500

1000

33% RH

500 88% RH 94% RH Cumulative water stress (MPa 0

0 80 160 240 320 400 Drying time (h)

(b) 2000 33% RH h)

88% RH 1500 94% RH

1000

500 Cumulative water stress (MPa 0

0.0 0.6 1.2 1.8 2.4 3.0 Water content (g g–1 dw)

Fig. 2.10. (a) Cumulative water stress during desiccation as the function of drying time. The cumulative water stress is calculated by integrating the /time function (see text for details). (b) Cumulative water stress as the function of tissue water content under different desiccation conditions. Cumulative stress is much higher in slow-drying conditions because the dehydration time increases exponentially as drying rate decreases.

between water and many other biomole- Instead, a general account will be offered, cules will not be discussed in detail. These so that readers can be conﬁdent about topics will be covered in other chapters on choosing the appropriate method to quan- desiccation damage and mechanisms of tify particular water properties in studies desiccation tolerance (see Chapters 9–12). on desiccation tolerance. 02 Dessication - Chap 2 18/3/02 1:54 pm Page 72

72 W.Q. Sun

2.6.1. General considerations the study on water in skeletal muscle as an example, fast techniques (e.g. laser Raman 2.6.1.1. Time scale spectroscopy, infrared spectroscopy and dielectric relaxation) could not find any Any study on the state of water by a bio- intramolecular differences in hydrogen physical technique involves measuring bond lengths, angles or strength between parameters of time scale directly or indi- muscle water and pure water (Beall, 1981). rectly. Biophysical techniques often use the However, slower techniques, such as diffusional correlation time as the time nuclear magnetic resonance (NMR) (Fung scale to make comparisons of the measure- and McGaughy, 1974), electron paramag- ments on water. Many of the physical prop- netic resonance (EPR) (Belagyi, 1975) (see erties of water are theoretically related to Chapter 4), fluorescence polarization the diffusional correlation time ( D) of (Knight and Wiggins, 1979) and freezing water, the average time between jumps in behaviour (Rustgi et al., 1978), showed a position for water molecules in the system. restricted motion of at least a portion of cell Two critical numbers on this time scale ( D) water. This situation is identical to pho- 5 11 are 10 s and 10 s. The D of water in ice tographing moving objects with different is 10 5 s and in pure liquid is 10 11 s, one shutter speeds. If the shutter speed is very million times faster. Under normal condi- high relative to the velocities of two moving tions, water in biological systems exists in a objects (e.g. 1/800 s), the photo will proba- state somewhere between the solid state of bly not record any information as to crystalline ice and the liquid hydrogen- whether one object is moving faster than bonded lattice of pure water. In low- the other. On the other hand, if the shutter moisture systems, however, the D of water speed is too slow (e.g. 1/2 s), the images of molecules in the intracellular glasses would both moving objects will be blurred and no be much slower than 10 5 s. Readers may meaningful information can be obtained refer to a series of studies on molecular from such a photo. Only with a proper mobility in seeds and pollen by Buitink et shutter speed can the photo reflect the dif- al. (1998a, 1999, 2000a,b,c; Chapter 10 of ference in the velocity between the two this volume). moving objects. The ability of a biophysical technique to yield useful information about the physical 2.6.1.2. Structural complexity and dynamics state of water largely depends on how fast a of molecular ordering measurement can be made. Slow techniques which require measurement times Hydration of protein is a good example, greater than the diffusional correlation time illustrating the complexity of structures yield an average over all molecules in the and functions for water in biological sys- population with a kinetic contribution from tems. When a mole of lysozyme is hydrated diffusion, whereas fast techniques can yield by 60 moles of water (~ 0.07 g g1 dry pro- instantaneous information about intramole- tein), water is primarily located to charged cular factors such as H–O bond lengths and groups, and its mobility is reduced at least hydrogen bond angles (geometric factors). by 100 times relative to pure water. At this To choose the appropriate technique, one low hydration level, no structural differ- has to bear in mind that the type of prop- ence is observed in the protein. As hydra- erty or structure that a technique can probe tion increases to 220 moles of water per is related to the time scale. Generally speak- mole of protein (~ 0.25 g g1 dry protein), ing, fast techniques would probably pro- water begins to form clusters of various duce data of instantaneous structures of sizes and arrangements around the charged water and other biomolecules, but slow and polar sites of the protein. Internal pro- techniques would provide more informa- tein motion (H exchange) increases by 1000 tion about the interactions between water times to be comparable to that of solution, molecules and their environment. Taking while the protein sample is still a solid. At 02 Dessication - Chap 2 18/3/02 1:54 pm Page 73

Methods for Studying Water Relations Under Stress 73

a hydration level of 300 moles of water per tions that a model allows, have to be taken mole of protein (~ 0.38 g g1 dry protein), into consideration for experimental design enzymatic activity, mobility of bound lig- and implementation. If possible, additional and and fast water motion become easily experiments should be conducted to con- detectable. Dielectric relaxation measure- firm the results and to examine whether the ments show two water relaxation times, assumptions are satisfied. Unfortunately, one of 2 1011 s, close to that of bulky biophysical models or equations have fre- pure water, and the other of 109 s, indicat- quently been used to analyse the actual ing the heterogeneous nature of water data without checking their assumptions. behaviours and functions (Rupley et al., Worse still, sometimes a model was 1983). At the hydration level of 0.38 g g1 selected after the entire experiment was dry protein, each water molecule covers, on completed (Beall, 1983). average, 20 Å2 of protein surface, which is Conceptual models have been used for twice the effective area of a water molecule. the interpretation of the data on water in Yet several populations of water molecules biological systems, with many pitfalls. For are observed at such low hydration. example, the ‘two-fraction fast-exchange The surfaces of membranes, proteins model’ (Zimmermann and Brittin, 1957) and other macromolecules impose geomet- assumes that there is a small fraction of ric limitations on the possible arrange- highly immobilized cell water on the sur- ments of hydration water. Interfacial water face of macromolecules (ice-like) and a molecules, being part of the network of bio- large fraction of cell water that behaves like logical interfaces, are dynamically oriented bulk water. Rapid exchange between the and exhibit restricted motion (i.e. are two populations yields reduced average ‘bound’). The ordering of molecules on var- properties. This two-fraction model can be ious biological surfaces is strictly local, written as: and may fluctuate rapidly between possible 1 X (1 X) (27) arrangements. Ideally, a biophysical tech- * T Tslow TH O nique used to study the state of water in 2 biological systems should have the resolu- where T* is measured (average) relaxation tion to differentiate closely related struc- time; X and (1X) are the fraction of tures or populations. However, as discussed immobilized water and the fraction of cell earlier, kinetic measurements reflect only water that is like bulk water, respectively; T and T are the relaxation times of average or time-average properties over all slow H2O molecules in the system and, in many the slow fraction and bulk water. In this cases, do not provide definitive answers to equation, there is only one measured para- the questions of interest. To interpret the meter (T *), but three unknown quantities (X, T and T ). To estimate X, T data of kinetic measurements, the investiga- slow H2O slow and T must be arbitrarily assigned. This tor must impose a conceptual model, which H2O may be controversial (Beall, 1983). Such model represents a simplistic view on the studies are often misunderstood and misin- dynamics of water in biological systems, terpreted by readers who are less familiar which is still in use by some workers. If

with the biophysical techniques used. one intends to solve Tslow, then X must be estimated through other methods. When X is equated to the ‘non-freezable fraction’ or 2.6.1.3. The model-dependent interpretation: ‘osmotically inactive fraction’, additional the pitfalls assumptions are made. By redefining X as The selection of a theoretic model or the an adjustable parameter in different sys- development of a new model is an impor- tems, a new model is established (Beall, tant step in any kinetic study, which 1983). This example clearly shows the should be done before actual measure- uncertainty of biophysical interpretation. ments are made. The assumptions that a Simply because the model is easy to use model contains, and the specific predic- and fits the data well it does not necessarily 02 Dessication - Chap 2 18/3/02 1:54 pm Page 74

74 W.Q. Sun

mean that it represents the true state of measurements is briefly summarized in water in a system. Of course, all models are Table 2.1. Readers are advised to consult open to interpretation. other references, including those cited However, it is possible to measure above, and Chapter 4 in this volume. In this changes in the properties of water in living chapter, only a brief introduction will be systems that correlate with physiological provided on several techniques that have functions (Clegg et al., 1982; Clegg, 1986; been increasingly used in recent years. Bruni et al., 1989). Changes in dynamic properties of water at different hydration 2.6.2.1. Differential scanning calorimetry levels indicate the existence of different fractions of water, which may vary in struc- Differential scanning calorimetry (DSC) is ture and property and presumably play dif- probably the most commonly used thermal ferent biological roles. Studies have analysis technique. It has been used by a identified the existence of at least four or number of workers to study the possible five fractions of water, presumably relating relationship between freezing, desiccation to different interactions between water and tolerance and water properties in plant tis- cellular constituents (Clegg, 1986; sues (Williams and Leopold, 1989; Ratkovic, 1987; Vertucci, 1990; Pissis et al., Vertucci, 1990; Pammenter et al., 1991; 1996; Sun, 2000). Hydration levels corre- Berjak et al., 1992; Sun et al., 1994; Vertucci sponding to these fractions of cellular et al., 1994, 1995; Buitink et al., 1998b; water are associated with the onset of vari- Pritchard and Manger, 1998; Sun and ous metabolic activities in organisms Davidson, 1998; Sun, 1999). DSC measures (Clegg, 1986). the heat flow of plant tissues associated with various thermal events during cooling and/or heating scans. Such thermal events 2.6.2. Biophysical techniques include phase transitions (e.g. freezing, (see also Chapter 4) melting, glass transition, etc.), polymorphism, thermochemistry and the kinetics Kinetic properties and functions of water for a variety of complex reactions (e.g. in have been studied, using calorimetry vivo protein denaturation). The key idea (Ruegg et al., 1975; Bakradze and Balla, involved in DSC measurement of water sta- 1983; Vertucci, 1990; Sun, 1999), infrared tus is that thermal changes of water and (IR) and Raman spectroscopy (Careri et al., their corresponding quantities of energy 1979; Cameron et al., 1988), NMR spec- are greatly affected by the presence of other troscopy (Fung and McGaughy, 1974; biomaterials in plant tissues, and that ther- Mathur-de Vre, 1979; Seewaldt et al., 1981; mal behaviours of other biomaterials in Rorschach and Hazlewood, 1986; Ratkovic, plant tissues are affected by water content. 1987), quasi-elastic neutron-scattering For example, as water content decreases, spectroscopy (Lehmann, 1984; Trantham et the onset freezing and melting temperature al., 1984) and dielectric relaxation tech- of water decreases due to solute concentra- niques (Harvey and Hoekstra, 1972; tion, while at the same time glass transition Kamiyoshi and Kudo, 1978; Clegg et al., temperature of the tissue increases due to 1982; Pissis et al., 1987, 1996; Bruni and the reduced plasticization effect by water. Leopold, 1992). These techniques differ By analysing thermal behaviours of water greatly in how and what they measure with and biomaterials as a function of water respect to the dynamic properties and content, temperature and time, the status of structures of water and other biomolecules. water in plant tissues can be studied and A great deal of confusion over the physical the water status correlated to its biological state of water in biological systems has functions. resulted from the separation of information Technically, DSC is really a quite simple obtained with diverse techniques applied method. There are two cells in the DSC to similar systems. The nature of different detector, one reference cell and one sample 02 Dessication - Chap 2 18/3/02 1:54 pm Page 75

Methods for Studying Water Relations Under Stress 75

cell. An empty crucible is placed into the 2.6.2.2. Thermally stimulated current (TSC) reference cell and a crucible containing the method tissue sample is placed into the sample Different dielectric relaxation techniques cell. During a DSC experiment, both refer- had been used previously to study the ence cell and sample cell are cooled and/or properties of water in biological systems heated at a constant rate over a range of (Harvey and Hoekstra, 1972; Kamiyoshi temperatures. When a thermal event occurs and Kudo, 1978; Clegg et al., 1982; Careri in the tissue, it releases or absorbs heat and Giansanti, 1984). More recently, the energy (i.e. heat flow). A plot of heat flow TSC technique has been employed to study as a function of temperature is called a the mode of hydration and water organiza- thermogram, from which the thermal tion in plant tissues (Pissis et al., 1987, behaviour of the tissue can be deduced. 1996; Bruni and Leopold, 1992; Sun et al., Glass transition is usually marked by a 1994; Sun, 2000). This technique is capable stepwise shift in the baseline of a thermo- of providing information concerning the gram. This distinguishes it from freezing mobility and rotational freedom of hydra- and melting transitions, which produce tion water, hydration sites and mechanisms peaks. A melting event is accompanied by (Mascarenhas, 1980; Pissis et al., 1987, an endothermic peak and a freezing event 1996; Pissis, 1990; Bruni and Leopold, is accompanied by an exothermic peak. 1992). The TSC technique is based upon: The area under the particular peak repre- (i) the dependence of the microdynamics of sents the total heat energy or enthalpy water dielectric relaxation on their sur- change (H) for the event. DSC is a highly roundings resulting in different dielectric informative tool for analysing biological relaxation times for water in different frac- materials. Other information such as transitions; and (ii) the influence of water on the tion temperature and heat capacity change dielectric relaxation mechanisms of other ( Cp) of the tissue upon cooling or heating biomolecules (similar to those used for can also be calculated from a thermogram. DSC measurements). The difficulty in analysing DSC thermo- The TSC method measures the tiny grams lies in the correct identification of current generated by the thermally acti- origin for thermal events in heterogeneous vated release of stored dielectric polariza- biological samples. For example, lipid tran- tion during controlled heating and sition in seeds can mask the actual glass basically consists of three steps: (i) the transition (Williams and Leopold, 1989) polarization of a sample by a strong d.c. and interfere with the accurate calculation electric field at a particular temperature; of freezing and melting enthalpies (Sun, (ii) ‘freeze-in’ the polarization by cooling 1999). down to a sufficiently low temperature

Table 2.1. Biophysical techniques used to study the dynamic and structural properties of water and macromolecules in biological systems. Type of information Information about Time scale Time- Techniques (s) average Dynamic Structural Water Macromolecule Thermal analysis (DSC, DTA) 101 ~103 ++ X-ray diffraction 101 ~102 ++++ Spectroscopy (NMR, EPR) 104 ~100 + (+) (+) + Relaxation (NMR, EPR, dielectric) 1011 ~100 ++(+) Ultrasonic absorption 1010 ~105 +++ Quasi-elastic neutron scattering 1013 ~107 +++ Infrared and Raman spectroscopy 1016 ~1012 +++ +

DSC, differential scanning calorimetry; DTA, differential thermal analysis; NMR, nuclear magnetic resonance; EPR, electron paragmagnetic resonance. 02 Dessication - Chap 2 18/3/02 1:54 pm Page 76

76 W.Q. Sun

(e.g. liquid nitrogen temperature) while magnetic field at the nucleus.) Since the the field is still on; and (iii) the measure- electron density around each nucleus in a ment of the TSC spectrum during heating molecule varies according to the type of after the d.c. field is disconnected (Bruni nuclei and its molecular environment, the and Leopold, 1992). When a polarized tis- opposing field and thus the effective field sue reaches a temperature at which dipole at each nucleus will differ, which is called molecules (such as water) relax (lose their ‘chemical shift’. The chemical shift of a fixed orientation), a tiny current is gener- nucleus is the difference between the reso- ated and recorded. From a TSC spectrum, nance frequency of the nucleus and a stan- several important physical parameters can dard (relative to the standard, expressed in be obtained, including the intensity of p.p.m., ). The chemical shift is a very pre- depolarization charge (peak size, related cise measure of the chemical environment to the size of the water pool), depolariza- around a nucleus. tion temperature, its activation energy and The major frustration for many biolo- static permittivity. The measurement of gists wishing to understand and to use dielectric relaxation properties of water NMR is the complexity of the subject. and water-plasticized biomolecules offers However, as with other physical tech- valuable insight into the organization of niques used in studies of biological sys- water in plant tissues and the molecular tems, NMR may be used in an ‘empirical’ interactions between water and other bio- mode, simply examining the variation of molecules during desiccation (Bruni and an NMR parameter with the change of Leopold, 1992; Sun, 2000). experimental variable (e.g. water content) (James, 1993). Figure 2.11 shows 1H-NMR spectra of mung bean seeds at three water 2.6.2.3. Nuclear magnetic resonance (NMR) contents. The 1H-NMR spectra were broad, NMR spectroscopy studies the interaction with the line width in the order of 103 Hz. of electromagnetic radiation with matter. It Two NMR peaks were easily identifiable. is a powerful tool for the studies of kinetic The peak of water in the immobile fraction motion of water in tissues and of macro- had a peak maximum (chemical shift) molecule/water or membrane/water inter- value of 4.3 p.p.m. relative to the proton in

actions. Solid-state NMR can be used to D2O, which was used as a standard refer- determine the molecular structure of solid ence. The peak of water in the mobile frac- tissue samples. Solid-state H-NMR is often tion had a peak maximum value at the used to investigate the relaxation character- same place as the proton in D2O (i.e. = 0 istics of the protons of water molecules in p.p.m.). At a water content of 0.07 g g1 low-moisture biological systems (Mathur- dw, water appeared to exist primarily in de Vre, 1979; Seewaldt et al., 1981; the immobile fraction. The very small pro- Rorschach and Hazlewood, 1986; Ratkovic, portion of water in the mobile fraction 1987; Chapter 4). The basic principle of H- appeared as a shoulder in the spectrum. NMR is that each of two hydrogen nuclei The amount of water in the mobile fraction in a water molecule possesses a single spin increased rapidly as water content proton, which will cause the nucleus to increased. At 0.24 g g1 dw, two peaks produce an NMR signal. When an atom is merged almost completely as one peak, placed in a magnetic field, the spin of its centred at = 0 p.p.m. The relative propor- electrons will orient toward the direction tion of the immobile and mobile water frac- of the applied magnetic field. This orienta- tions may be estimated using the standard tion produces a small local magnetic field signal processing techniques. at the nucleus that opposes the externally Two important spin relaxation parame-

applied field, resulting in a smaller mag- ters are T1, the spin–lattice relaxation, and netic field (i.e. effective field) at the T2, the spin–spin relaxation time. The nucleus than the applied field. (Note that spin–lattice relaxation (T1) involves the in some cases it might also enhance the exchange of energy with the environment 02 Dessication - Chap 2 18/3/02 1:54 pm Page 77

Methods for Studying Water Relations Under Stress 77

(the lattice), and is caused by fluctuating come into resonance with monochromatic local magnetic fields arising from the radiation. The magnetic field of most com- motion of the molecules. The spin–spin mercial ESR spectrometers is about 0.3 T,

relaxation (T2) characterizes interactions corresponding to resonance with an elec- between spins and is related to the width tromagnetic frequency of ~10 GHz and

of the NMR peak (Fig. 2.11). T1 and T2 can wavelength of ~3 cm. Therefore, the range be used to study chemical kinetics and of applicability of ESR is narrower than rotational and conformational motion of that of NMR. ESR is basically a microwave molecules. technique, and is one of the fastest-growing areas in analytical instrumentation because of recent and remarkable achievements in 2.6.2.4. Electron spin resonance microwave technology. ESR consists of a (see also Chapter 4) microwave source, a cavity, a microwave Electron spin resonance (ESR) or electron detector and an electromagnet. The sample paramagnetic resonance (EPR) is related to is placed in a glass or quartz tube, which is NMR. It is the study of molecules with inserted into the cavity. The ESR spectrum unpaired electrons (free radicals, transition is obtained by measuring the microwave metal complexes, triplet states, etc.) by absorption as the magnetic ﬁeld strength is observing the magnetic ﬁelds at which they continuously changed. This method

0.24 g g–1 Signal amplitude

0.14 g g–1

0.07 g g–1

D2O

–16 –8 0 8 16 24 Chemical shift (, p.p.m.)

Fig. 2.11. The 1H-NMR spectra of water in mung bean seeds at three water contents. The width of all 1 spectra was 4000 Hz. D2O was used as a standard reference. The inset shows the assignment of H-NMR signal into two different water fractions: mobile water (fraction 1) and immobile water (fraction 2). The spectrum was recorded with FX90QNMR (JEOL Ltd, Japan). The powdered sample, weighing approx. 1–2 g, was loaded into the standard 5 mm NMR tube. A 90° 36-µs electromagnetic pulse was applied to the sample. A total of eight scans were used to improve the resolution. A repeat time was 20 s to re-establish the equilibrium via spin–lattice and spin–spin relaxation before the next scan (W.Q. Sun, unpublished data). 02 Dessication - Chap 2 18/3/02 1:54 pm Page 78

78 W.Q. Sun

detects the number of ‘unpaired spins’ of cals) is commercially available. By incor- electronic charges. The ‘strange’ ESR spec- porating some probes such as nitroxide trum is the first derivative of the derivatives into tissues before drying, microwave energy absorption (Fig. 2.12). detailed studies may be undertaken on the The hyperfine structure (splitting of indi- changes in the aqueous and non-aqueous vidual resonance lines into components) of intracellular environments upon desicca- an ESR spectrum is a fingerprint that helps tion. The ESR technique provides a fairly to identify free radical species in the sam- direct measurement of the change in cyto- ple and characterize their environments. plasmic viscosity, which probably plays The ESR technique does not directly an important role in metabolic down- measure water properties in tissues; how- regulation in desiccation tolerance (see ever, it can be used to study many ques- Chapter 10). tions that are related to desiccation. Using an ESR spin-labelling technique, Belagyi (1975) reported that a portion of cell water 2.7. Concluding Remarks in muscles exhibited a restricted motion. In recent years, Hoekstra and his co-workers Comparative studies play a key role in have used this technique to study mem- understanding the mechanisms or strate- brane behaviours, molecular mobility, cyto- gies of various organisms in the survival of plasmic viscosity and partitioning of desiccation. The water status of tissues in amphiphilic molecules of desiccation-tol- desiccation tolerance studies should be erant and desiccation-intolerant plant tis- expressed precisely by preferred thermody- sues upon desiccation (Golovina et al., namic parameters to permit the compari- 1998; Buitink et al., 1999, 2000a,b,c; son of data from different biological Leprince et al., 1999; Chaper 4). A variety systems. The commonly used parameter, of molecular spin probes (stable free radi- water content, is not adequate for the )

A (a)

) Microwave absorption ( (b) B /d A First derivative (d

Magnetic field (B)

Fig. 2.12. (a) Microwave energy absorption. (b) The peculiar appearance of the electron spin resonance (ESR) spectrum. The ESR spectrum is the ﬁrst derivative signal of microwave energy absorption. The peak of absorption corresponds to the point where the ﬁrst derivative passes through zero (dashed lines). (See Figures in Chapter 4.) 02 Dessication - Chap 2 18/3/02 1:54 pm Page 79

Methods for Studying Water Relations Under Stress 79

expression of tissue water status in most of desiccation stresses would certainly cases. Whenever possible, the researcher improve the mechanistic studies of desic- should first study the components of the cation tolerance. Water plays an important water relations of cells or tissues and role in maintaining the structural integrity obtain important reference parameters of biological systems. Although the kinetic about their water status. The Höfler dia- properties of water in many biological sys- gram and PV curve can be applied to most tems have been extensively studied, the well-hydrated plant tissues, whereas the organization of cellular water and its rela- isothermal sorption study can be applied to tion to desiccation tolerance or desiccation intermediate- to low-moisture systems. damage are not fully understood. Further Drying rate and desiccation stress can be studies on the interactions between water quantified by introducing thermodynamic and macromolecular structures by biophys- concepts into the study of water-loss ical techniques are essential to identify dynamics during desiccation (drying fundamental cellular or metabolic compo- curve). The quantitative (instead of qualita- nents that are associated with desiccation tive) analysis of physico-chemical aspects damage or desiccation tolerance.

2.8. References

Bakradze, N.G. and Balla, Y.I. (1983) Crystallization of intracellular water in plant tissues. Biophysics 28, 125–128. Beall, P.T. (1981) The water for life. The Sciences January 1, 6–29. Beall, P.T. (1983) States of water in biological systems. Cryobiology 20, 324–334. Beckett, R.P. (1997) Pressure–volume analysis of a range of poikilohydric plants implies the existence of negative turgor in vegetative cells. Annals of Botany 79, 145–152. Belagyi, J. (1975) Water structure in striated muscle by spin labeling techniques. Acta Biochimica et Biophysica; Academiae Scientiarum Hungaricae 10, 63–70. Bell, L.N. and Labuza, T.P. (2000) Moisture Sorption: Practical Aspects of Isotherm Measurement and Use. Eagan Press, Eagan, Minnesota, 123 pp. Berjak, P. and Pammenter, N.W. (1994) Recalcitrance is not an all-or-nothing situation. Seed Science Research 4, 263–264. Berjak, P., Pammenter, N.W. and Vertucci, C. (1992) Homoiohydrous (recalcitrant) seeds: developmental status, desiccation sensitivity and the state of water in axes of Landolphia kirkii Dyer. Planta 186, 249–261. Brunauer, S., Emmett, P.H. and Teller, E. (1938) Adsorption of gases in multimolecular layers. Journal of American Chemical Society 60, 309–319. Brunauer, S., Deming, L.S., Deming, W.E. and Teller, E. (1940) On a theory of the van der Waals absorption of gasses. Journal of American Chemical Society 62, 1723. Bruni, F. and Leopold, A.C. (1991) Hydration, protons and onset of physiological activities in maize seeds. Physiologia Plantarum 81, 359–366. Bruni, F. and Leopold, A.C. (1992) Pools of water in anhydrobiotic organisms: a thermally stimulated depolarization current study. Biophysical Journal 63, 663–672. Bruni, F., Careri, G. and Clegg, J.S. (1989) Dielectric properties of Artemia cysts at low water contents: evidence for a percolative transition. Biophysical Journal 55, 331–338. Buitink, J., Claessens, M.M.A.E., Hemminga, M.A. and Hoekstra, F.A. (1998a) Inﬂuence of water content and temperature on molecular mobility and intracellular glasses in seed and pollen. Plant Physiology 118, 531–541. Buitink, J., Walters, C., Hoekstra, F.A. and Crane, J. (1998b) Storage behaviour of Typha latifolia pollen at low water contents: interpretation on the basis of water activity and glass concepts. Physiologia Plantarum 103, 145–153. Buitink, J., Hemminga, M.A. and Hoekstra, F.A. (1999) Characterization of molecular mobility in seed tissues: an EPR spin probe study. Biophysical Journal 76, 3315–3322. 02 Dessication - Chap 2 18/3/02 1:54 pm Page 80

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Rorschach, H.E. and Hazlewood, C.F. (1986) Protein dynamics and the NMR relaxation time T1 of water in biological systems. Journal of Magnetic Resonance 70, 79–88. Ruegg, M., Moor, U. and Blanc, B.H. (1975) Hydration and thermal denaturation of ß-lactoglobulin: calorimetric study. Biochimica et Biophysica Acta 400, 334–342. Rupley, J.A., Gratton, E. and Careri, G. (1983) Water and globular proteins. Trends in Biochemical Sciences 8, 18–22. Rustgi, S.N., Peemoeller, H., Thompson, R.T., Kydon, D.W. and Pintar, M.M. (1978) A study of molecular dynamics and freezing phase transition in tissues by proton spin relaxation. Biophysical Journal 22, 439–452. Sakurai, M., Kawai, K., Inoue, Y., Hino, A. and Kobayashi, S. (1995) Effects of trehalose on the water structure in yeast cells as studied by in vivo 1H NMR spectroscopy. Bulletin of Chemical Society of Japan 68, 3621–3627. Scheidegger, C., Schroeter, B. and Frey, B. (1995) Structural and functional processes during water vapour uptake and desiccation in selected lichens with green algal photobionts. Planta 197, 399–409. Seewaldt, V., Priestley, D.A., Leopold, A.C., Feigenson, W. and Goodsaid-Zalduondo, F. (1981) Membrane organization in soybean seeds during hydration. Planta 52, 19–23. Sinclair, T.R. and Ludlow, M.M. (1985) Who taught plants thermodynamics? The unfulfilled potential of plant water potential. Australian Journal of Plant Physiology 12, 213–217. Stadelmann, E.J. (1984) The derivation of the cell wall elasticity function from the cell turgor potential. Journal of Experimental Botany 35, 859–868. Stannett, V.T., Ranade, G.R. and Koros, W.J. (1982) Characterization of water vapor transport in glassy polyacrylonitrile by combined permeation and sorption techniques. Journal of Membrance Sciences 10, 219–233. Steudle, E., Zimmermann, U. and Luttge, U. (1977) Effect of turgor pressure and cell size on the wall elasticity of plant cells. Plant Physiology 59, 285–289. Sun, W.Q. (1997) Glassy state and seed storage stability: the WLF kinetics of seed viability loss at T > Tg and the plasticization effect of water on seed storage stability. Annals of Botany 79, 291–297. Sun, W.Q. (1998) Function of the glassy state in seed storage stability. In: Taylor, A.G. and Huang, X.L. (eds) Progress in Seed Research. New York State Agricultural Experiment Station, Cornell University, Geneva, New York, pp. 169–179. Sun, W.Q. (1999) State and phase transition behaviors of Quercus rubra seed axes and cotyledonary tissues: relevance to the desiccation sensitivity and cryopreservation of recalcitrant seeds. Cryobiology 38, 372–385. Sun, W.Q. (2000) Dielectric relaxation of water and water-plasticized biomolecules in relation to cellular water organization, cytoplasmic viscosity and desiccation tolerance in recalcitrant seed tissues. Plant Physiology 124, 1203–1215. Sun, W.Q. and Davidson, P. (1998) Protein stability in the amorphous carbohydrate matrix: relevance to anhydrobiosis. Biochimica et Biophysica Acta 1425, 245–254. Sun, W.Q. and Gouk, S.S. (1999) Preferred parameters and methods for studying moisture content of recalcitrant seeds. In: Marzalina, M., Khoo, K.C., Jayanthi, N., Tsan, F.Y. and Krishnapillay, T.M. (eds) Recalcitrant Seeds: Proceedings of the IUFRO Seed Symposium. Forest Research Institute of Malaysia, Kuala Lumpur, pp. 403–430. Sun, W.Q., Irving, T.C. and Leopold, A.C. (1994) The role of sugar, vitrification and membrane phase transition in seed desiccation tolerance. Physiologia Plantarum 90, 621–628. Sun, W.Q., Koh, D.C.Y. and Ong, C.M. (1997) Correlation of modified water sorption properties with the decline of storage stability of osmotically-primed seeds of Vigna radiata (L.) Wikzek. Seed Science Research 7, 391–397. Thomson, W.W. and Platt, K.A. (1997) Conservation of cell order in desiccation mesophyll of Selaginella lepidophylla ([Hook and Grev.] Spring). Annals of Botany 79, 439–447. Tompsett, P.B. and Pritchard, H.W. (1998) The effect of chilling and moisture status on the germination, desiccation tolerance and longevity of Aesculus hippocastarum L. seeds. Annals of Botany 82, 249–261. Trantham, E.C., Rorschach, H.E., Clegg, J.S., Hazlewood, C.F., Nicklow, R.M. and Wakabayashi, N. (1984) The diffusive properties of water in Artemia cells determined by quasi-electron neutron scattering. Biophysical Journal 45, 927–938. 02 Dessication - Chap 2 18/3/02 1:54 pm Page 83

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Appendix: Solutions for Controlled are generally accurate within 2%. RHs of Dehydration and Rehydration commonly used salt solutions between 5 and 40°C are compiled in Table A2.2. RHs Saturated salt solutions of additional salt solutions at 25°C are given in Table A2.3. These data are taken In a closed container, a saturated salt solu- from earlier works of Rockland (1960), tion (with excess salt present) produces a Winston and Bates (1960) and Young constant water vapour pressure at a given (1967), tested and corrected by the author. temperature. Relative humidity (RH) in the Users are advised to avoid salts that release container is calculated by: salt vapours into the atmosphere. When autoclaving is required, the decomposition RH A exp(B/T) (1) temperature of a salt should be checked. where A and B are constants, and T is the After autoclaving, the closed container temperature in kelvin (Wexler, 1997). The should be allowed sufﬁcient time to equili- values of A and B as well as the valid tem- brate (i.e. avoiding condensation and perature range are given in Table A2.1 for supersaturation). The supersaturation in various salts. For example, RH of saturated the liquid phase and the condensation of KCl solution at 25°C is equal to 49.38 water on the wall in the container affect exp(159/298) = 84.2 (%). Calculated values RH signiﬁcantly.

Table A2.1. Commonly used salts, their vapour pressure constants A and B, and the valid temperature range. Data are taken from Wexler (1997). Temperature range Relative humidity Compound (°C) at 25°C AB

NaOH.H2O 15–60 6 5.48 27 LiBr.2H2O 10–30 6 0.23 996 ZnBr2.2H2O 5–30 8 1.69 455 KOH.2H2O 5–30 9 0.014 1924 LiCl.H2O 20–65 11 14.53 75 CaBr2.6H2O 11–22 16 0.17 1360 LiI.3H2O 15–65 18 0.15 1424 CaCl2.6H2O 15–25 29 0.11 1653 MgCl2.6H2O 5–45 33 29.26 34 NaI.2H2O 5–45 38 3.62 702 Ca(NO3) 2.4H2O 10–30 51 1.89 981 Mg(NO3) 2.6H22O 5–35 53 25.28 220 NaBr.2H2O 0–35 58 20.49 308 NH4NO3 10–40 62 3.54 853 KI 5–30 69 29.35 254

SrCl2.6H2O 5 –30 71 31.58 241 NaNO3 10–40 74 26.94 302 NaCl 10–40 75 69.20 25

NH4Cl 10–40 79 35.67 235 KBr 5–25 81 40.98 203

(NH4) 2SO4 10–40 81 62.06 79 KCl 5–25 84 49.38 159

Sr(NO3) 2.4H2O 5–25 85 28.34 328 BaCl2.2H2O 5–25 90 69.99 75 CsI 5–25 91 70.77 75

KNO3 0–50 92 43.22 225 K2SO4 10–50 97 86.75 34 02 Dessication - Chap 2 18/3/02 1:54 pm Page 85

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Table A2.2. Equilibrium relative humidities of saturated salt solutions at different temperatures. Data are taken from Rockland (1960), Winston and Bates (1960) and Young (1967). Temperature Saturated salt solution 5°C 10°C 15°C 20°C 25°C 30°C 35°C 40°C

H2SO4 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 ZnCl2 5.5 – 5.5 – 5.5 – 5.5 – NaOH 6.0 – 6.0 6.0 7.2 – 7.5 – LiBr 9.0 – 8.0 – 7.0 – 7.0 – KOH 13.0 – 9.0 – 8.0 – 8.0 –

LiCl.H2O 14.0 13.5 13.0 12.5 12.0 11.5 11.5 11.0 CaBr2 23.0 – 20.0 18.5 16.5 – 15.0 – KAc 24.8 24.0 23.5 23.0 23.0 23.0 22.0 23.0

MgBr2 32.0 31.0 31.5 31.0 30.5 30.0 30.0 30.0 MgCl2 34.0 33.5 33.5 33.0 32.5 32.0 32.5 32.0 CaCl2 40.0 38.0 35.0 32.5 30.0 – 30.0 – K2CO3 43.0 47.0 44.0 44.0 43.0 42.0 41.5 40.0 NaI 43.5 – 38.0 38.5 38.0 36.0 34.0 32.5

Zn(NO3) 2 45.0 43.0 40.7 40.0 32.5 24.0 21.0 19.0 KCNS 54.0 52.0 50.0 47.0 46.5 43.5 41.5 41.0

Mg(NO3) 2 55.0 53.0 53.7 53.0 52.5 52.0 50.5 51.0 Na2Cr2O7.H2O 59.5 60.0 56.5 54.5 53.0 52.5 51.0 50.0 NaBr 60.5 58.0 58.0 57.8 57.2 57.0 57.0 57.0

Ca(NO3) 2 61.0 66.0 58.0 56.0 52.2 51.0 45.5 46.0 NaBr.2H2O 63.0 61.0 59.0 57.5 56.0 54.5 53.0 51.5 CuCl2 66.7 68.0 68.0 68.5 67.0 67.0 67.0 67.0 LiAc 72.0 72.0 71.0 70.0 68.0 66.0 65.0 64.0

NH4NO3 73.0 75.0 70.0 65.5 62.5 59.5 56.8 53.0 NaCl 76.0 75.8 75.5 75.3 75.1 75.0 75.0 75.0

NaNO3 79.0 77.5 76.5 76.0 74.0 72.5 71.0 70.5 (NH4) 2SO4 81.7 81.2 80.0 79.8 79.7 79.5 79.2 79.0 NH4Cl 82.0 79.0 79.5 79.0 78.0 77.5 75.5 74.0 Li2SO4 84.0 84.0 84.0 85.0 85.0 85.0 85.0 81.0 KBr 85.0 86.0 85.0 83.5 83.0 82.0 81.0 80.0 KCl 87.8 86.7 86.0 85.3 85.0 83.5 83.0 83.0

K2CrO4 89.0 89.0 88.0 88.0 87.0 86.0 84.0 82.0 BaCl2 95.0 93.0 92.0 90.7 90.0 89.0 88.0 87.0 ZnSO4 95.0 93.0 92.0 90.0 88.0 86.0 85.0 84.0 KNO3 96.5 95.5 95.0 94.0 92.5 91.5 89.5 88.5 K2SO4 98.0 97.0 97.0 97.0 97.0 97.0 96.0 96.0 Na2HPO4 98.0 98.0 98.0 98.0 97.0 96.0 93.0 91.0 Pb(NO3) 3 99.0 98.5 98.5 98.5 96.2 95.5 95.2 94.7

Non-saturated salt solutions during equilibration, because the equilibration between the tissue, vapour phase and Non-saturated salt solutions can also be solution phase results in a slight decrease or used. This method allows for the creation of increase in salt concentration. This problem a precise and evenly graded series of RHs (or can be minimized using high mass ratios water potentials) with the same salt. The dis- between the solution and the sample. A advantage of using non-saturated salt solu- mass ratio of 150–200 (i.e. 150–200 g solutions is that they have limited buffering tion g1 tissue) is sufﬁcient. Water potentials capacity relative to saturated salt solutions. of NaCl solutions at different concentrations RHs inside the container are not constant and temperatures between 5 and 40°C are 02 Dessication - Chap 2 18/3/02 1:54 pm Page 86

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Table A2.3. A list of saturated salt solutions (with the presence of excess salt) and their equilibrium relative humidities (RHs) at 25°C. Saturated salt solution RH (%) Saturated salt solution RH (%)

ZnBr2 8.6 SrBr2.6H2O58.5 H3PO4 9.0 FeCl2.4H2O60.0 CaAc2.H2O + sucrose 13.0 NaMnO4.3H2O61.5 CaAc2.H2O 17.0 NH4NO3+ AgNO3 61.5 Ca(CNS) 2.3H2O 17.5 CuBr2 62.5 LiI.3H2O 18.0 CoCl2 64.0 KHCO2 (Formate) 21.5 NaNO2 64.2 KAc.1.5H2O 22.2 K2S2O3 66.0 NiBr2.3H2O 27.0 CuCl2.2H2O67.7 MgBr2.6H2O 31.5 NaCl + NaNO3 69.0 Sr(CNS) 2.3H2O 31.5 SrCl2.6H2O71.0 SrI2.6H2O 33.0 SrCl2 71.0 MnBr2.6H2O 34.5 NH4Cl + KNO3 71.2 Cu(NO3) 2.6H2O 35.0 NaCl + KCl 71.5 Ca(MnO4) 2.4H2O 37.5 NaAc.3H2O73.0 FeBr2.6H2O 39.0 NaCl + Na2SO4.7H2O74.0 NaI.2H2O 39.2 BaBr2 74.5 Mg(ClO4) 2.6H2O 41.0 K tartrate 75.0 CoBr2 41.5 NH4Br2 75.0 CrCl3 42.5 Zn(CNS) 2 80.5 BaI2.2H2O 43.0 NaH2PO4 81.0 K2CO3.2H2O 43.0 AgNO3 82.0 CeCl3 45.5 KCl + KClO3 85.0 LiNO3.3H2O 47.0 KNa tartrate 87.0 Mg(CNS) 2 47.5 Na2CO3.10H2O87.0 KNO2 48.1 MgSO4.7H2O89.0 K4P4O7.3H2O 49.5 BaCl2.2H2O90.3 Co(NO3) 2.6H2O 49.8 Na tartrate 92.0 NH4NO3 + NaNO3 50.0 (NH4)H2PO4 92.7 KBr + urea 51.0 NH4HPO4 93.0 Zn(MnO4) 2.6H2O 51.0 CaH4(PO4) 2.H2O96.0 NiCl2.6H2O 53.0 KH2PO4 96.0 Na2Cr2O7.2H2O 53.7 CaHPO4.2H2O97.0 Ba(CNS) 2.2H2O 54.5 CuSO4.5H2O97.2 Pb(NO3) 2 + NH4NO3 55.0 K2Cr2O7 98.0 MnCl2.4H2O 56.0 KClO3 98.0

given in Table A2.4. Besides NaCl solutions, tial or RH are often not available. PEG is

CaCl2 and KCl solutions offer good RH con- inexpensive and not corrosive, whereas trol. Water potentials of CaCl2 and KCl solu- many salt solutions are corrosive and tions are listed in Table A2.5. volatile. Effects of PEG concentration and temperature on water potential were studied by Michel and Kaufmann (1973). An empiri- Polyethylene glycol (PEG) solutions cal equation has been derived to calculate the water potential of PEG solutions at given PEG solutions are widely used in controlled concentrations and temperatures: dehydration and rehydration. PEG solutions have several advantages over salt solutions. (1.18 103C) – (1.18 105 C2) Salt solutions at high speciﬁc water poten- (2.67 105CT) (8.39 108C 2T) (2) 02 Dessication - Chap 2 18/3/02 1:54 pm Page 87

Methods for Studying Water Relations Under Stress 87

Table A2.4. Water potentials of sodium chloride (NaCl) solutions at different concentrations and temperatures. Mass (%); g solute per 100 g solution. Water potential (MPa) Molality Mass (mol kg1) (%) 5°C 10°C 15°C 20°C 25°C 30°C 35°C 40°C 0.05 0.29 0.22 0.22 0.23 0.23 0.23 0.24 0.24 0.25 0.1 0.58 0.43 0.44 0.45 0.45 0.46 0.47 0.48 0.49 0.2 1.16 0.85 0.87 0.88 0.90 0.92 0.93 0.95 0.96 0.3 1.72 1.27 1.30 1.32 1.34 1.37 1.39 1.42 1.44 0.4 2.28 1.69 1.73 1.76 1.79 1.82 1.86 1.89 1.92 0.5 2.84 2.12 2.16 2.20 2.24 2.28 2.32 2.36 2.40 0.6 3.39 2.54 2.59 2.64 2.69 2.74 2.79 2.84 2.89 0.7 3.93 2.97 3.03 3.09 3.15 3.21 3.27 3.33 3.39 0.8 4.47 3.40 3.47 3.54 3.61 3.68 3.75 3.82 3.89 0.9 5.00 3.83 3.92 4.00 4.08 4.16 4.23 4.31 4.39 1.0 5.52 4.27 4.37 4.46 4.55 4.64 4.73 4.82 4.90 1.1 6.04 4.71 4.82 4.92 5.03 5.13 5.23 5.32 5.42 1.2 6.55 5.16 5.28 5.39 5.51 5.62 5.73 5.84 5.94 1.3 7.06 5.61 5.74 5.87 5.99 6.12 6.24 6.35 6.47 1.4 7.56 6.07 6.21 6.35 6.49 6.62 6.75 6.88 7.01 1.5 8.06 6.53 6.68 6.84 6.99 7.13 7.28 7.41 7.55 1.6 8.55 7.00 7.16 7.33 7.49 7.65 7.81 7.95 8.11 1.7 9.04 7.46 7.64 7.82 8.00 8.17 8.33 8.49 8.65 1.8 9.52 7.94 8.13 8.33 8.52 8.70 8.88 9.04 9.21 1.9 9.99 8.43 8.63 8.84 9.04 9.24 9.43 9.60 9.78 2.0 10.46 8.92 9.13 9.36 9.57 9.78 9.98 10.16 10.35

where C is the concentration of PEG (molec- water potential is calculated with Equation ular weight 6000) in g kg1 water and T is (2). The PEG solution can be dried at 105°C temperature (°C). Water potential of PEG in an oven to a constant dry weight. The solutions is curvilinearly related to the con- problem with submerging the tissue in a centration and increases linearly with tem- PEG solution is that PEG can enter the inter- perature. The error of calculated water cellular spaces. PEG is considered a non- potential is generally within 0.03 MPa in penetrating polymer because it does not comparison with the psychrometric mea- cross the membrane! Our calorimetric study surements. Water potentials of PEG-6000 reveals a PEG melting peak in submerged solutions at concentrations ranging from 10 tissues, even after extensive washing. to 400 g kg1 water and at temperatures between 5 and 40°C are given in Table A2.6. PEG solutions are very viscous, especially at Glycerol solutions high concentrations; hence it takes a longer time to achieve the equilibrium between the Glycerol can also be used to create a pre- PEG solution and the tissue. Caution is cise and evenly graded series of RHs needed to prevent bacterial and fungal cont- between 30 and 98%. Glycerol solutions amination during the study. The equilibra- are safe to use and less corrosive (except tion can be accelerated by placing closed for tin) than salt solutions. Microbial containers in a gently shaking incubator and growth can be effectively inhibited by

in some cases submerging the tissue in solu- adding four drops of saturated CuSO4 solution. The change in PEG concentration after tion to each 100 ml of glycerol solution

the experiment can be determined by the (ASTM, 1983). The CuSO4-treated solution gravimetric method, and the equilibrium can be used repeatedly after the required 02 Dessication - Chap 2 18/3/02 1:54 pm Page 88

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Table A2.5. Water potentials of potassium chloride (KCl) and calcium

chloride (CaCl2) solutions at 20 and 25°C. Mass (%): g solute per 100 g solution.

KCl CaCl2 Mass (%) 20°Ca 25°Cb 20°Ca 25°Cb 0.5 0.28 0.31 0.27 0.28 1 0.56 0.61 0.54 0.57 2 1.12 1.22 1.07 1.16 3 1.68 1.85 1.62 1.81 4 2.26 2.48 2.22 2.50 5 2.83 3.13 2.87 3.24 6 3.42 3.80 3.58 4.02 7 4.02 4.48 4.36 4.89 8 4.64 5.18 5.23 5.77 9 5.25 5.90 6.15 6.73 10 5.87 6.65 7.16 7.76 12 7.18 8.21 9.40 10.09 14 9.89 12.00 12.85 16 11.68 14.99 16.13 18 13.62 18.45 20.02 20 15.69 22.34 24.60 22 26.50 24 20.34 30.89 36.20 26 36.26 28 42.37 51.67 30 50.06 32 60.68 71.78 36 97.33 40 129.12 a Derived from Handbook of Chemistry and Physics, 78th edn (Lide, 1997). Water potential is calculated according to the freezing-point depression. b Data were derived from Robinson and Stokes (1959).

concentration adjustment. The composi- The ASTM’s method uses the refractive tion of glycerol solutions can be accurately index at 25°C to express glycerol concen- determined using speciﬁc gravity or the tration. The relationship between RH, refractive index. Equilibrium RHs of glyc- refractive index and temperature is erol solutions at 24°C were reported by described by the following equations: Braun and Braun (1958). Using the same (R A)2 (100 A)2 A2 (RH A)2 (3) set of data, Forney and Brandl (1992) 0 derived an empirical equation to calculate A 25.6 0.195T 0.0008T 2 (4) equilibrium RHs of glycerol solutions. R 1.3333 (1.398 103 R ) (5) Equilibrium RHs and water potentials of 0 glycerol solutions at 20°C were derived where RH is the desired relative humidity according to the freezing-point depression. (%), R is the refractive index of the glycerol These data are listed in Table A2.7. One solution, and T is temperature (°C). The can calculate the required concentration of value of A is calculated using Equation (4).

a glycerol solution for a desired RH at a Ro is calculated by substituting A and RH given temperature, using the ASTM’s stan- in Equation (3). The refractive index at dard recommended practice (ASTM, 1983). 25°C is calculated using Equation (5). For a 02 Dessication - Chap 2 18/3/02 1:54 pm Page 89

Methods for Studying Water Relations Under Stress 89

glycerol solution of the known refractive where A is deﬁned by Equation (4), and Ro index (R), equilibrium RH at different tem- is calculated from Equation (5). The mea- peratures can be calculated by rearranging surement of refractive index is quite Equations (3), (4) and (5) (Sun and Gouk, tedious. The concentration (mass %) of the 1999). The relationship is: desired glycerol solution has been derived from concentrative properties of aqueous + 2 +−2 +2 − RH =()100 AARAA()0 (6) glycerol solutions (Lide, 1997) by the

Table A2.6. Water potentials of polyethylene glycol (MW 6000) solutions. Data were derived according to Michel and Kaufmann (1973). Water potential (MPa) at different temperatures PEG 1 (g kg H2O) 5°C 10°C 15°C 20°C 25°C 30°C 35°C 40°C 10 0.012 0.010 0.009 0.007 0.006 0.005 0.003 0.002 20 0.025 0.023 0.020 0.017 0.014 0.011 0.008 0.006 30 0.042 0.037 0.033 0.028 0.024 0.020 0.015 0.011 40 0.060 0.054 0.048 0.042 0.036 0.030 0.024 0.018 50 0.081 0.073 0.065 0.058 0.050 0.042 0.034 0.027 60 0.104 0.094 0.085 0.075 0.066 0.056 0.047 0.037 70 0.129 0.118 0.106 0.095 0.083 0.072 0.061 0.049 80 0.157 0.143 0.130 0.116 0.103 0.090 0.076 0.063 90 0.186 0.171 0.156 0.140 0.125 0.109 0.094 0.078 100 0.22 0.20 0.183 0.166 0.148 0.131 0.113 0.096 110 0.25 0.23 0.21 0.194 0.174 0.154 0.134 0.114 120 0.29 0.27 0.25 0.22 0.20 0.179 0.157 0.135 130 0.33 0.30 0.28 0.26 0.23 0.21 0.182 0.157 140 0.37 0.34 0.32 0.29 0.26 0.24 0.21 0.181 150 0.41 0.38 0.35 0.32 0.30 0.27 0.24 0.21 160 0.46 0.43 0.39 0.36 0.33 0.30 0.27 0.23 170 0.51 0.47 0.44 0.40 0.37 0.33 0.30 0.26 180 0.56 0.52 0.48 0.44 0.41 0.37 0.33 0.29 190 0.61 0.57 0.53 0.49 0.45 0.41 0.37 0.33 200 0.66 0.62 0.58 0.53 0.49 0.45 0.40 0.36 210 0.72 0.68 0.63 0.58 0.54 0.49 0.44 0.40 220 0.78 0.73 0.68 0.63 0.58 0.53 0.48 0.43 230 0.84 0.79 0.74 0.68 0.63 0.58 0.53 0.47 240 0.91 0.85 0.79 0.74 0.68 0.63 0.57 0.51 250 0.97 0.91 0.85 0.79 0.73 0.67 0.62 0.56 260 1.04 0.98 0.92 0.85 0.79 0.73 0.66 0.60 270 1.11 1.05 0.98 0.91 0.85 0.78 0.71 0.65 280 1.19 1.11 1.04 0.97 0.90 0.83 0.76 0.69 290 1.26 1.19 1.11 1.04 0.96 0.89 0.82 0.74 300 1.34 1.26 1.18 1.10 1.03 0.95 0.87 0.79 310 1.42 1.34 1.25 1.17 1.09 1.01 0.93 0.85 320 1.50 1.41 1.33 1.24 1.16 1.07 0.99 0.90 330 1.58 1.49 1.41 1.32 1.23 1.14 1.05 0.96 340 1.67 1.58 1.48 1.39 1.30 1.20 1.11 1.01 350 1.76 1.66 1.56 1.47 1.37 1.27 1.17 1.07 360 1.85 1.75 1.65 1.54 1.44 1.34 1.24 1.13 370 1.95 1.84 1.73 1.62 1.52 1.41 1.30 1.20 380 2.04 1.93 1.82 1.71 1.60 1.48 1.37 1.26 390 2.14 2.02 1.91 1.79 1.68 1.56 1.44 1.33 400 2.24 2.12 2.00 1.88 1.76 1.64 1.52 1.40 02 Dessication - Chap 2 18/3/02 1:54 pm Page 90

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Table A2.7. Equilibrium relative humidity (RH) and water potential of glycerol solutions at 20°C and 24°C. Mass (%): g glycerol per 100 g solution. 20°C 24°C Mass (%) Speciﬁc gravity a % RH b b (MPa) % RH c (MPa) 10 1.0215 97.9 2.79 98.0 2.77 12 1.0262 97.4 3.45 97.5 3.47 14 1.0311 96.9 4.16 97.0 4.18 16 1.0360 96.4 4.89 96.5 4.89 18 1.0409 95.8 5.69 95.9 5.74 20 1.0459 95.2 6.52 95.4 6.46 24 1.0561 93.9 8.35 94.1 8.34 28 1.0664 92.4 10.42 92.6 10.55 32 1.0770 90.7 12.71 91.0 12.94 36 1.0876 88.9 15.28 89.2 15.68 40 1.0984 86.9 18.19 87.2 18.79 44 1.1092 84.9 22.46 48 1.1200 82.5 26.39 52 1.1308 79.7 31.13 56 1.1419 76.6 36.57 60 1.1530 73.2 42.78 64 1.1643 69.4 50.11 68 1.1755 65.2 58.68 72 1.1866 60.6 68.71 76 1.1976 55.5 80.77 80 1.2085 49.8 95.64 84 1.2192 43.6 113.88 88 1.2299 36.7 137.51 a Taken from Handbook of Chemistry and Physics, 78th edn (Lide, 1997). b Calculated according to the freezing-point depression. c The relationship between concentration and equilibrium RH was reported by Braun and Braun (1958). The equation derived by Forney and Brandl (1992) was used to determine equilibrium RH of solutions at other concentrations.

author. RHs of glycerol solutions at concen- ASTM’s method is 0.2% at 25°C, and trations between 10 and 92% (mass %) and increases as temperature deviates from at temperatures between 5 and 35°C has 25°C (ASTM, 1983). Data in Table A2.8 are been calculated according to the ASTM’s consistent with those in Table A2.7, which method (Table A2.8). The accuracy of were derived by different methods. 02 Dessication - Chap 2 18/3/02 1:54 pm Page 91

Methods for Studying Water Relations Under Stress 91

Table A2.8. Equilibrium relative humidity (RH) of glycerol solutions at different temperatures. Values were derived, using the refractive index of the solution at 25°C. Mass (%): g glycerol per 100 g solution. RH (%) at different temperatures Mass (%) 5°C 10°C 15°C 20°C 25°C 30°C 35°C 10 98.1 98.1 98.2 98.2 98.3 98.3 98.3 12 97.6 97.7 97.7 97.8 97.8 97.9 97.9 14 97.1 97.2 97.2 97.3 97.4 97.4 97.5 16 96.6 96.6 96.7 96.8 96.9 96.9 97.0 18 96.0 96.1 96.1 96.2 96.3 96.4 96.4 20 95.4 95.5 95.5 95.6 95.7 95.8 95.9 24 94.0 94.1 94.2 94.3 94.4 94.5 94.6 28 92.5 92.6 92.8 92.9 93.0 93.1 93.2 32 90.8 91.0 91.1 91.3 91.4 91.5 91.7 36 88.9 89.1 89.3 89.4 89.6 89.7 89.9 40 86.8 87.0 87.2 87.4 87.6 87.7 87.9 44 84.5 84.7 84.9 85.1 85.3 85.5 85.7 48 82.0 82.2 82.5 82.7 82.9 83.1 83.3 52 79.2 79.5 79.7 80.0 80.2 80.4 80.6 56 76.2 76.5 76.7 77.0 77.2 77.4 77.7 60 72.8 73.0 73.3 73.6 73.8 74.1 74.3 64 69.0 69.3 69.6 69.9 70.1 70.4 70.6 68 64.9 65.2 65.5 65.8 66.1 66.3 66.6 72 60.3 60.6 60.9 61.2 61.5 61.8 62.1 76 55.2 55.6 55.9 56.2 56.5 56.8 57.1 80 49.5 49.8 50.2 50.5 50.8 51.1 51.4 84 43.0 43.4 43.7 44.0 44.4 44.7 45.0 88 35.5 35.9 36.2 36.5 36.9 37.2 37.5

References

ASTM (1983) Maintaining constant relative humidity by means of aqueous solutions. In: 1983 Annual Book of ASTM Standards (Standard E104). American Society for Testing and Materials, Philadelphia, pp. 572–575. Braun, J.V. and Braun, J.D. (1958) A simpliﬁed method of preparing solutions of glycerol and water for humidity control. Corrosion 14, 117–118. Forney, C.F. and Brandl, D.G. (1992) Control of humidity in small controlled environment chambers using glycerol–water solutions. HortTechnology 2, 52–54. Lide, D.R. (ed.) (1997) Handbook of Chemistry and Physics, 78th edn. CRC Press, New York, pp. 8, 57–81. Michel, B.E. and Kaufmann, M.R. (1973) The osmotic potential of polyethylene glycol 6000. Plant Physiology 51, 914–916. Robinson, R.A. and Stokes, R.H. (1959) Electrolyte Solutions, 2nd edn. Butterworths Scientiﬁc Publications, London, 559 pp. Rockland, L.B. (1960) Saturated salt solutions for static control of relative humidity between 5 and 40°C. Analytical Chemistry 32, 1375–1376. Sun, W.Q. and Gouk, S.S. (1999) Preferred parameters and methods for studying moisture content of recalcitrant seeds. In: Marzalina, M., Khoo, K.C., Jayanti, N., Tsan, F.Y. and Krishnapillay, B. (eds) Recalcitrant Seeds. Proceedings of IUFRO Seed Symposium 1998. Forest Research Institute Malaysia, Kuala Lumpur, pp. 404–430. Wexler, A. (1997) Constant humidity solutions. In: Lide, D.R. (ed.) Handbook of Chemistry and Physics, 78th edn. CRC Press, New York, pp. 15, 24–25. Winston, P.W. and Bates, D.H. (1960) Saturated solutions for the control of humidity in biological research. Ecology 41, 232–237. Young, J.F. (1967) Humidity control in the laboratory using salt solutions – a review. Journal of Applied Chemistry 17, 241–245. 02 Dessication - Chap 2 18/3/02 1:54 pm Page 92 Dessication - Chap 03 18/3/02 1:55 pm Page 93

3 Experimental Aspects of Drying and Recovery

Norman W. Pammenter,1 Patricia Berjak,1 James Wesley-Smith1 and Clare Vander Willigen2 1School of Life and Environmental Sciences, University of Natal, Durban 4041, South Africa; 2Department of Botany, University of Cape Town, Private Bag, Rondebosch 7701, South Africa

3.1. Introduction 94 3.2. Drying Rate 94 3.2.1. Commonly employed drying techniques 94 3.2.1.1. Seed material 95 3.2.1.2. Vegetative tissue 97 3.2.2. Quantiﬁcation and modelling of drying rates 98 3.2.3. Effects of different drying rates 99 3.2.3.1 Desiccation-sensitive tissue 99 3.2.3.2. Desiccation-tolerant tissue 100 3.2.4. ‘Ultradrying’ of desiccation-tolerant material 101 3.3. Inﬂuence of Rehydration Technique 102 3.4. Length of Time in the Partially Dehydrated State 102 3.5. Methods of Assessing Response to Rehydration 103 3.5.1. ‘Germination’ 103 3.5.2. Resurrection plants 104 3.5.3. Electrolyte leakage 104 3.5.4. Tetrazolium test 104 3.5.5. Other responses 104 3.6. Expression of Water Content Data 105 3.6.1. Mass basis 105 3.6.2. Water potential 105 3.6.3. Relative water content 106 3.7. Conclusion 106 3.8. References 106

94 N.W. Pammenter et al.

3.1. Introduction dration may have adverse metabolic and other deleterious consequences unrelated When plant material is subjected to dehy- to drying. dration, the observed response (in terms of 3. The rate of air movement across the tis- damage accumulation and survival) can sue; this affects the thickness of the bound- vary with the techniques used to assess the ary layer and the ability of water vapour to response. This has the potential to cause diffuse through it. Forced ventilation will confusion in the interpretation of experi- lead to faster drying than will still air. mental data. It is the objective of this chap- 4. The size and shape of the tissue; this ter to outline the range of techniques that affects the surface area-to-volume ratio and have been used, to note the effects each can the distance water must diffuse from the have on the observed response and, as far interior of the tissue to the surface. Small as possible, to suggest underlying causes of tissue pieces will dry faster than large these effects. ones, and ‘flat’ tissue will dry faster than Experimental or technical aspects that ‘bulky’ tissue. can influence the response to dehydration 5. The chemical and physical composition include drying rate, the length of time the of the outer layer of the tissue; this will tissue is maintained in the (partially) dry affect the permeability of the tissue to state, the rehydration and recovery tech- water, in either the liquid or vapour form. niques, and the method used to assess the Tissues with lignified, suberized or waxy response. The developmental status of the outer layers will dry more slowly than tissue may also influence the response to those without such layers. drying. The information presented in this 6. The amount of material to be dried; this chapter is derived almost exclusively from will affect factors such as surface area-to- studies on seeds and vegetative tissue (vas- volume ratios and boundary layers. cular and non-vascular species). The dis- Generally, small quantities of material will cussion has not been extended to pollen dry faster than will a large mass. and spores, largely because their size is These factors are, to a greater or lesser such that they normally dehydrate rapidly extent, under the control of the experi- and so effects of drying conditions are less menter, who can thus alter (but not control frequently studied (see Chapter 6). with precision) the rate at which the material dries. It should be noted that the terms ‘rapid’, ‘intermediate’ and ‘slow’ are com- 3.2. Drying Rate parative only within a single study; there are no absolute boundaries to these terms. During drying, water molecules diffuse A drying rate that in one study might be from the tissue to the surrounding air. described as ‘rapid’ might be considered to There are a number of factors that can be ‘slow’ in another. affect the rate at which this diffusion occurs and hence the rate at which the tissue will dry. Some of these factors are: 3.2.1. Commonly employed drying 1. The vapour pressure of the surrounding techniques air; this affects the difference in free energy of water between the tissue and the air, and Commonly used drying techniques vary in hence the drying rate. Tissue will obvi- the degree to which temperature and ously dehydrate faster in dry than in humidity are controlled and the extent of humid air. air movement across the material being 2. Temperature; this will also influence the dried. In the case of seeds they also vary in water free energy difference, which will be whether or not outer coverings are greater at higher temperatures. However, removed. The choice of method is often use of elevated temperatures during dehy- influenced by the amount of material that Dessication - Chap 03 18/3/02 1:55 pm Page 95

Experimental Aspects of Drying and Recovery 95

is being dried, the facilities available and will vary amongst species. Cromarty et al. the reason for which the material is being (1985) have given details of the design of dried. Some of the methods are specific for seed bank facilities for orthodox seeds, seeds, and some for resurrection plants, but including detailed consideration of drying many can be used for any small piece or protocols. Drying times using these proto- pieces of tissue. Commonly used tech- cols are of the order of days. niques are summarized in Table 3.1. As 4. Air flow in a laminar flow cabinet drying conditions can have marked effects (Grout et al., 1983; Normah et al., 1986; on the response of tissue to drying (Section Pence, 1992; Chandel et al., 1995). This 3.2.3), it is critical that the drying condi- technique can be used for small seeds and tions (e.g. temperature, relative humidity parts of seeds (excised embryos or embry- (RH), light conditions, air flow) used in any onic axes) and has the advantages that it investigation are reported. introduces an air flow over the material (forced ventilation) and reduces the introduction of further microbial contaminants. 3.2.1.1. Seed material However, the temperature and RH of the air Techniques that have been used to dry seed are not controlled, and will vary with material are outlined below: locality and between seasons, but drying rates of the order of hours can be achieved 1. Air drying of seeds or fruits in sun or with small tissue pieces. shade (e.g. Albrecht, 1993). Of all the tech- 5. Drying seeds by burying them in acti- niques used this one offers the least control vated silica gel (Pammenter et al., 1998). of the various factors affecting drying, but This will yield the most rapid rate of dry- it is commonly used in the field where ing in the absence of forced ventilation, facilities are limited, samples are large and and has been adopted as the standard tech- drying shortly after collection is desired. nique by the IPGRI–Danida Forest Seed When drying seeds, the usual practice is to Centre sponsored project on the handling spread them in an even layer, but, if the and storage of recalcitrant tropical tree layer is more than one seed deep, the sam- seeds (IPGRI/DFSC, 1996). ple must be turned frequently to prevent 6. Rapid air flow over material in a small uneven drying. A similar practice is chamber, i.e. flash drying. A common prac- employed for the whole fruits, for particu- tice is to place the material on a grid in a lar species. Drying using this technique can small container and pass air into the bot- take several days. tom, over the sample, and vent from the 2. Drying under ambient laboratory condi- top of the chamber (Berjak et al., 1990; tions (e.g. Wu et al., 1998) has the same Pammenter et al., 1991). The air may or disadvantages as the first method, except may not be dried. If air from a gas cylinder that ambient conditions probably vary less or a compressor is used, it should be dry. in the laboratory than outside. Drying (As a word of warning, not all compressors times will be similar to those achieved in are properly maintained, and a compressed the first method. air line to a laboratory bench may have 3. Walk-in chambers with temperature droplets of water in it and is obviously and RH control. These facilities are nor- unsuitable.) An improved system designed mally available only in large-scale com- by one of us (J.W.-S.) in which air is circu- mercial or research organizations and are lated through silica gel and over excised generally used to dry substantial quanti- embryonic axes is illustrated in Fig. 3.1; ties of material. Recommended practice is this equipment yields the fastest drying to dry at 10–25°C and 10–15% RH rates of all the techniques we have used (International Plant Genetic Resources (several minutes to hours). Institute (IPGRI), 1994). This approach 7. Drying excised axes under partial vac- permits standard and repeatable drying uum (Fu et al., 1993). Although this tech- conditions, but the drying rate achieved nique yielded rapid drying (faster than Dessication - Chap 03 18/3/02 1:55 pm Page 96

96 N.W. Pammenter et al. ., ., ., et al et al ., 1999 et al ., 1986 et al et al ., 1998; Farrant ., 1991 ., 1985; IPGRI, 1994; et al et al ., 1990; Farrant , 1983; Normah ., 1983; Pammenter et al ., 1992 ., 1997 ., 1992; Vander Willigen, ., 1992; Vander ., 1998 et al et al et al. et al et al et al et al Grout Cromarty Wu 1999 2001 Seel of seeds 1998 Quantity of Drying twigs, moss clumps Drying methodStill air at controlled temperature and RH material Days to weeks chambers time Reference Forced ventilation at controlled temperature and RH MonolayerBuried in silica gel Hours to days Ntuli to hundreds Tens Grout Flash-drying – forced ventilation with or without of minutes Tens Pammenter silica gel to hours Excised plant parts or moss clumps on bench top Segments of leaves, Hours Bartels Small chambers at controlled temperature and RH Hetherington and Smillie, 1982; Forced ventilation in laminar ﬂow hood or small Bochicchio Summary of drying methods and approximate times used in studies on responses to desiccation. Excised axes Laminar ﬂow hood Monolayer Hours Table 3.1. Table Seeds Sun drying Kilograms Days Albrecht, 1993 Vegetative tissueVegetative Withdrawal of watering from whole plant Whole tracheophytes Days Gaff Dessication - Chap 03 18/3/02 1:55 pm Page 97

Experimental Aspects of Drying and Recovery 97

storage above silica gel), none of the axes axes excised from seeds (Grout et al., 1983; survived the treatment. Consequently, this Normah et al., 1986; Pritchard and technique is not recommended without Prendergast, 1986; Berjak et al., 1990). This further investigation. has the advantage of permitting drying 8. Drying to equilibrium at constant RH. rates of the order of tens of minutes to a This is generally done by placing the mate- few hours, but can be very laborious if rial over saturated solutions of salts, within large numbers of axes are required. If this a small chamber. The air can be still or may approach is adopted, it is important to be stirred by a fan in the chamber (Ntuli et minimize damage whilst excising the axes. al., 1997). The advantage of this technique is that the equivalent water potential 3.2.1.2. Vegetative tissue at equilibrium is known ( = (RT/V)*ln(RH/100), where R is the univer- A similar wide range of drying techniques sal gas constant, T the absolute tempera- has been applied to vegetative material, ture, V molal volume of water and RH although in this case the drying is gener- relative humidity). However, under differ- ally for experimental purposes and only ent RH conditions the material will dry at small quantities are dried. One of the few different rates and will reach equilibrium examples of drying large quantities concerns the alga Porphyra (Ooshua, 1993). As after different times, ranging up to several is the case with seeds, the various tech- days. niques differ in the degree of control and Whatever drying method is adopted, the the drying rates achieved. rate of drying is often ultimately deter- In experiments on tracheophytes, a com- mined by the size of the tissue; large seeds mon method is to induce desiccation by the will always dry more slowly than small simple expedient of withholding water (e.g. ones. To overcome this limitation, it has Gaff et al., 1992; Reynolds and Bewley, become common practice to dry embryonic 1993; Quartacci et al., 1997; Vander

Embryonic axes

Plastic mesh

Fan

Silica gel

Fig. 3.1. The improved apparatus for ﬂash-drying of excised embryonic axes. A computer fan circulates air through the silica gel and over the axes. Dessication - Chap 03 18/3/02 1:55 pm Page 98

98 N.W. Pammenter et al.

Willigen et al., 2001). This technique mim- 3.2.2. Quantification and modelling of ics conditions and drying rates (usually drying rates days to weeks) occurring in the natural habitat of the plants, most drying appearing The factors determining drying rate are to occur only after the soil has lost virtually manifold, many of them are difficult to all its water (Sherwin et al., 1998; Norwood quantify and they change during the drying et al., 1999). The size and nature of the process. Consequently, mechanistic model- material (younger leaves enclosed or cov- ling of the drying process is extremely diffi- ered by older ones) often results in uneven cult. During the drying process, water is drying within a single plant. converted from the liquid form in the tissue Faster drying rates (hours to days) have to the vapour phase in the surrounding air. been achieved by using excised tissues The driving force is the difference in the (twigs, leaves or callus) on the laboratory free energy of water between the tissue and bench top, with forced ventilation in a lam- the air. As the tissue dries the free energy of inar flow cabinet, or rapid air flow in a the water decreases, leading to a decrease in small chamber (e.g. Bartels et al., 1990; the free energy differential and so to non- Reynolds and Bewley, 1993; Quartacci et linear drying kinetics. There are a number al., 1997; Farrant et al., 1999). Drying in of resistances retarding the diffusion of small chambers over silica gel or saturated water from the tissue to the air. These salt solutions, with or without stirring the include the boundary layer (the layer of still air, has also been employed (Hetherington air in immediate contact with the tissue), and Smillie, 1982; Bochicchio et al., 1998; the outer layer of the tissue (the nature and Farrant et al., 1999). Non-vascular plants permeability of which vary) and the resis- dry in the field within a few hours tance to the movement of water from the (although this rate could be retarded by the interior of the tissue to the surface. The site clumped nature of the growth form). of evaporation is not known, and so it is Laboratory drying techniques are often unclear whether water moves from the inte- chosen to simulate natural rates, within rior to the tissue surface in the liquid or small enclosed chambers at RHs ranging vapour phase. The boundary layer will be from 50 to 85% at constant temperature influenced by the speed of the air moving (e.g. Seel et al., 1992; Oliver et al., 1993; over the tissue and by the size and shape of Tuba et al., 1996). Very rapid drying has the tissue. With forced ventilation, the air been achieved by the use of silica gel or of flow is probably turbulent, rather than lami- a lyophilizer (Oliver and Bewley, 1984; nar, and so difficult to model. The size and Oliver et al., 1998). shape of seeds and excised axes are often An extremely important experimental irregular and variable, and so their effect on aspect of drying vegetative tissue is the the boundary layer is also difficult to model. light conditions during dehydration, as During the drying process, as the tissue photo-oxidation can be an important com- dehydrates, the resistance to the transfer of ponent of desiccation damage in photosyn- water from the interior to the surface will thetic tissue (Seel et al., 1992; Sherwin and probably change, complicating the analysis. Farrant, 1998; Tuba et al., 1998). Although All this complexity means that mechanistic many seeds have green cotyledons that pre- modelling of drying rates must, by necessity, sumably contain chlorophyll, the light con- be based on simplifying assumptions. ditions during drying have not attracted Cromarty et al. (1985) have suggested a the attention of seed biologists. It is also model for use in seed banks, based on satu- possible that temperature could affect the ration vapour pressure at seed temperature, response; seeds of Zizania palustris are velocity of air over the seed lot, seed mass more tolerant of desiccation when dried at and oil content. The model assumes a thin 25°C than at lower (Kovach and Bradford, layer of seeds and a spherical shape. 1992; Berjak et al., 1994; Ntuli et al., 1997) Empirical models of drying rates are also or higher (Ntuli et al., 1997) temperatures. not always successful. Depending on the Dessication - Chap 03 18/3/02 1:55 pm Page 99

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methods used, and the drying rates heterophyllus (J. Wesley-Smith, unpub- obtained, different factors could be deter- lished data), C. australe (Govindasamy, mining the rate of drying, and the rate-deter- 1997) and T. dregeana (Govindasamy, mining factor could be changing during the 1997; Pammenter et al., 1999) were dried at drying process. Although exponential rela- 96% RH (slowly), drying was exponential; tionships sometimes can be fitted to the data when axes of these species were dried over from a drying time course, particularly for silica gel (rapidly), the initial drying was seeds or excised axes, it is our experience faster than predicted by an exponential that in many cases the same form of equa- rate. Similarly, when seeds of Ekebergia tion cannot be used to describe the data for capensis were dried slowly (seeds with different drying rates (e.g. Pammenter et al., endocarp buried in silica gel; dehydration 1998). As a generalization, if tissue is dried over 10 days), drying was exponential; relatively slowly, the relationship between when seeds were dried more rapidly (seeds water content and drying time is exponen- without endocarp buried in silica gel; tial. However, for material dried rapidly, the dehydration over 1 day), initial drying was initial water loss is considerably faster than faster than predicted by an exponential that predicted by an exponential relation- relationship (Fig. 3.2; Pammenter et al., ship. It must be re-emphasized that the 1998). These authors suggested that the terms ‘slowly’ and ‘rapidly’ are relative. drying kinetics indicated that uneven dry- There is no drying rate common across ing of the tissue might be occurring under species where drying changes from faster rapidly dehydrating conditions. It does than exponential to exponential; a fast dry- appear, however, that excised axes of ing rate in one experiment could be the Theobroma cacao show exponential drying equivalent of slow in another. over a range of drying rates (see Chapter 2). For example, during ‘slow’ drying of whole seeds of Landolphia kirkii (Pammenter et al., 1991), and of Camellia 3.2.3. Effects of different drying rates sinensis (Berjak et al., 1993), the water content of the axes within the seeds followed It is beyond the scope of this chapter to an exponential relationship with time, but discuss in detail the consequences of dehy- ‘rapid’ drying of excised axes did not drating plant tissue. However, reference (curve-fitting exercises were not reported must be made to the subject to understand in the original publications; the data have the influence of drying rate on the response been re-analysed). Similarly, initial faster- to dehydration. The effect appears to than-exponential drying rates have been depend on whether the tissue is inherently observed in rapidly dried excised axes of desiccation-sensitive or -tolerant. Syzigium guiniense, Castanospermum australe, Trichilia dregeana, Artocarpus het- 3.2.3.1. Desiccation-sensitive tissue erophyllus, Azadirachta indica, and the radicle tips of axes of Podocarpus henkelii. Certainly with desiccation-sensitive (recal- By way of contrast, axes of Avicennia citrant) seeds, or embryonic axes excised marina and entire axes of P. henkelii (the from these seeds, material that is dried embryonic axes of both species are rela- rapidly (of the order of tens of minutes to tively large) show exponential drying hours) can survive to lower water contents (N.W. Pammenter, P. Berjak and J. Wesley- before viability is lost than material that is Smith, unpublished observations). It might dried slowly (over a period of days) be argued that the same relationship can- (Normah et al., 1986; Pritchard and not be used to describe ‘slow’ and ‘rapid’ Prendergast, 1986; Farrant et al., 1989; drying because different material is often Pammenter et al., 1991, 1998; Pritchard, used to obtain different drying rates: seeds 1991; Berjak et al., 1993; Pritchard and for slow drying and excised axes for rapid Manger, 1998). The rapid drying is not drying. However, when excised axes of A. actually increasing desiccation tolerance as Dessication - Chap 03 18/3/02 1:55 pm Page 100

100 N.W. Pammenter et al.

2.5 (a) (b) 2.0 dry mass) –1 1.5

1.0

0.5

0.0 02468100 1020304050 Time (days) Time (h) Axis water content (g g

Fig. 3.2. Data illustrating that although the rate of water loss of slowly dried seeds (a) can be described by an exponential equation, that of rapidly dried seeds (b) is not exponential. In (a) the line is an exponential ﬁt to the data; in (b) the dotted line is an exponential ﬁt, the solid line is drawn by eye. Note the different time scales. Data from Pammenter et al. (1998).

such; it is simply that, if the tissue is dried state, it rapidly loses viability (Walters et fast enough, low water contents can be al., 2001). achieved before sufficient time elapses for Studies on the effects of dehydration of it to die. It has been suggested that some of desiccation-sensitive vegetative tissue are the processes leading to viability loss are more limited. However, if much of the vol- aqueous-based and so occur at relatively ume of the cells is occupied by fluid-filled high (intermediate) water contents, of the vacuoles, mechanical damage associated order of 1.0–0.3 g water g1 dry mass, cor- with volume reduction consequent upon responding to water potentials of about drying might be important (Iljin, 1957; 1.5 to 14 MPa (Vertucci and Farrant, Vertucci and Farrant, 1995; Farrant et al., 1995; Farrant et al., 1997; Pammenter and 1997), in which case drying rate might Berjak, 1999; Walters et al., 2001). Material have little effect. that is dried very rapidly passes through these intermediate water contents so fast 3.2.3.2. Desiccation-tolerant tissue that the damage caused by the deleterious processes does not have time to accumu- In tissue that is, or becomes, desiccation- late; thus viability loss does not occur as a tolerant, the effect of drying rate depends consequence (Pammenter et al., 1998; upon the stage of development and/or the Pammenter and Berjak, 1999). Although nature of the tolerance mechanisms. rapid drying does permit viability retention Developing orthodox seeds start to to low water contents, those tolerated by acquire desiccation tolerance concomitant desiccation-sensitive seeds or axes (mini- with, or slightly preceding, reserve accu- mum of about 0.2 g water g1 dry mass) are mulation, and the population as a whole never as low as water contents usually is generally tolerant by the end of this occurring naturally in dry desiccation-tol- stage (e.g. Bewley and Black, 1994). The erant (orthodox) seeds (< 0.05 g water g1 response to rate of drying of orthodox dry mass). Also, survival of these low seeds that are still in the desiccation-sen- water contents by embryonic axes of recal- sitive stage of development is in almost citrant seeds is apparent only if the mater- direct contrast to the response of recalci- ial is assessed for survival immediately trant seeds. If, after histodifferentiation, after drying. If the material is maintained prior to the acquisition of desiccation tol- (at room temperature) in the partially dry erance, a developing orthodox seed is Dessication - Chap 03 18/3/02 1:55 pm Page 101

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dried rapidly, it will not survive; if it is chlorophyll during drying, whereas dried slowly, it will (Kermode and Bewley, species that are poikilochlorophyllous lose 1985; Bewley and Black, 1994). This is chlorophyll and dismantle thylakoid mem- probably because, during slow drying, suf- branes (Hetherington and Smillie, 1982; ficient time elapses for the development of Sherwin and Farrant, 1998), although it is the tolerance mechanisms. possible that some of the observed abnor- The response of desiccation-tolerant malities are a consequence of the fixation vegetative tissue (‘resurrection’ plants) to method (see Platt et al., 1997). drying rate appears to vary with the Poikilochlorophylly appears to preclude nature of the tolerance mechanism rapid drying rates and, although some (reviewed by Oliver and Bewley, 1997). In homoiochlorophyllous plants can survive non-vascular resurrection plants, toler- faster drying rates, there are other species ance seems to be achieved predominantly that cannot (Farrant et al., 1999). by an ability to repair damage caused by Excluding old leaves, which would natu- desiccation, and is thus primarily based rally senesce, all tissues of most resurrec- on constitutive mechanisms (Oliver and tion angiosperms are tolerant; however, Bewley, 1997). Drying rate appears to have there are species in which only the young, little influence on ultimate survival, but immature tissues are tolerant (Gaff and recovery takes longer in rapidly dried tis- Ellis, 1974; Vander Willigen et al., 2001), sue, perhaps suggesting the existence of suggesting that developmental stage is some inducible protection mechanisms another compounding factor. Leaf tissues (Schonbeck and Bewley, 1981a). It has been of some species show tolerance whether observed that non-vascular resurrection attached or detached from the parent plants tend to become less desiccation- plant, whereas others survive only tolerant if kept in the hydrated state for attached, or after an initial drying phase extended periods compared with daily on the parent plant, during which they dehydration/rehydration ‘hardening’ cycles presumably acquire the necessary signals (Schonbeck and Norton, 1979; Schonbeck for tolerance (Gaff and Loveys, 1992). A and Bewley, 1981b). detailed discussion of these responses, Resurrection tracheophytes have been and their underlying causes, is beyond the classified as ‘modified desiccation-tolerant scope of this chapter. Suffice it to say that plants’, in comparison with non-vascular these complexities must be borne in mind resurrection plants (‘fully desiccation-tol- by the investigator when designing and erant plants’), because their ability to sur- interpreting the results of experiments. vive desiccation is rate-dependent (Oliver and Bewley, 1997). The responses of resurrection angiosperms to drying technique 3.2.4. ‘Ultradrying’ of desiccation-tolerant appears to be complex. Rapid desiccation material is generally lethal, although Bochicchio et al. (1998) have shown that it is water con- There has been recent interest in, and tent, rather than drying rate, that affects lively debate on, the effects of storing survival of detached leaves of the resurrec- orthodox seeds at water contents below tion plant Boea hygroscopica. The general those normally used in gene banks. It is not effect of drying rate on the response of res- intended to review that debate here, but urrection tracheophytes is a consequence simply to point out that, even in desicca- of an inducible desiccation tolerance, this tion-tolerant tissue, the experimental tech- tolerance being based on protection during nique – the extent to which the material is desiccation, rather than repair on rehydra- dried – may have an influence on the tion. This group can be further subdivided response to drying (and subsequent stor- into two, based on the strategy to prevent age). For more information, the reader is light-associated damage on drying. The referred to Ellis (1998), Walters (1998) and homoiochlorophyllous species retain Walters and Engels (1998). Dessication - Chap 03 18/3/02 1:55 pm Page 102

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3.3. Influence of Rehydration Technique or more slowly in misting chambers (Seel et al., 1992, Tuba et al., 1996). As with It is well known that if dry biological mate- dehydration, lighting and temperature rial is immersed in water a number of sub- must be carefully controlled. stances of low molecular weight will leak from the tissue (discussed by Hoekstra et al., 1999). If the tissue is desiccation-toler- 3.4. Length of Time in the Partially ant, this leakage will subside with rehydra- Dehydrated State tion, although, if the tissue is initially very dry, leakage can be extensive and could be When desiccation-sensitive tissue is dehy- damaging, this damage being exacerbated drated, it is subjected to a number of by imbibition at low temperatures (Pollock, stresses as it dries. The type of damage that 1969; Hobbs and Obendorf, 1972). Because potentially can occur will change as the of this, it is common in studies on seeds or water content decreases (see Chapter 9); as excised axes to ‘prehumidify’ tissue by the intensity of the stress increases, the maintaining it in a saturated atmosphere or effect on the tissue generally becomes more placing on damp paper, before immersion severe. However, the effect of a stress, par- in water. However, many recalcitrant seeds ticularly a mild stress, is not instantaneous. are damaged at water contents far in excess If a stress induces a metabolic disorder, it of those at which ‘imbibitional damage’ takes time for the damage consequent upon occurs, and there is little evidence to sug- that disorder to accumulate. Thus, the gest that such damage upon imbibition is effect of a stress depends not only on its an important factor in desiccation-sensitive intensity, but also on the time for which the seed material. Kovach and Bradford (1992) stress is applied. It is this concept of ‘inten- initially ascribed the response to desicca- sity’ versus ‘duration’ of a stress that under- tion of seeds of Z. palustris to imbibitional lies the confusion that has obscured the damage, although Vertucci et al. (1995) interpretation of the effects of drying rates suggested that the damage was a direct on desiccation-sensitive seed material. result of desiccation, rather than imbibi- To unravel the confounding issues of tion. Sacandé et al. (1998) have demon- water content and time in drying experi- strated imbibitional damge exacerbated by ments, it is necessary to dry tissue almost low-temperature imbibition and increased instantaneously to a range of water con- storage time in seeds of neem (A. indica) at tents and then to maintain the material at water contents < 8% (fresh mass basis), but these water contents. In practice, this is not this is a water content considerably lower a simple experimental achievement. Most than most recalcitrant seeds will survive. seeds are too large to dry rapidly and, if In vegetative tissues, rehydration tech- isolated embryonic axes are dried, it is dif- niques are generally even less well ficult to ‘store’ them in the partially dehy- described and assessed than are the dehydrated state without some other deleterious dration techniques, and consequently the conditions (such as anoxia or microbial effects of various methods of rehydration proliferation) occurring. Maintaining vege- are relatively unknown. In the tracheo- tative tissue in the partially dehydrated phytes, whole plants are generally rewa- state is probably even more difficult and no tered to field capacity with (Norwood et experiments are known where this has al., 1999) and with or without (Sherwin been attempted. and Farrant, 1998) additional aerial spray- Despite these difficulties, some informa- ing. Dehydrated excised leaves are rehy- tion is available. Walters et al. (2001) have drated by floating on or in water (Gaff and shown that partially dehydrated embryonic Loveys, 1992; Reynolds and Bewley, 1993; axes of tea lose viability within a few days, Bochicchio et al., 1998; Dace et al., 1998). and that the rate at which viability is lost Mosses are rehydrated by immersion in depends on the water content. Similarly, distilled water (Oliver and Bewley, 1984) isolated axes of T. dregeana dried over sil- Dessication - Chap 03 18/3/02 1:55 pm Page 103

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ica gel lost viability as the water content 3.5.1. ’Germination’ reached the level in equilibrium with the desiccant, whilst axes dried at 96% RH lost With seeds, the most common assessment viability some days after the tissue had method is to set them out to germinate. reached equilibrium (Pammenter et al., However, as pointed out by Hong and Ellis 1999). Similarly, in the relatively long-lived (1996), it is possible that the treatment may seeds of Araucaria huntsteinii, longevity at induce a dormancy and that seeds that do 6°C was reduced as water content was not germinate may not actually be dead. reduced below about 45% (Pritchard et al., Those authors recommended that the dura- 1995). As an aside, this raises questions tion of germination tests be extended until concerning the concept of ‘degree of recal- all non-germinated seeds have been posi- citrance’. Would this be assessed on the tively identified as dead by the fact that they basis of the minimum water content to rot. Another problem is that a seed may pro- which the seed (or embryonic axis) can be duce a radicle, and so be scored as having dried without loss of viability, or on the germinated, but be so damaged as to be time for which it survives in equilibrium unable to establish a viable seedling (Fu et with some predetermined water potential? al., 1993; Berjak et al., 1999). The precision The concept of ‘intensity’ versus ‘duration’ of a germination test can be increased by fol- of a stress has practical applications. It has lowing the time course of germination. An been suggested that, in the case of recalci- increased lag before the first seed germi- trant seeds that germinate in storage, partial nates or a decrease in the rate of germina- dehydration may prevent this and so tion (increased time to 50% germination) increase storage life span (Hong and Ellis, may indicate damage that is repaired during 1996). However, it appears that even a mild the lag phase. A simple assessment of final water stress applied to seeds of the tropical germination would not reveal this damage. species T. dregeana is deleterious (Drew et A number of suggestions have been made al., 2000), and relatively mild partial drying concerning fitting equations to germination of the temperate seeds of A. huntsteinii data (Brown and Mayer, 1988), but they gen- (Pritchard et al., 1995) and Aesculus hip- erally require more samples than are often pocastanum (Tompsett and Pritchard, 1998) available when undertaking studies on can reduce longevity. Thus ‘sub-imbibed recalcitrant seeds from wild species. storage’ should be approached with caution When experiments are conducted on as it can actually reduce life span. excised embryonic axes, ‘germination’ can In passing, it should be noted that even be assessed by placing the axes on damp desiccation-tolerant organisms do not have paper in an enclosed chamber (such as a an infinite life span in the dehydrated Petri dish), although it is common to use in state; they accumulate damage in this state, vitro growth media. As many recalcitrant and so desiccation could be considered to seeds, particularly from the tropics, har- be a stress, even in tolerant tissues. bour fungal propagules (Berjak, 1996; Calistru et al., 2000), their removal by suitable pretreatment is essential under these 3.5. Methods of Assessing Response to conditions. As with seeds, care must be Dehydration taken in assessing ‘germination’. Swelling and/or greening of an axis suggests that it A variety of techniques have been used to is not dead (although a dehydrated axis assess damage in response to drying. will swell on rehydration), but it does not Different techniques measure different phe- necessarily imply that it is capable of pro- nomena (membrane characterisics, respira- ducing an independent plantlet. tory competence, photosynthetic activity), Assessment can also be complicated by but the ultimate test is whether an indepen- choice of the medium, as this may have an dent functioning organ(ism) can be re-estab- influence on the ‘growth’ of the tissue (as it lished after dehydration and rehydration. does in normal in vitro multiplication and Dessication - Chap 03 18/3/02 1:55 pm Page 104

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propagation). Material that has been dam- damage associated with imbibition. With aged, but not killed, by the dehydration seeds or excised axes there is generally treatment may take a considerable time to good agreement between leakage character- show signs of growth, and so care should istics and other signs of damage such as be taken not to discard it too early. loss of vigour or viability (McKersie and Tomes, 1980; Pammenter et al., 1991; Berjak et al., 1992, 1993). Older techniques 3.5.2. Resurrection plants to assess membrane integrity involve the use of vital dyes, which leak from damaged With whole resurrection plants, the criteria cells (Gaff and Loveys, 1992). for determining whether the organism is ‘functional’ may vary. This may simply be on physical appearance (particularly the 3.5.4. Tetrazolium test greening of poikilochlorophyllous tissue), measurement of chlorophyll ﬂuorescence The reduction of colourless tetrazolium

characteristics (Fv/Fm), which indicates the chloride (2,3,5-triphenyl tetrazolium chlo- photochemical efficiency of photosystem ride (TTZ)) to a pink/red formazan dye is II), or assessment of the ability to assimi- taken as a measure of respiratory activity, as late carbon dioxide photosynthetically, or TTZ is reduced by components of the mito- to respire. chondrial electron transport chain. Although the tetrazolium test is used extensively to assess the quality of orthodox 3.5.3. Electrolyte leakage seeds, its use in the study of desiccation response has been limited. Ntuli et al. A technique that is commonly employed (1997) showed that considerable differences with small pieces of tissue (small seeds, occurred between the ability to germinate excised axes, leaves of resurrection tra- and apparent viability as a result of tetra- cheophytes, ‘pieces’ of non-vascular zolium tests, when investigating the desic- plants) is to measure leakage of electrolytes cation response of Z. palustris, indicating or of specific ions such as K+. An advan- that the two tests were not equivalent or not tage of this technique is its simplicity and necessarily measuring the same thing. A rapidity, especially using multiple-cell further caveat in using the TTZ test as a via- electrical conductivity meters, which are bility assay is that a dead seed supporting a commercially available. The extent of elec- vigorous mycelium internally will test posi- trolyte leakage is considered to assess the tive as a result of fungal respiration. degree of membrane damage (Bramlage et al., 1978; McKersie and Tomes, 1980). As the rate of leakage over the first few min- 3.5.5. Other responses utes is often higher than the steady rate established later, the steady-state rate of A number of biochemical and biophysical leakage is often taken as an indication of responses to dehydration have been membrane damage (McKersie and Stinson, assessed by a variety of workers. Examples 1980). An alternative approach is to assess include the activities of antioxidants leakage (or rate of leakage) after a given (Tommasi et al., 1999), accumulation of late time as a proportion of total leakage (or embryogenesis abundant proteins (Finch- maximum rate), which occurs when all Savage et al., 1994; Gee et al., 1994), ethyl- membranes are fully disrupted by treat- ene production, respiration and protein ments such as homogenizing, boiling, auto- synthesis (Salmen Espindola et al., 1994), claving or repeated freeze/thaw cycles. It is partitioning of amphipathic molecules common practice to prehumidify tissue in between cytosol and membranes (Golovina a saturated atmosphere or on damp filter et al., 1998) and changes in cytoplasmic vis- paper prior to immersion to reduce any cosity (Leprince and Hoekstra, 1998). These Dessication - Chap 03 18/3/02 1:55 pm Page 105

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are not covered here because often it was change in ‘water content’ reﬂects the pro- the details of these responses that were the portional change in the amount of water in objectives of the investigations, and so they the tissue; if the water content changes do not fall within the scope of a discussion from 1.0 to 0.5 g water g1 dry weight, the of general experimental approaches. tissue has lost half its water. If the data are expressed on a fresh mass basis, for tissue at 1.0 g water g1 dry mass that loses half 3.6. Expression of Water Content Data its water, water content on a fresh mass (see also Chapter 2) basis changes from 50% to 33.3%.

3.6.1. Mass basis 3.6.2. Water potential The amount of water in plant tissue has been expressed in a number of different The responses of tissue to drying are deter- ways, but generally most commonly on mined by the processes that occur during some form of ‘mass’ basis. This is probably drying. The processes that occur at any because it is the simplest measure to water content are influenced by the free obtain; the hydrated/partially hydrated tis- energy status of the water, and so it is bio- sue is weighed, dried for some predeter- logically more meaningful to express tissue mined time, and reweighed. The water in terms of water potential rather International Seed Testing Association rec- than water content. Water potential of ommends drying at 103°C for 17 h (ISTA, small pieces of tissue can be measured by 1999); an alternative approach, particularly thermocouple psychrometry, but this is if using small amounts of tissue, is to dry at limited to higher potentials (above about a lower temperature (to reduce loss of non- 5 MPa). However, recent technical water volatile material) to constant weight. advances permit dewpoint psychrometric The data can then be expressed on a fresh measurements to much lower water poten- mass basis (mass of water per unit fresh tials. Sorption isotherms (equilibrating tis- mass, often presented as a percentage); this sue at known fixed RH to constant water indicates the proportion of the hydrated or content) can be used to establish the rela- partially dehydrated tissue that is water. tionship between water content and water Alternatively, the data can be expressed on potential (Vertucci et al., 1994), although a dry mass basis (mass of water per unit this is difficult at high water potentials dry mass). Strictly speaking, when express- because the relationship between RH and ing the data on a mass basis, the term water potential changes rapidly in this ‘water content’ is incorrect; this should be region. Soaking tissue in solutions of reserved for expressing the absolute known concentrations (and hence known amount of water in the tissue, irrespective water potentials) of non-penetrating solutes of the quantity of tissue. What is generally such as polyethylene glycol (PEG) 8000 has described as ‘water content’ is actually a been used to assess the water ‘water concentration’ (the amount of water content/water potential relationship at per unit amount of fresh or dry tissue). high water potentials (Vertucci et al., 1994; Thus, water content on a dry mass basis Pritchard et al., 1995; Tompsett and should be termed ‘dry mass-specific water Pritchard, 1998). A complication of estab- concentration’. However, use of the term lishing sorption isotherms is the phenome- ‘water content’ to describe a ‘water concen- non of hysteresis; the equilibrium water tration’ is so deeply entrenched that it is content at any RH depends on whether dry unlikely to change. We prefer data to be tissue is being hydrated or hydrated tissue expressed on a dry mass basis. In this case, is losing water (for a discussion, see Eira et the basis to which values are being normal- al., 1999). There is an additional problem ized does not change as the amount of when working with recalcitrant seeds or water changes, and the proportional axes of tropical species. This material gen- Dessication - Chap 03 18/3/02 1:55 pm Page 106

106 N.W. Pammenter et al.

erally harbours very high levels of fungal complication when calculating RWC is the propagules, and fungal proliferation almost estimate of water content at ‘full turgor’. If invariably accompanies attempts to equili- recalcitrant seeds are immersed in water, brate tissue with atmospheres of RH above they will take up water as they germinate, about 80%; it is therefore impossible to and so full turgor cannot be equated with discriminate between the contribution of water content of tissue imbibed in water. the seed material and the fungal mycelium Data could be expressed relative to the to the derived isotherm. Under these con- water content at shedding, but this can be ditions, use of concentrated solutions of very variable among seeds within a harvest, PEG 8000 is advised. as well as between collections. With vegetative tissue it is possible to over-hydrate tissue such that liquid water occupies some of 3.6.3. Relative water content the intercellular air spaces in leaves, or exists as intercellular or surface water in Relative water content, RWC (amount of non-vascular plants (Beckett, 1997). These water in the tissue/amount of water at full effects lead to overestimates of water con- turgor), has also been used to express the tent at full turgor. water status of tissue (see Grange and Finch- Savage (1992) for seed tissue, and Vander Willigen et al. (2001) for vegetative tissue). 3.7. Conclusion It is more meaningful than simple water contents, although relative cell volume When investigating the response of tissue (which is based on symplastic water only) is to dehydration, a range of techniques are probably a better measure of direct stress to available. However, the observed response which the tissue is subjected. To assess the is likely to depend on factors such as the proportions of apoplastic and symplastic drying method, and possibly the rehydra- water in tissue requires the construction of tion technique and method of assessment. pressure–volume curves. Not only is this The techniques adopted will depend on difﬁcult (because of the difﬁculty of measur- the size or amount of tissue being dried, ing water potential at low water contents), the facilities available and, importantly, the but it is possible that the assumptions purpose of the investigation. The investiga- underlying the analysis of pressure–volume tor should be aware of the technical com- curves (Tyree and Hammel, 1972) may not plications when assessing and interpreting hold at low water contents. An additional the data obtained.

3.8. References

Albrecht, J. (ed.) (1993) The Tree Seed Handbook of Kenya. GTZ, Nairobi, Kenya, 264 pp. Bartels, D., Schneider, K., Terstappen, G., Piatkowski, D. and Salamini, F. (1990) Molecular cloning of abscisic acid-modulated genes which are induced during desiccation of the resurrection plant Craterostigma plantagineum. Planta 181, 27–34. Beckett, R.P. (1997) Pressure–volume analysis of a range of poikilohydric plants implies the existence of negative turgor in vegetative cells. Annals of Botany 79, 145–152. Berjak, P. (1996) The role of microorganisms in deterioration during storage of recalcitrant and intermediate seeds. In: Ouédraogo, A.S., Poulsen, K. and Stubsgaard, F. (eds) Intermediate/ Recalcitrant Tropical Forest Tree Seeds. IPRGI, Rome, pp. 121–126. Berjak, P., Farrant, J.M., Mycock, D.J. and Pammenter, N.W. (1990) Recalcitrant (homoiohydrous) seeds: the enigma of their desiccation-sensitivity. Seed Science and Technology 18, 297–310. Berjak, P., Pammenter, N.W. and Vertucci, C.W. (1992) Homoiohydrous (recalcitrant) seeds: developmental status, desiccation sensitivity and the state of water in axes of Landolphia kirkii Dyer. Planta 186, 249–261. Berjak, P., Vertucci, C.W. and Pammenter, N.W. (1993) Effects of developmental status and dehydra- Dessication - Chap 03 18/3/02 1:55 pm Page 107

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tion rate on characteristics of water and desiccation-sensitivity in recalcitrant seeds of Camellia sinensis. Seed Science Research 3, 155–166. Berjak, P., Bradford, K.J., Kovach, D.A. and Pammenter, N.W. (1994) Differential effects of temperature on ultrastructural responses to dehydration in seeds of Zizania palustris. Seed Science Research 4, 111–121. Berjak, P., Walker, M., Watt, M.P. and Mycock, D.J. (1999) Experimental parameters underlying failure or success in germplasm cryopreservation: a case study on zygotic axes of Quercus robur L. Cryo-Letters 20, 251–262. Bewley, J.D. and Black, M. (1994) Seeds: Physiology of Development and Germination, 2nd edn. Plenum Press, New York. Bochicchio, A., Vazzana, C., Puliga, S., Alberti, A., Cinganelli, S. and Vermieri, P. (1998) Moisture content of the dried leaf is critical to desiccation tolerance in detached leaves of the resurrection plant Boea hygroscopica. Plant Growth Regulation 24, 163–170. Bramlage, W.J., Leopold, A.C. and Parrish, D.J. (1978) Chilling stress to soybeans during imbibition. Plant Physiology 61, 525–529. Brown, R.F. and Mayer, D.G. (1988) Representing cumulative germination. 2. The use of the Weibull function and other empirically derived curves. Annals of Botany 61, 127–138. Calistru, C., McLean, M., Pammenter, N.W. and Berjak, P. (2000) The effects of mycoﬂoral infection on the viability and ultrastructure of wet-stored recalcitrant seeds of Avicennia marina (Forssk.) Vierh. Seed Science Research 10, 341–353. Chandel, K.P.S., Chaudhury, R., Radhamani, J. and Malik, S.K. (1995) Desiccation and freezing sensitivity in recalcitrant seeds of tea, cocoa and jackfruit. Annals of Botany 76, 443–450. Cromarty, A.S., Ellis, R.H. and Roberts, E.H. (1985) Design of Seed Storage Facilities for Genetic Conservation. International Board for Plant Genetic Resources (now IPGRI), Rome. Dace, H., Sherwin, H.W., Illing, N. and Farrant, J.M. (1998) Use of metabolic inhibitors to elucidate mechanisms of recovery from desiccation stress in the resurrection plant Xerophyta humilis. Plant Growth Regulation 24, 171–177. Drew, P.J., Pammenter, N.W. and Berjak, P. (2000) ‘Sub-imbibed’ storage is not an option for extending longevity of recalcitrant seeds of the tropical species, Trichilia dregeana Sond. Seed Science Research 10, 355–363. Eira, M.T.S., Walters, C. and Caldas, L.S. (1999) Water sorption properties in Coffea spp. seeds and embryos. Seed Science Research 9, 321–330. Ellis, R.H. (1998) Longevity of seeds stored hermetically at low moisture contents. Seed Science Research 8 (Suppl. 1), 9–10. Farrant, J.M., Pammenter, N.W. and Berjak, P. (1989) Germination-associated events and the desiccation sensitivity of recalcitrant seeds – a study on three unrelated species. Planta 178, 189–198. Farrant, J.M., Pammenter, N.W., Berjak, P. and Walters, C. (1997) Subcellular organization and metabolic activity during the development of seeds that attain different levels of desiccation tolerance. Seed Science Research 7, 135–144. Farrant, J.M., Cooper, K., Kruger, L.A. and Sherwin, H.W. (1999) The effect of drying rate on the survival of three desiccation-tolerant angiosperm species. Annals of Botany 84, 371–379. Finch-Savage, W.E., Pramanik, S.K. and Bewley, J.D. (1994) The expression of dehydrin proteins in desiccation-sensitive (recalcitrant) seeds of temperate trees. Planta 193, 478–485. Fu, J.R., Xia, Q.H. and Tang, L.F. (1993) Effects of desiccation on excised embryonic axes of three recalcitrant seeds and studies on cryopreservation. Seed Science and Technology 21, 85–95. Gaff, D.F. and Ellis, R.P. (1974) Southern African grasses with foliage that revives after dehydration. Bothalia 11, 305–308. Gaff, D.F. and Loveys, B.R. (1992) Abscisic acid levels in drying plants of a resurrection grass. Transactions of Malaysian Society of Plant Physiology 3, 286–287. Gaff, D.F., Bartels, D., Gaff, J.L. and Schneider, K. (1992) Gene expression at low RWC in two hardy tropical grasses. Transactions of Malaysian Society of Plant Physiology 3, 238–240. Gee, O.H., Probert, R.J. and Coomber, S.A. (1994) ‘Dehydrin-like’ proteins and desiccation tolerance in seeds. Seed Science Research 4, 135–141. Golovina, E.A., Hoekstra, F.A. and Hemminga, M.A. (1998) Drying increases intercellular partioning of amphiphilic substances into the lipid phase: impact on membrane permeability and signiﬁ- cance for desiccation tolerance. Plant Physiology 118, 975–986. Dessication - Chap 03 18/3/02 1:55 pm Page 108

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Govindasamy, R. (1997) Desiccation rate, desiccation response and damage accumulation: can desiccation sensitivity be quantiﬁed? Unpublished BSc (Hons) thesis, University of Natal, Durban, South Africa, 63 pp. Grange, R.I. and Finch-Savage, W.E. (1992) Embryo water status and development of the recalcitrant species Quercus robur L.: determination of water relations parameters by pressure–volume analysis. Journal of Experimental Botany 43, 657–662. Grout, B.W.W., Shelton, K. and Pritchard, H.W. (1983) Orthodox behaviour of oil palm seed and cryopreservation of the excised embryo for genetic conservation. Annals of Botany 52, 381–384. Hetherington, S.E. and Smillie, R.M. (1982) Humidity-sensitive degreening and regreening of leaves of Borya nitida Labill. as followed by changes in chlorophyll ﬂuorescence. Australian Journal of Plant Physiology 9, 587–599. Hobbs, P.R. and Obendorf, R.L. (1972) Interaction of initial seed moisture and imbibitional temperature on germination and productivity of soybean. Crop Science 12, 664–667. Hoekstra, F.A., Golovina, E.A., van Aelst, A.C. and Hemminga, M.A. (1999) Imbibitional leakage from anhydrobiotes revisited. Plant Cell and Environment 22, 1121–1131. Hong, T.D. and Ellis, R.H. (1996) A Protocol to Determine Seed Storage Behaviour. IPGRI, Rome, 64 pp. Iljin, W.S. (1957) Drought resistance in plants and physiological processes. Annual Review of Plant Physiology 3, 341–363. IPGRI (International Plant Genetic Resources Institute) (1994) Genebank Standards. FAO/IPGRI, Rome, 13 pp. IPGRI/DFSC (1996) The project on handling and storage of recalcitrant and intermediate tropical forest tree seeds. Newsletter 1, July 1996. ISTA (International Seed Testing Association) (1999) Rule 9.5.8, Low constant temperature oven method. Seed Science and Technology 27 (Suppl. International Rules for Seed Testing, Rules 1999), 49. Kermode, A.R. and Bewley, J.D. (1985) The role of maturation drying in the transition from seed development to germination. 1. Acquisition of desiccation-tolerance and germinability during development of Ricinus communis L. seeds. Journal of Experimental Botany 36, 1906–1915. Kovach, D.A. and Bradford, K.J. (1992) Imbibitional damage and desiccation tolerance of wild rice (Zizania palustris) seeds. Journal of Experimental Botany 43, 747–757. Leprince, O. and Hoekstra, F.A. (1998) The responses of cytochrome redox state and energy metabolism to dehydration support a role for cytoplasmic viscosity in desiccation tolerance. Plant Physiology 118, 1253–1264. McKersie, B.D. and Stinson, R.H. (1980) Effect of dehydration on leakage and membrane structure in Lotus corniculatus L. seeds. Plant Physiology 66, 316–320. McKersie, B.D. and Tomes, D.T. (1980) Effects of dehydration treatments on germination, seedling vigour, and cytoplasmic leakage in wild oats and birdsfoot trefoil. Canadian Journal of Botany 58, 471–476. Normah, M.N., Chin, H.F. and Hor, Y.L. (1986) Desiccation and cryopreservation of embryonic axes of Hevea brasiliensis Muell.-Arg. Pertanika 9, 299–303. Norwood, M., Truesdale, M.R., Richter, A. and Scott, P. (1999) Metabolic changes in leaves and roots during dehydration of the resurrection plant Craterostigma plantagineum (Hochst). South African Journal of Botany 65, 421–427. Ntuli, T.M., Berjak, P., Pammenter, N.W. and Smith, M.T. (1997) Effects of temperature on the desiccation responses of seeds of Zizania palustris. Seed Science Research 7, 145–160. Oliver, M.J. and Bewley, J.D. (1984) Plant desiccation and protein synthesis. IV. RNA synthesis, stability, and recruitment of RNA into protein synthesis during desiccation and rehydration of the desiccation-tolerant moss, Tortula ruralis. Plant Physiology 74, 21–25. Oliver, M.J. and Bewley, J.D. (1997) Desiccation-tolerance of plant tissues: a mechanistic overview. Horticultural Reviews 18, 171–213. Oliver, M.J., Mischler, B.D. and Quisenberry, J.E. (1993) Comparative measures of desiccation-tolerance in the Tortula ruralis complex. I. Variation in damage control and repair. American Journal of Botany 80, 127–136. Oliver, M.J., Wood, A.J. and O’Mahony, P. (1998) ‘To dryness and beyond’ – preparation for the dried state and rehydration in vegetative desiccation-tolerant plants. Plant Growth Regulation 24, 193–201. Dessication - Chap 03 18/3/02 1:55 pm Page 109

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Ooshua, T. (1993) The cultivation of Porphyra ‘Nori’. In: Ohno, M. and Critchley, A.T. (eds) Seaweed Cultivation and Marine Ranching. Japan International Co-operation Agency, Nagai, Japan, pp. 57–74. Pammenter, N.W. and Berjak, P. (1999) A review of recalcitrant seed physiology in relation to desiccation-tolerance mechanisms. Seed Science Research 9, 13–37. Pammenter, N.W., Vertucci, C.W. and Berjak, P. (1991) Homeohydrous (recalcitrant) seeds: dehydration, the state of water and viability characteristics in Landolphia kirkii. Plant Physiology 96, 1093–1098. Pammenter, N.W., Greggains, V., Kioko, J.I., Wesley-Smith, J., Berjak, P. and Finch-Savage, W.E. (1998) Effects of differential drying rates on viability retention of Ekebergia capensis. Seed Science Research 8, 463–471. Pammenter, N.W., Berjak, P. and Walters, C. (1999) The effect of drying rate, and processes leading to viability loss in recalcitrant seeds. In: Marzalina, M., Khoo, K.C., Jayanthi, N., Tsan, F.Y. and Krishnapillay, B. (eds) IUFRO Seed Symposium 1998 Recalcitrant Seeds. Forestry Research Institute Malaysia, Kuala Lumpur, Malaysia, pp. 14–24. Pence, V.C. (1992) Desiccation and the survival of Aesculus, Castanea and Quercus embryo axes through cryopreservation. Cryobiology 29, 391–399. Platt, K.A., Oliver, M.J. and Thomson, W.W. (1997) Importance of ﬁxative for reliable ultrastructural preservation of poikilohydric plant tissues. Observations on dry, partially, and fully hydrated tissues of Selaginella lepidophylla. Annals of Botany 80, 599–610. Pollock, B.M. (1969) Imbibitional temperature sensitivity of Lima bean seeds controlled by initial seed moisture. Plant Physiology 44, 907–911. Pritchard, H.W. (1991) Water potential and embryonic axis viability in recalcitrant seeds of Quercus rubra. Annals of Botany 67, 43–49. Pritchard, H.W. and Manger, K.R. (1998) A calorimetric perspective on desiccation stress during preservation procedures with recalcitrant seeds of Quercus robur L. Cryo-Letters 19 (Suppl. 1), 23–30. Pritchard, H.W. and Prendergast, F.G. (1986) Effects of desiccation and cryopreservation on the in vitro viability of embryos of the recalcitrant seed species Araucaria huntsteinii K. Schum. Journal of Experimental Botany 37, 1388–1397. Pritchard, H.W., Tompsett, P.B., Manger, K. and Smidt, W.J. (1995) The effect of moisture content on the low temperature responses of Araucaria huntsteinii seed and embryos. Annals of Botany 76, 79–88. Quartacci, M.F., Forli, M., Rascio, N., Dalla Vecchia, F., Bochicchio, A. and Navari-Izzo, F. (1997) Desiccation-tolerant Sporobolus staphiainus: lipid composition and cellular ultrastructure during dehydration and rehydration. Journal of Experimental Botany 48, 1269–1279. Reynolds, T.L. and Bewley, J.D. (1993) Characterization of protein synthetic changes in a desiccation- tolerant fern, Polypodium virginianum. Comparison of the effects of drying, rehydration and abscisic acid. Journal of Experimental Botany 44, 921–928. Sacandé, M., Hoekstra, F.A., van Pijlen, J.G. and Groot, S.P.C. (1998) A multifactorial study of conditions inﬂuencing the longevity of neem (Azadirachta indica) seeds. Seed Science Research 8, 473–482. Salmen Espindola, L., Noin, M., Corbineau, F. and Côme, D. (1994) Cellular and metabolic damage induced by desiccation in recalcitrant Araucaria angustifolia embryos. Seed Science Research 4, 193–201. Schonbeck, M.W. and Bewley, J.D. (1981a) Responses of the moss Tortula ruralis to desiccation treatments. I. Effects of minimum water content and rates of dehydration and rehydration. Canadian Journal of Botany 59, 2698–2706. Schonbeck, M.W. and Bewley, J.D. (1981b) Responses of the moss Tortula ruralis to desiccation treatments. II. Variations in desiccation tolerance. Canadian Journal of Botany 59, 2707–2712. Schonbeck, M.W. and Norton, T.A. (1979) Drought-hardening in the upper-shore seaweeds Fucus spi- ralis and Pelvetia canaliculata. Journal of Ecology 67, 687–696. Seel, W.E., Hendry, G.A.F. and Lee, J.A. (1992) The combined effect of desiccation and irradiance on mosses from xeric and hydric habitats. Journal of Experimental Botany 43, 1023–1030. Sherwin, H.W. and Farrant, J.M. (1998) Protection mechanisms against excess light in the resurrection plants Craterostigma wilmsii and Xerophyta viscosa. Plant Growth Regulation 24, 203–210. Sherwin, H.W., Pammenter, N.W., February, E., Vander Willigen, C. and Farrant, J.M. (1998) Xylem Dessication - Chap 03 18/3/02 1:55 pm Page 110

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hydraulic characteristics, water relations and wood anatomy of the resurrection plant Myrothamnus ﬂabellifolius Welw. Annals of Botany 81, 567–575. Tommasi, F., Paciolla, C. and Arrigoni, O. (1999) The ascorbate system in recalcitrant and orthodox seeds. Physiologia Plantarum 105, 193–198. Tompsett, P.B. and Pritchard, H.W. (1998) The effect of chilling and moisture status on the germination, desiccation tolerance and longevity of Aesculus hippocastanum L. seed. Annals of Botany 82, 249–261. Tuba, Z., Csintalan, Z. and Proctor, M.C.F. (1996) Photosynthetic responses of a moss, Tortula ruralis, ssp. ruralis, and the lichens Cladonia convoluta and C. furcata to water deﬁcit and short periods

of dessication, and their ecophysiological signiﬁcance: a baseline study at present-day CO2 concentration. New Phytologist 133, 353–361. Tuba, Z., Proctor, M.C.F. and Csintalan, Z. (1998) Ecophysiological responses of homoiochlorophyllous and poikilochlorophyllous desiccation tolerant plants: a comparison and an ecological perspective. Plant Growth Regulation 24, 211–217. Tyree, M.T. and Hammel, H.T. (1972) The measurement of the turgor pressure and water relations of plants by the pressure-bomb technique. Journal of Experimental Botany 23, 267–282. Vander Willigen, C., Pammenter, N.W., Mundree, S.G. and Farrant, J.M. (2001) Some physiological comparisons between the resurrection grass, Eragrostis nindensis, and the related desiccation- sensitive species, E. curvula. Plant Growth Regulation 35, 121–129. Vertucci, C.W. and Farrant, J.M. (1995) Acquisition and loss of desiccation tolerance. In: Kigel, J. and Galili, G. (eds) Seed Development and Germination. Marcel Dekker, New York, pp. 237–271. Vertucci, C.W., Crane, J., Porter, R.A. and Oelke, E.A. (1994) Physical properties of water in Zizania embryos in relation to maturity status, water content and temperature. Seed Science Research 4, 211–224. Vertucci, C.W., Crane, J., Porter, R.A. and Oelke, E.A. (1995) Survival of Zizania embryos in relation to water content, temperature and maturity status. Seed Science Research 5, 31–40. Walters, C. (1998) Ultra-dry technology: perspective from the National Seed Laboratory, USA. Seed Science Research 8 (Suppl. 1), 11–14. Walters, C. and Engels, J. (1998) The effects of storing seeds under extremely dry conditions. Seed Science Research 8 (Suppl. 1), 3–8. Walters, C., Pammenter, N.W., Berjak, P. and Crane, J. (2001) Desiccation damage, accelerated ageing and respiration in desiccation tolerant and sensitive seeds. Seed Science Research 11, 135–148. Wu, X.-M., Wu, N.-F., Qian, X.-Z., Li, R.-G., Huang, F.-H. and Zhu, L. (1998) Phenotypic and geno- typic changes in rapeseed after 18 years of storage and regeneration. Seed Science Research 8 (Suppl. 1), 55–64. Dessication - Chap 04 18/3/02 1:55 pm Page 111

4 Biochemical and Biophysical Methods for Quantifying Desiccation Phenomena in Seeds and Vegetative Tissues

Olivier Leprince1 and Elena A. Golovina2,3 1UMR Physiologie Moléculaire des Semences, Institut National d’Horticulture, 16 Bd Lavoisier, F49045, Angers, France; 2Laboratory of Plant Physiology, Department of Plant Sciences, University of Wageningen, Arboretumlaan 4, 6703 BD Wageningen, The Netherlands; 3Timiryazev Institute of Plant Physiology, Botanicheskaya 35, Moscow 127276, Russia

4.1. Introduction 112 4.2. Caveats: the Consequences of Being Dry 112 4.3. How to Study Biochemical Responses to Drying 114 4.3.1. Responses of gas exchange and volatile emission to drying 114 4.3.1.1. Headspace analysis 114 4.3.1.2. Laser photoacoustic spectroscopy (PA) 114 4.3.2. NMR applications to measure steady-state concentrations and to assess metabolic responses to drying 115 4.3.3. Photosynthesis studies 116 4.3.4. Oxidative stress and anhydrobiosis 116 4.4. Spectroscopy Techniques 119 4.4.1. Electron paramagnetic resonance (EPR) 119 4.4.1.1. General description 119 4.4.1.2. Applications of EPR methods 120 4.4.2. Nuclear magnetic resonance 127 4.4.2.1. General description 127 4.4.2.2. The NMR study of water in living systems 128 4.4.2.3. NMR imaging 130 4.4.2.4. High-resolution multinuclear NMR spectroscopy 131 4.4.2.5. Structure and dynamics of cellular membranes 133 4.4.3. Fourier transform infrared (FTIR) spectroscopy 134 4.4.3.1. General description of infrared spectroscopy 134 4.4.3.2. Biological applications 134 4.5. Additional Techniques to Study Biochemical and Biophysical Aspects of Desiccation Tolerance 136 4.5.1. Differential scanning calorimetry (DSC) 136 4.5.2. Electron microscopy 136 4.6. Acknowledgements 137 4.7. References 137

112 O. Leprince and E.A. Golovina

4.1. Introduction resort to non-invasive techniques that enable the investigator to assess any bio- In recent years, the development of physi- chemical phenomenon in drying tissues cal techniques has brought substantial without introducing water during the insights into the physical state of water and analysis. The few techniques available are cellular components of desiccated systems. briefly presented; and (ii) characterize These techniques cover a large range of mutants and/or transgenic plants the phe- spectroscopic techniques such as nuclear notypic traits of which can be associated magnetic resonance (NMR), electron para- both with a particular biochemical path- magnetic resonance (EPR, also referred to way and the level of desiccation tolerance as electron spin resonance (ESR)), Fourier (Chapter 12; Wolkers et al., 1998a; Shiota transform infrared (FTIR) spectroscopy as and Kamada, 2000; Weber et al., 2000; well as dielectric measurements and Wehmeyer and Vierling, 2000). For exam- calorimetry. The success of these tech- ple, the antisense inhibition of ADP-glu- niques and spectroscopy in particular orig- cose pyrophosphorylase, a key enzyme in inated in their versatility and in their starch synthesis, was found to increase the ability to assess the physical state of desic- sucrose and nitrogen content of mature cated systems by non-invasive means. seeds of transgenic Vicia narbonensis. Below, the resourcefulness and limitations Also, the decrease in ADP-glucose of the physical techniques are reviewed. pyrophosphorylase activity altered the cel- In contrast to physical aspects, most of lular volume and water relations during the fundamental questions pertaining to the seed-filling phase (Weber et al., 2000). the biochemical aspects of desiccation These observations show that a specific tolerance in anhydrobiotes remain to be alteration in carbon metabolism has answered. The lack of non-destructive pleiotropic effects on seed development and sensitive techniques has greatly and illustrate the potential of molecular impeded our understanding of the role of biology to assess non-destructively the role metabolism in desiccation tolerance. of various biochemical and biophysical phe- Furthermore, all of our biochemical assays nomena related to desiccation tolerance. and isolation of organelles have been set up in dilute solutions using water or organic compounds as solvents. However, 4.2. Caveats: the Consequences of Being they have been indiscriminately applied to Dry drying and desiccated specimens. Here it is argued that the experimental approach Since water acts as a solvent and substrate of grinding dry or nearly dried specimens in the cell in a variety of ways, its reduced in aqueous buffers and measuring meta- availability in dried tissues will induce a bolic activities and biological markers of set of physical and biochemical responses oxidative stress in vitro in dilute solutions that may disappear during an invasive is unlikely to reflect the in vivo situation. measurement, thereby confusing the inter- This methodology has complicated the pretation of the data. Before ascertaining a interpretation of data regarding biochemi- cause–effect relationship between desicca- cal activities associated with different tion tolerance and a specific biochemical hydration levels (Lynch and Clegg, 1986; and biophysical process, the following Vertucci and Leopold, 1986). Hoekstra and remarks should be taken into account: van Roekel (1983) have clearly illustrated how isolation-inflicting injury of isolated 1. To adequately link the response of mitochondria in germinating pollen can metabolism to drying with desiccation tol- confuse the interpretation of results erance, it is necessary to map metabolic obtained in vitro. To partially overcome activities as a function of water content or this technical bottleneck, two strategies water potential of the drying cell and not can be adopted: (i) whenever possible, as a function of time of drying (Vertucci Dessication - Chap 04 18/3/02 1:55 pm Page 113

Methods for Quantifying Desiccation Phenomena 113

and Leopold, 1986; Leprince et al., 1999, oxidative reactions) occurring during nat-

2000). This necessity is more acute when ural ageing (i.e. below Tg) are compared accumulation of dry matter occurs during with those occurring during accelerated

development. Often a sensitive microbal- ageing (between Tc and Tg (for example, ance is needed to determine fresh and dry 75–85% relative humidity (RH) and tem- weights of milligram quantities of samples. peratures between 35 and 50°C) or above

Furthermore, fast measuring techniques Tc (100% RH, 41°C)). This is illustrated in should be preferred over time-consuming the study of Lievonen et al. (1998) on non- assays so that the rates of water loss match enzymatic browning reaction rates around

the time necessary to acquire the data. the Tg of mixtures made of water, glycerol Furthermore, Hendry (1993) argued that and maltodextrin. It is also clearly illus- attempts to characterize desiccation phe- trated in the kinetics of seed and pollen nomena in drying tissues should be set ageing (see, for example, Buitink et al., against a range of desiccation-induced 2000g and references therein). damage and not only against percentages of 3. To be quantiﬁed and characterized, survival after drying. metabolites, proteins, DNA, organelles, 2. It is important to recognize that during etc., must be extracted and puriﬁed drying the cytoplasmic viscosity increases beforehand. This procedure strongly dramatically until glass formation depends on the tissue water content. This (Leprince and Hoekstra, 1998; Buitink et is valid for both water-soluble compounds al., 2000e; Chapters 2 and 10). Unless using aqueous extraction and lipid-solu- enzymatic activities are assessed in an ble compounds using organic solvents. environment similar to that found in the For lipid extraction and separation using drying cells, we do not know how the rise the Folch’s method, Hamilton et al. (1992) in viscosity during drying will affect meta- recommended that the amount of water bolic rates and/or pathways. We can present in the tissues should be calculated

already predict that O2-processing systems and taken into account during the differ- will be altered by the loss of water since ent washing and partitioning procedures.

O2 solubility is known to decrease with For aqueous extraction, we do not know rising viscosity (Gros et al., 1992; Leprince whether the extraction and puriﬁcation of and Hoekstra, 1998). Biochemical events water-soluble metabolites or proteins dif- during drying should obey different laws fer quantitatively and qualitatively when of diffusion since the cytoplasm will tissues are ground either fresh or dried. undergo a physical transformation from a This point is particularly relevant for liquid state to a solid-like state (i.e a glass). amphiphilic molecules such as phenolic The moisture contents at which these compounds, which are likely to partition changes in diffusion characteristics occur into the membranes and/or oil bodies dur- during drying should preferably be deter- ing drying and vice versa during rehydra- mined. It has been suggested that this tion (Chapter 10; Golovina et al., 1998; moisture content corresponds to the glass Buitink et al., 2000e). Thus, the desicca- formation that is measured by the tempera- tion-induced changes in metabolite con- ture at which the drying cytoplasm forms a centrations and in protein conformation

glass (Tg). Below Tg, solid state should be interpreted with caution if these physics/chemistry prevails (Chapter 10). changes are assessed from crude extracts. However, Buitink et al. (2000f) suggested 4. Most of the methods used so far are that the most important change in the averaging techniques. However, it is physical properties of the cytoplasm likely that there is a gradient of water

occurs 50°C above Tg, at a temperature cor- within seed tissues during dehydration responding to the so-called collapse tem- and rehydration. Therefore, a qualitative

perature (Tc). A distinction between liquid and quantitative gradient of responses state and solid state should be made when within the tissues submitted to drying chemical and biochemical events (such as might be expected. Dessication - Chap 04 18/3/02 1:55 pm Page 114

114 O. Leprince and E.A. Golovina

4.3. How to Study Biochemical ter (Rogerson and Matthews, 1977; Vertucci Responses to Drying and Leopold, 1986, 1987). In these techniques, the gas to be analysed has to accu- Two technically different strategies are mulate over time in a closed environment available to probe the responses of metabo- before taking the measurement (i.e. static lism to drying by non-invasive means: the headspace analysis). To reach a detectable detection and analysis of gases that concentration, the gas accumulation may emanate from, or are absorbed by, the dry- take some time, particularly in drying sam- ing tissues (so-called headspace analysis) ples in which the metabolism and gas diffu- and the resort to in vitro or in vivo NMR. In sion are greatly reduced by the lack of photosynthetic anhydrobiotes, fluorescence water. Consequently, the assay may be too spectroscopy is also a convenient tool to slow in comparison with the rate of water characterize energy metabolism during dry- loss. Furthermore, an additional problem is ing (see Chapter 7). The methods that reliably maintaining the specimen at the assess free-radical-induced damage in des- same water content during the measure- iccation tolerance are also reviewed. ment. Thus, unless the kinetics of water loss matches the time frame needed to assess the metabolic rates, the relation 4.3.1. Responses of gas exchange and between hydration levels and metabolic volatile emission to drying activities in drying tissues may not be accurate. Therefore, it is best to adapt a flow- 4.3.1.1. Headspace analysis through system coupled to an active trapping system (i.e. dynamic headspace A wide variety of gaseous metabolites can analysis). A flow of dry or humidified air

be studied in drying tissues: O2 and CO2 passes over the sample acting as both a gas exchange as markers of respiration or photo- carrier and dehydrating agent. The volatiles synthesis, ethanol and acetaldehyde as are then absorbed by a compound located markers of fermentation, alkanes, alkones, in the exit ﬂow and analysed by GC (Wilson alkenes, aldehydes (volatile) as markers of and McDonald, 1986; Zhang et al., 1994). oxidative stress. Several instruments such For dried tissues that are in a glassy state,

as infrared CO2 analysers, gas-phase O2 elec- the release of volatiles may take several trodes or O2 analysers can detect and days or weeks because of the slow diffusion analyse CO2 and O2 exchanges. These types of molecules. To overcome this problem, of analysers have mainly been used to several authors have used a thermal desorp- describe the effects of desiccation on photo- tion technique consisting of heating the dry synthetic activities of resurrection plants specimens to at least 60°C. This procedure (Schwab et al., 1989) and photosymbiotic is thought to purge the volatiles that are lichens (Nash et al., 1990; Scheidegger et trapped in the glassy matrix (Wilson and al., 1995). However, they are not always McDonald, 1986; Hailstones and Smith, suited to assaying low respiration rates from 1989; Zhang et al., 1994; Degoussée et al., nearly dried material because of their low 1995). However, it is not always possible to sensitivity (O. Leprince, unpublished data). determine whether the production of Gas exchange rates and volatile produc- volatiles after desorption results from the tion can also be measured by gas chro- heat treatment per se or not (Wilson and matography (GC) (Kimmerer and McDonald, 1986). Kozlowski, 1982; Gorecki et al., 1984; Klein and Sachs, 1992; Leprince and Hoekstra, 4.3.1.2. Laser photoacoustic spectroscopy 1998; Leprince et al., 1999), gas chromatog- (PA) raphy–mass spectrometry (GC-MS) (Zhang et al., 1994), high-pressure liquid chro- PA is an emerging technique that has over- matography (HPLC) (Degoussée et al., 1995) come the problems associated with head- or by using a Gilson differential respirome- space analysis. A PA set-up consists mainly Dessication - Chap 04 18/3/02 1:55 pm Page 115

Methods for Quantifying Desiccation Phenomena 115

of two components: a line-tunable CO laser appears to be the most appropriate tech- that excites gas molecules specifically nique for this purpose (Shachar-Hill and according to their infrared fingerprint Pfeffer, 1996; Roberts, 2000). Several strate- absorption, and parallel resonators. Each gies can be adopted using 13C-, 31P-, 14N- or resonator is coupled to a sensitive micro- 15N-NMR, depending on the nature of the phone in which the concentration of gases metabolite to be analysed. NMR studies is sequentially measured based on an may or may not be destructive. The princi- acoustic phenomenon (Harren and Reuss, ples of NMR spectroscopy and the advan- 1997; see Zuckerman et al., 1997, tages and disadvantages of in vivo www.sci.kun.nl/tracegasfac/experime.htm applications as a non-invasive technique for technical details of the experimental will be described below in Section 4.4 (see set-up). This technique permits the probing also Shachar-Hill and Pfeffer, 1996). of metabolic processes non-invasively that A first strategy is to monitor dynamic result in the emission and/or absorption of changes of natural or enriched nuclei in the

ethylene, acetaldehyde, ethanol and CO2 samples over different intervals during dry- from drying tissues without altering the ing. This approach may or may not be water content (Leprince et al., 2000). destructive. In both cases, it yields dynamic Furthermore, technical improvements are information about metabolic fluxes and fast being made to allow the analysis of lipid responses of metabolism to physiological peroxidation products such as ethene, perturbations. The natural abundance of 13C ethane, pentane and hexane. PA techniques is only 1.1%. Thus 13C-NMR is not a sensi- offer two important advantages over con- tive method and requires concentrations in ventional headspace analysis: (i) biologi- the mM range. However, one can take cally relevant gases can be detected with a advantage of this low sensitivity to trace sensitivity limit of 100- to 1000-fold higher some metabolic changes by the detection of than GC; and (ii) the time response is less compounds that accumulate to high levels. than 1 min. The PA is set up as a flow- These metabolites include compatible through system. The gas employed to dry solutes that accumulate in cyanobacteria the tissues is also the carrier of the (Reed et al., 1985), sugars and oil in seeds gas/volatile to be analysed. The disadvan- (Rutar, 1989; Ishida et al., 1990, 1996; tages are: (i) to our knowledge, the access to Koizumi et al., 1995), and trehalose in fun- PA is restricted to a handful of laboratories gal spores (Bécard et al., 1991). The non- in The Netherlands (www.sci.kun.nl/ invasive character of NMR may allow the tracegasfac/), Germany, Italy and the US; (ii) time-course of metabolic events to be the equipment is not commercially avail- directly followed and the subcellular local- able and the current prototypes require the ization of some metabolites to be deter- assistance of physicists and engineers to mined, for instance, in maturing or take and process the measurements; (iii) a germinating seeds (Colnago and Seidl, limited range of biologically relevant gases 1983; Ishida et al., 1990, 1996). can be measured; and (iv) water vapour Alternatively, since the natural abun- strongly interferes with the measurement dance of 13C is low, it is possible to label and must be totally removed from the gas. specific metabolites and monitor their fate through the cellular network of metabolic pathways in vivo or in vitro with crude 4.3.2. NMR applications to measure steady- extracts (Dieuaide-Noubhani et al., 1995; state concentrations and to assess metabolic Roberts, 2000; Roscher et al., 2000). responses to drying (see also Section 4.4.2) Commercially n-13C-labelled sugars are available for almost every carbon position To determine the effects of desiccation on of sucrose, glucose and fructose molecules. the dynamics of metabolic pathways, the Similarly 31P- and 14N-labelled compounds flux of metabolites through the different can be used to monitor the dynamics of paths must be known. NMR spectroscopy phosphorylated metabolites and amino Dessication - Chap 04 18/3/02 1:55 pm Page 116

116 O. Leprince and E.A. Golovina

acids, respectively. Whether in vivo NMR 4.3.4. Oxidative stress and anhydrobiosis is applicable to drying tissues remains to be ascertained. A particular problem is to Whether oxidative stress is a cause or an maintain reliably the specimen in the same effect of desiccation sensitivity has yet to functional state and with the same water be resolved due to a large body of conflict- content during the NMR measurements. ing evidence in the literature. The core of A second strategy, which is not mutu- the problem is two fold. From a physiologi- ally exclusive to that above, is to screen cal point of view, Hendry (1993) argued chemical fingerprints of crude extracts that attempts to correlate free radical obtained from different hydration levels. processes with desiccation tolerance must This application will yield analytical infor- be done in relation to the characterization mation about metabolites in a steady state. of other desiccation-induced damage. This By comparing spectra of crude extracts must be done in order to assess whether obtained during drying, it should be possi- free radicals generated during drying are a ble to pinpoint the metabolites, the concen- cause or an effect of the loss of viability. trations of which are mostly sensitive to Echoing the earlier review of Gutteridge changes in water content during drying and Halliwell (1990) and Leprince et al. (Fan, 1996; Noteborn et al., 2000). Roughly (1990), he pointed out that free-radical- 0.5–1 g of fresh material is often required mediated injury can occur before or after to take an NMR spectrum, which could be the time of death during drying (Hendry, a limiting factor if the amount of biological 1993). Thus, great caution should be exer- material is restricted. cised in ascertaining whether oxidative stress plays a role in desiccation-induced injury and loss of desiccation tolerance. 4.3.3. Photosynthesis studies Oxidative injury in both animals and plants generally results from stress-induced meta- The recent methods to assess photosyn- bolic disturbances, particularly in the elec- thetic activities have become non-destruc- tron transport chains. Considering the tive. They exploit the interactions between technical difficulties in estimating meta- light, gas exchange, operation of the photo- bolic activities during drying (see Section synthetic electron transport and ambient 4.3, p. 114), even greater care should then conditions. For these reasons, they are be taken in attempts to link oxidative dam- widely used to study the photosynthetic age to desiccation-induced metabolic per- responses to environmental stresses turbation. Various pathologies and (Bukhov et al., 1989; Foyer et al., 1994). degenerative diseases have been linked to They have also been applied to compare the mitochondrial dysfunction and generation photosynthetic responses of anhydrobiotes of reactive O2 species (ROS) in mammals with those of desiccation-sensitive plants (Yates and van Houten, 1997; Esposito et (Schwab and Heber, 1984; Schwab et al., al., 1999; Wallace, 1999). Thus, it should 1989; see Chapter 7). The most applied be interesting to see whether parallels exist technique is chlorophyll a fluorescence, between animal and plant anhydrobiotes. which assesses the efficiency of electron The second problem regarding the puta- transport through photosystem II and non- tive role of oxidative stress in desiccation photochemical quenching processes associ- tolerance and ageing is the methodology ated with it. Light-induced electron that has been employed so far. A survey of transport in photosystem II can be studied methods employed to detect oxidative stress using fluorescence induction kinetics in seeds can be found in Benson (1990). (Vertucci and Leopold, 1986). Light-scattering measurements at 535 nm are also useful 1. The array of techniques that have been to gain insights into the physical and chem- employed to assess the role of free-radical- ical events associated with the formation of induced injury in anhydrobiotes is very a membrane potential in thylakoids. small. A survey of the literature on molecu- Dessication - Chap 04 18/3/02 1:55 pm Page 117

Methods for Quantifying Desiccation Phenomena 117

lar markers used to estimate free-radical- function and cell death (Yates and van induced damage during drying or in the Houten, 1997). Oxidation of proteins by dry state shows that the results are based ROS can induce protein fragmentation or mostly on a thiobarbituric acid-reactive enzyme inactivation, leading to the disrup- substances (TBARS) assay and a non-inva- tion of glycolysis (Hyslop et al., 1988) and sive EPR spectroscopy technique (Table the Calvin cycle (Kaiser, 1979). Protein oxi- 4.1). The former measures malonyldialde- dation has been linked to various patholo- hyde (MDA) as a breakdown product of gies in humans (Dean et al., 1993; Berlet lipid peroxidation using a simple and and Stadtman, 1997) and to seed ageing rapid assay (Heath and Packer, 1968) and (Zhang et al., 1997). the latter estimates a carbon free radical of 3. Analytical procedures to estimate lipid unknown origin (Hendry, 1993). As dis- hydroperoxides in crude extracts are cussed below, doubts have been cast as to fraught with potential artefacts (Gutteridge whether these two assays are reliable and Halliwell, 1990; Hageman et al., 1992; enough for all anhydrobiotic material. Meagher and Fitzgerald, 2000). The prob- 2. Table 4.1 also shows that, in 98% of lems are numerous, ranging from the pres- these studies, lipid peroxidation was mea- ence of contaminants (metal ions) that sured as a marker of free-radical-induced initiate lipid peroxidation during tissue injury. It must be recognized that proteins grinding to instability of peroxidized lipids (Dean et al., 1993; Berlet and Stadtman, during the extraction and lack of sensitiv- 1997) and DNA (von Sonntag and ity. Furthermore, the nature of peroxidative Schuchmann, 1987; Hageman et al., 1992; damage depends on the type of free-radical Wiseman and Halliwell, 1996) are also sen- initiator and the membrane or lipid compo- sitive to free radicals, albeit less than fatty sition (McKersie et al., 1990). Thus, the acids. Interestingly, mitochondrial DNA is lipid peroxidation assays currently used in more sensitive to ROS than nuclear DNA. seed science cannot be indiscriminately Free-radical-induced DNA damage can sig- applied to all seeds. Unfortunately, the niﬁcantly contribute to mitochondrial dys- studies surveyed in Table 4.1 did not

Table 4.1. Occurrence and types of methods employed to determine free- radical damage in drying and/or ageing seeds, pollen and vegetative tissues. Examples of free-radical damage specific to DNA are oxidized nucleotides such as thymine glycol, 8-hydroxy-2-deoxyguanine and methylguanine (Hageman et al., 1992). Examples of free-radical damage to protein are iminopeptides, carbonyl content and glutamyl-semialdehyde residues of oxidatively modified proteins (Berlet and Stadtman, 1997). Number of studies over the past three Methods decades MDA or TBARS determinationa 17 Determination of an organic free radical by in vivo EPR spectroscopy 11 Free fatty acid determination 6 Chemical modifications of lipids (e.g. conjugated dienes, fatty acid composition) 5 Other techniques (determination of breakdown products resulting from lipid peroxidation, chemiluminescence, fluorescent probes) 9 Free-radical damage specific to DNA 0 Free-radical damage specific to protein 2 aMDA, malonyldialdehyde; TBARS, thiobarbituric acid-reactive substances. Dessication - Chap 04 18/3/02 1:55 pm Page 118

118 O. Leprince and E.A. Golovina

assess whether the marker used as an index known to accumulate in desiccation-toler- of oxidative injury was sensitive or appro- ant systems. Anthocyanins accumulate to priate for the biological material or experi- large concentrations in leaves of resurrec- mental conditions. For example, in oily tion plants. Hodges et al. (1999) have intro- seeds, lipid peroxidation is dependent on duced several modifications to overcome the triacylglycerol composition and struc- this interference in leaf extracts. However, ture of oil reserves (Neff et al., 1992). the suggested improvements did not Furthermore, in an assay of peroxidized appear to be reliable for the results shown lipids of a crude extract obtained from oily in Table 4.2. seeds, the triacylglycerol fraction may 5. An organic stable free radical has been mask more important and significant linked to respiration, oxidative stress and changes occurring in membrane lipids. desiccation tolerance (Hendry et al., 1992; Another example further illustrates the Hendry, 1993; Leprince et al., 1995). point. Table 4.2 compares two methods However, measurements of this organic estimating the level of peroxidized lipids radical using non-invasive EPR have gener- in germinating pea axes in relation to the ated conflicting evidence concerning its loss of desiccation tolerance. Using the link to desiccation tolerance (Hendry, assay developed by Jiang et al. (1991), 1993). Its chemical nature and localization results suggest that there is a link between should be identified to ascertain whether an increase in oxidative damage following this is a reliable method to estimate oxida- drying and the loss of desiccation toler- tive stress in seeds. The fact that the EPR ance. In contrast, the TBARS assay pro- signal is sensitive to liquid water makes it vides inconclusive evidence. difficult to use in drying tissues. Studying frozen specimens could lessen the problem 4. The limitations of the TBARS test have but this approach will not be able to tell been known for several years (Gutteridge whether the radical is generated by drying and Halliwell, 1990; Hodges et al., 1999) or by freezing. and include a lack of sensitivity and specificity and a tendency to overestimate MDA From these remarks, we conclude that contents. Recently, it has been shown that positive results from a lipid peroxidation several compounds commonly found in assay will provide evidence that a free-radi- plant extracts (e.g. sugars, oligosaccharides, cal reaction has occurred either during anthocyanins) also react with thiobarbi- drying or during drying and extraction. A turic acid, thereby interfering strongly with negative result will not provide any evi- the peroxidized products (Table 4.3). Non- dence one way or the other. Therefore, it is reducing sugars and oligosaccharides are suggested that a range of assays should be

Table 4.2. Comparison of two methods measuring lipid peroxidation as a marker of oxidative damage in crude extracts of germinating axes of pea before and after fast drying. TBARS were measured as in Leprince et al. (1990) and calculated as in Du and Bramlage (1992) to take into account interference by sugars. Lipid hydroperoxide levels were measured using the xylenol orange/ammonium sulphate reagent

according to the method of Jiang et al. (1991) and quantiﬁed using H2O2 as a standard. Data are the average (± SE) of 3–5 replicates (O. Leprince, J. Fajerman and F.A. Hoekstra, unpublished observations). TBARSa Lipid peroxide 1 1 Sensitivity to drying Treatment ( mol mg dw) ( mol equiv. H2O2 mg dw) Tolerant Fresh 2.76 ± 1.93 15 ± 3 Dried 9.89 ± 1.79 16 ± 2 Intolerant Fresh 33.05 ± 15.01 22 ± 2 Dried 27.12 ± 9.00 71 ± 15 aTBARS, thiobarbituric acid-reactive substances. dw, dry weight. Dessication - Chap 04 18/3/02 1:55 pm Page 119

Methods for Quantifying Desiccation Phenomena 119

tested as a necessary prerequisite to 4.4. Spectroscopy Techniques address the role of free-radical processes in desiccation tolerance and seed As follows from a consideration of quan- longevity. To be validated, these assays tum mechanics, an atom or molecule has should be carried out on material that has discrete energy states. Spectroscopy is the been treated with agents to generate oxida- measurement of the energy differences tive stress such as ultraviolet (UV) radia- between these states. The energy differ- tion and free-radical generators such as ences E can be measured by the absorp-

paraquat or H2O2. tion spectra of electromagnetic radiation. An increasing number of methodologies In conventional spectroscopy, the fre- are currently being developed to analyse quency is varied and the frequency at free-radical-induced damage in vitro and in which maximal absorption occurs reflects vivo. Most of these methods are derived the difference between the states. The fre- from studies on animals, humans or food- quencies vary from the MHz range for NMR stuffs. The challenge will be to adapt these to the GHz (microwave) range for EPR techniques to seed science and anhydrobi- spectroscopy. The frequencies for absorp- 12 ology, keeping in mind the caveats tion spectroscopy range from 10 Hz for IR 16 described in this chapter and the guide- to 10 Hz for UV light. The frequencies of X-rays and -irradiation are 1019 Hz and lines provided by studies aimed at unravel- 1021 Hz, respectively. ling the free-radical chemistry occurring in living tissues. Among emerging methodologies, PA (see above), the use of specific flu- 4.4.1. Electron paramagnetic resonance (EPR) orescent probes (LeBel et al., 1992) and (see also Chapter 2) fluorescence spectroscopy, spin-trapping techniques and non-invasive EPR spec- 4.4.1.1. General description troscopy (see Section 4.4) and new spectroscopy assays (Jiang et al., 1991; LeBel et The energy differences that are studied al., 1999; Junqua et al., 2000) warrant fur- with EPR spectroscopy are the result of the ther investigation. interaction of unpaired electrons with a

Table 4.3. Interference of various compounds in the thiobarbituric acid-reactive substances (TBARS) assay in crude extracts of plant and seed tissues. The TBARS assay was performed using the procedure of Heath and Packer (1968) in the presence of various amounts of sugars and anthocyanins. TBARS concentrations were calculated from the difference between absorbance values at 532 and 600 nm and expressed as a relative increase compared to extracts without interfering compounds. Absorbance peak of the interfering Relative Compound compound (nm) increase (%) Apple peel extract +a 2.5 mM sucrose 440 + 9% 1 mM fructose 440 + 5% Cabbage leaves extract +a anthocyanins 540 + 272% Germinating pea axes +b 0.3% (w:v) rafﬁnose 441 + 35% 1% (w:v) rafﬁnose + 79% aData derived from Du and Bramlage (1992) and Hodges et al. (1999). bUnpublished data of O. Leprince, J. Fajerman and F.A. Hoekstra. Dessication - Chap 04 18/3/02 1:55 pm Page 120

120 O. Leprince and E.A. Golovina

static magnetic field (Zeeman splitting). In Sample preparation is relatively easy. The EPR spectroscopy the electromagnetic fre- sample is incubated in an aqueous solution quency is kept constant and the magnetic of spin-probe or spin-labelled molecules field is scanned (due to limitations of for a short time and then transferred into microwave electronics). There are four fre- the resonator of an EPR spectrometer for quencies available in EPR spectrometers: spectra recording. Usually, neutral spin- 1.1 GHz (L-band), 3.0 GHz (S-band), 9.5 probe molecules can readily pass through GHz (X-band) and 35 GHz (Q-band). membranes and partition within cells over Among them, the X-band is the most com- the polar and apolar phases according to monly used. The interaction between their partition coefficients. The EPR signal nuclei and the electron (hyperfine interac- of the spin-probe molecules outside cells tions) causes the hyperfine splitting of the can be eliminated by broadening agents via EPR spectrum. The spectral shape can give spin–spin interaction. Paramagnetic metal information about the sample under study. ion complexes such as chromium oxalate Only systems that contain non-paired or ferricyanide are often used as broaden- electrons will give an EPR signal. Pairing ing agents. Thus, the EPR spectrum of spin gives zero net electron magnetic moment. probes in the sample in the presence of Since a paired spin system is energetically broadening agents is exclusively derived favourable, chemical bonding normally from the inside of cells. Different aspects of results in molecules that have no unpaired desiccation tolerance can be studied by the electrons and, hence, no EPR signal occurs. analysis of EPR spectra of spin probes Exceptions to this rule are transition-metal introduced into the cells. ions, free radicals and free electron centres Spin labels are stable free radicals. The such as those produced by high-energy unpaired electron belongs to the nitroxide irradiation of macromolecules. Free radi- group, which is flanked by quaternary car- cals produced in biological systems usually bon atoms of methyl groups, protecting cannot be detected by EPR because of their the radical from recombination and short half-life times, resulting in low accounting for the high stability of the steady-state concentrations. label. The EPR spectra of nitroxide spin The introduction of spin-label/probe labels have a three-line nitrogen hyperfine methods has considerably increased the structure and are environmentally sensi- possibilities for the application of EPR in tive. The variety of spin probes of differ- biological systems. The spin-label group ent properties and the possibility of the that is almost exclusively used is the attachment of nitroxides to biological nitroxide moiety synthesized by Rozantzev molecules of interest have created exten- (1970) and introduced by McConnell sive applications of this method in biol- (McConnell and McFarland, 1970). ogy (Marsh, 1981; Morse, 1985). There is a limitation to the hydrated sam- A problem that often arises in experi- ple size that can be used for spectra record- ments with biological samples is chemical ing because of dielectric losses caused by reduction of spin labels and spin probes. water. However, the high sensitivity of the Despite the high stability in aqueous and method allows the use of very small sample other media, nitroxides are susceptible to sizes of less than 1 mg. The relative simplic- (reversible) reduction by some biological ity of sample preparation and spectra metabolites (such as ascorbic acid and thi- recording enables the operator to handle ols), electron transport chains and other large numbers of samples per day. redox systems, resulting in the disappearance of paramagnetism. Ferricyanide is 4.4.1.2. Applications of EPR methods usually effective at limiting the rate of reduction or reoxidizing reduced label GENERAL REMARKS. The great variety of spin (Kaplan et al., 1973). Oxygenation, aeration probes allows a multiplicity of information or the use of specific inhibitors can also be about biological systems to be obtained. used to protect nitroxides against reduction Dessication - Chap 04 18/3/02 1:55 pm Page 121

Methods for Quantifying Desiccation Phenomena 121

or to restore them from the reduced 4 daa wheat kernel hydroxylamines (Marsh, 1981). Another problem with the application of the spin-label technique is the disturbance Dead that might be caused by the guest molecules (a) tissue themselves (e.g. membrane spin labels). This is inherent in all methods using reporter groups, in contrast to spectroscopic methods that do not use guest molecules (e.g. NMR, FTIR). Because of the high sensitivity of the spin-label method, very low concentrations of label can be used, which Difference (viable cells) minimizes possible disturbance. However, the possibility of obtaining information about a speciﬁc environment of interest Oil makes the EPR method attractive in the (b) study of desiccation tolerance. In contrast, NMR and FTIR give information that is averaged along the sample, which can be complementary to the information from reporter group methods. Cytoplasm

CELLULAR VIABILITY BASED ON MEMBRANE INTEGRITY. The principle of the EPR spin-probe Fig. 4.1. (a) The electron paramagnetic resonance method for the estimation of the relative (EPR) spectrum of 4-oxo-2,2,6,6-tetramethyl-1- amount of viable cells is based on the fact piperidinyloxy (TEMPONE) in developing wheat that membranes of viable cells are imper- kernel that was harvested at 4 days after anthesis (4 daa) and dried on the ears. The thick line represents meable to some broadening agents, the broad component of the spectrum and whereas the membranes of damaged cells originates from TEMPONE in dead tissue; the thin are not (Keith and Snipes, 1974). Thus, the line represents the total spectrum. (b) Spectrum EPR signal of spin-probe molecules inside showing the difference between the total spectrum cells with disrupted membranes is and broad component, representing TEMPONE quenched by a broadening agent, and the located in viable cells. Peaks originating from total amplitude of the EPR signal from the TEMPONE in the aqueous cytoplasm of viable cells sample will correlate with the amount of and from oil bodies are indicated. Total scan width viable cells in a sample (Dobrucki et al., is 100 gauss. Spectra are reproduced from Golovina 1990). Because of the high sensitivity of the et al. (2001). method, it is possible to determine small amounts of viable cells in mostly dead tissue. This approach has been applied in the Many of the anhydrobiotic systems con- study of desiccation tolerance acquisition tain oil bodies as storage material. In this of proembryonic cells in wheat kernels, case, amphiphilic spin-probe molecules which were slowly dried on the ear at an will partition into lipid bodies as well, and early stage when proembryos could not be EPR spectra are composed of two compo- detected morphologically (Fig. 4.1) nents originating from spin-probe mole- (Golovina et al., 2001). Such an approach cules in aqueous (cytoplasm) and allows the developmental death of wheat hydrophobic (oil) environments. These endosperm cells during kernel develop- spectra differ in the distance between lines

ment (Golovina et al., 2000) and the (the isotropic splitting constant aiso is progress of cell death after cold (Fig. 4.2) or around 16 G for an aqueous environment imbibitional stress in neem seeds to be fol- and around 14 G for a lipid environment) lowed (Sacandé et al., 2001). and in the position of the central line Dessication - Chap 04 18/3/02 1:55 pm Page 122

122 O. Leprince and E.A. Golovina

plasmic (h ) and oil peaks (h ) can be Axis of cyt oil neem used for the quantitative assessment of the proportion of viable cells in a sample (Golovina and Tikhonov, 1994; Golovina et al., 1997a,b; Leprince et al., 1999). Oil (a) Control PLASMA MEMBRANE PERMEABILITY. Changes in plasma membrane permeability can be estimated by using the water-soluble nitroxide radicals in the presence of a broadening Cytoplasm agent (Miller and Barran, 1977; Golovina et al., 1998; Hoekstra et al., 1999). The method is based on the presence of tempo- (b) 7 days rary defects in membranes that allow ferricyanide ions to penetrate into the cell and broaden the signal arising from spin probes located in the cytoplasm. The line-height (c) 28 days ratio of the lipid peak to the water peak (L/W) will correlate with the number of ferricyanide ions that have penetrated the cell through the plasma membrane and can be Fig. 4.2. Electron paramagnetic resonance (EPR) used to characterize plasma membrane study of chilling damage of neem (Azadirachta permeability (Fig. 4.3). indica) seeds using 4-oxo-2,2,6,6-tetramethyl-1- piperidinyloxy (TEMPONE) as a spin probe. CELL VOLUME AND OSMOTIC EFFECT. The height Spectrum (a), mature, fresh axes used as control; ratio of cytoplasmic to lipid peaks in EPR spectra (b) and (c), whole embryos after, spectra can be used to determine cell vol- respectively, 7 and 28 days of storage under humid ume changes under osmotic stress. This conditions at 5°C. Spectra are plotted in the same scale to allow comparison. The oil and cytoplasmic follows from the fact that spin-probe mol- peaks are indicated in the high-ﬁeld region (right ecules are equally distributed inside and side) of spectrum (a). Total scan width is 100 gauss. outside the cells. The total cellular vol- Spectra are reproduced from Sacandé et al. (2001). ume that is not accessible to broadening agents will determine the amount of spin probe that is separated from the broaden- (g-factor), and can be resolved in the high ing agent and, hence, the line-height of ﬁeld (right-side) part of the spectrum the cytoplasmic component in the EPR (Marsh, 1981). In the case of loss of mem- spectrum. This total volume is the prod- brane integrity, ferricyanide ions that have uct of the number and volume of viable penetrated the cell only broaden the signal cells. Cell division and enlargement of of TEMPONE (4-oxo-2,2,6,6-tetramethyl-1- cells during imbibition and germination piperidinyloxy) molecules localized in the (Golovina et al., 2001) and osmotically aqueous cytoplasm. The signal of TEM- induced changes in cell volume (Miller, PONE from oil bodies remains unbroad- 1978) can cause changes in the line- ened, because ferricyanide cannot partition height of the cytoplasmic component. into the lipid phase. The intensity of this The ratio between the line-heights of hydrophobic signal can then be used as a cytoplasmic and lipid peaks can be used measure of the total amount of cells in the to quantify the osmotic effect. However, sample, whereas the intensity of the polar when the amount of oil changes during cytoplasmic component represents the seed development (Golovina et al., 2001) amount of cells with intact membranes or during germination (Sacandé et al., (Golovina et al., 1997a,b). The ratio R 2001), the height of the cytoplasmic line between the heights of the aqueous cyto- can be used instead. Dessication - Chap 04 18/3/02 1:55 pm Page 123

Methods for Quantifying Desiccation Phenomena 123

Typha latifolia 1998). Line-height differences arising from differentially broadened lines due to (a) 0 s slowed motion of spin-probe molecules (Fig. 4.4, spectra a and b) are used to esti- (b) 7 s mate viscosity (Keith and Snipes, 1974). The line-height ratio between lines can be converted to viscosity. Based on such an approach, the changes in cytoplasmic viscosity with drying of desiccation-tolerant (c) 30 s and sensitive samples (Leprince et al., 1999) and with the acquisition of desiccation tolerance during seed development (Golovina et al., 2001) have been established. When spin-probe motion slows further, L not only is a progressive increase in differ- (d) 60 s ential broadening observed, but also a dis- tortion of the line shape (Fig. 4.4, spectrum W c). Rigidly immobilized, randomly oriented radicals give a powder spectrum (Marsh, 1981), which can be used to characterize biological glasses (Buitink et al., 1998, 1999, 2000b,c,d,f). In this case, the viscosity must Fig. 4.3. Electron paramagnetic resonance (EPR) be estimated using saturation transfer EPR study of imbibitional leakage of Typha latifolia pollen using 4-oxo-2,2,6,6-tetramethyl-1- or pulsed EPR methods (see below). piperidinyloxy (TEMPONE) as a spin probe. Pollen was either directly incubated in a solution of PARTITIONING OF AMPHIPHILES INTO THE LIPID PHASE TEMPONE/ferricyanide (spectrum a) or after a WITH DRYING. The shape of spin-probe spec- previous rehydration in liquid germination medium tra depends on properties of the environ- for 7 s (spectrum b), 30 s (spectrum c) and 60 s ment; therefore amphiphilic spin probes (spectrum d). All the spectra exhibit contributions can be used to follow their partitioning from the aqueous cytoplasm (W) and from lipid (L) with drying (Golovina et al., 1998; Buitink (oil bodies) environments, which are resolved in the et al., 2000e; Hoekstra and Golovina, 2000; high-ﬁeld region (right side). The spectra were Golovina and Hoekstra, 2002). The samples normalized to the height of the lipid (L) peak. The ratio W/L was taken as a measure of plasma are preloaded with spin probes and allowed membrane permeability (see explanation in the text, to dry. The EPR spectra recorded from the Section 4.4.1). Total scan width is 80 gauss. Spectra samples at different moisture content will are based on data from Hoekstra et al. (1999). be composed of spectra originating from spin-probe molecules at different locations (Fig. 4.5). They can be decomposed, and the CYTOPLASMIC VISCOSITY. Because the shape of relative proportion of spin probes at the dif- EPR spectra of spin probes is sensitive to ferent locations can be estimated (Hoekstra molecular motion, cytoplasmic viscosity and Golovina, 2000; Golovina and can be studied with spin-probe techniques Hoekstra, 2001). (Keith and Snipes, 1974). To characterize the shape of the spectrum originating from PHYSICAL PROPERTIES OF MEMBRANES. The small, the cytoplasmic location of the spin probe, water-soluble spin probe TEMPO (2,2,6,6- other components (lipid, starch-like) of the tetramethyl-1-piperidinyloxy) or spin- spectrum have to be subtracted (Golovina labelled fatty acids, steroids or et al., 2000, 2001). Alternatively, charged phospholipids are used to study the physi- spin probes that do not partition into the cal properties of membranes (for refer- lipid phase can be applied (Buitink et al., ences, see Berliner, 1976; Marsh, 1981; Dessication - Chap 04 18/3/02 1:55 pm Page 124

124 O. Leprince and E.A. Golovina

(a) Cytoplasm Root (a)

h0 h–1

(b) Oil bodies h0/h–1= 1.19

Axis (b)

Membrane (c) surface Sucrose glass h0/h–1= 1.75

2Amax a:b:c = 5:10:85

Fig. 4.4. Electron paramagnetic resonance (EPR) Fig. 4.5. Typical electron paramagnetic resonance spectra of 4-oxo-2,2,6,6-tetramethyl-1- (EPR) spectra of 4-oxo-2,2,6,6-tetramethyl-1- piperidinyloxy (TEMPONE) in the cytoplasm of piperidinyloxy (TEMPONE) in aqueous cytoplasm wheat seedling root (spectrum a) and hydrated (spectrum a), oil bodies (spectrum b), and of spin- wheat axis (spectrum b). The ratio of the height of probe molecules immobilized at the membrane surface (spectrum c). Spectrum (d) is the sum of the central line (h0) to the height of the high-ﬁeld spectra (a), (b) and (c). Spectra were combined in line (h1) reﬂects cytoplasmic viscosity. The cytoplasmic viscosity is less in seedling root such a proportion that the relative amounts of spin probe in spectra (a), (b) and (c) were 5%, 10% and (h0/h1= 1.19) than that in hydrated wheat axis 85%, respectively. Spectrum (d) simulates the late (h0/h1= 1.75). The EPR spectrum of TEMPONE in air-dried sucrose glass (spectrum c) is typical for stage of TEMPONE partitioning during drying. Total rigidly immobilized, randomly oriented spin-probe scan width is 100 gauss. Spectra are based on data molecules. The distance between the outer extremes from Golovina and Hoekstra (2001).

2Amax (in gauss) can be used to characterize the slow motion of spin-probe molecules. Total scan width is 100 gauss. Spectra are based on data from Golovina and Hoekstra (2001). provides valuable information about membrane dynamics, because the stable free- radical doxyl group can be placed at Morse, 1985). Isolated membranes can be different positions along the acyl chain. labelled with TEMPO, which partitions Thus information can be obtained from dif- between water and the membranes. The ferent depths in membranes, from the sur- partitioning depends on membrane ﬂuidity face to the core. Labelling model membranes and aqueous-phase viscosity and can be poses no problem. The spin label is mixed at used for the determination of the mem- 1 mol % with the lipids in organic solvent, brane phase transition. However, the possi- which is subsequently removed by evapora- ble partitioning of TEMPO into oil bodies tion. The anhydrous mixture is then dis- complicates the in vivo membrane investi- persed in the appropriate amounts of gations. The use of spin-labelled fatty acids aqueous phase. Labelling isolated biological Dessication - Chap 04 18/3/02 1:55 pm Page 125

Methods for Quantifying Desiccation Phenomena 125

membranes poses more problems. Spin- teins and lipids in biological membranes labelled fatty acids have first to be dis- (for references, see Marsh, 1981; Hemminga, solved in ethanol and then added to the 1983) and glasses (Roozen et al., 1991). The membranes in such a quantity that the final method has been successfully applied in concentration of ethanol does not exceed 1 the study of biological glasses in anhydrobi- mol %. Other ways of membrane labelling otic systems (Buitink et al., 1998, 1999; have been reviewed by Marsh (1981). 2000b,c,d,f). This approach of measuring Labelling membranes with doxyl stearates slow rotational motion has given stunning in vivo following the same approach can be insight into the differences between biologi- used but is more tricky than with isolated cal glasses and sugar or polymer glasses. membranes. A small number of in vivo stud- For example, a remarkable observation orig- ies have been published. Among them only inating from the ST-EPR measurements was a few investigations have been conducted the occurrence of a second kinetic change on desiccation-tolerant systems (Golovina in mobility at a definite temperature above and Tikhonov, 1994; Vishnyakova et al., the glass transition temperature (Buitink et 2000; Golovina and Hoekstra, 2001). al., 2000f), which may have physiological relevance for survival in the dry state (Fig. ADDITIONAL EPR TECHNIQUES APPLIED TO DESICCATION- 10.3 in Chapter 10). TOLERANT SYSTEMS. When conventional EPR Pulsed EPR. CW-EPR often cannot give spectroscopy as described above yields the true values for the relaxation times of the rigid-limit powder line shape (see Fig. 4.4, spin label, because of the inhomogeneous spectrum c), it is insensitive to the rate of broadening of the lines. However, pulsed molecular motion. The following two EPR EPR (electron spin echo technique) pro- methods have been designed to overcome vides a direct method for the measurement this problem and adapted to anhydrobiotic of relaxation times that give insight into material. They are particularly suitable to the molecular dynamics of spin probes characterize a glassy state. (Morse, 1985). This method has been used to identify the glassy state in wheat seeds Saturation-transfer EPR. Saturation-transfer (Dzuba et al., 1993) and to characterize the EPR (ST-EPR) allows the measurement of motion of guest molecules in biological very slow molecular motion with rotational glasses of different moisture content correlation times between 107 and 103 (Buitink et al., 2000a). s. For comparison, conventional continuous wave (CW) EPR enables rotational correla- EPR imaging (EPRI). The potential of using tion times below 107 s to be resolved. In both NMR and EPR imaging was suggested conventional EPR, motion averaging of the by Lauterbur (1973). However, while NMR spectral anisotropy occurs within the time imaging (NMRI) has progressed into clinical

of spin–spin relaxation T2. In ST-EPR, this usage, the application of EPRI is restricted, averaging occurs within the time of particularly in biological systems. This is

spin–lattice relaxation T1, which is 300 caused by the severe dielectric losses and times longer than T2 for slow-moving consequent heating that occurs in aqueous nitroxide molecules (Marsh, 1981). Thus, samples at conventional EPR frequencies (X- ST-EPR extends the motional sensitivity of band). The dimension of the hydrated sam- the spin-label technique to one that moni- ple that can be used for imaging is limited to tors a 300-fold slower motion than with a few millimeters. This problem can be conventional EPR. ST-EPR spectra of partly overcome by using low-frequency EPR nitroxide spin probes can be analysed by (L-band) and a surface coil (Berliner and independent line shape parameters. Using a Fujii, 1985). EPRI is used for visualizing reference material of known viscosity, the paramagnetic centres in a sample. There are molecular rotation can be calculated in an several biological applications in which empirical way (Hemminga, 1983). ST-EPR EPRI has a signiﬁcant advantage over NMRI:

has been used to study the motion of pro- the spatial distribution of O2 and redox Dessication - Chap 04 18/3/02 1:55 pm Page 126

126 O. Leprince and E.A. Golovina

metabolism, mapping viable and non-viable this effect is proportional to the O2 concen- cells, the diffusion of paramagnetically tration (Swartz, 1987). labelled solutes, and mapping native free radi- pH measurements. Spin-labelled amine cals that are stable or trapped by incorporated and carboxylic acids have been used to spin traps at the sites of transient radical pro- determine the pH in vesicles and cells duction (Berliner and Fujii, 1986; Bacic et al., (Mehlhorn et al., 1982). The method is 1989; Dobrucki et al., 1990). In spite of the based on the differential membrane perme- potential advantage of EPRI, there are only a ability for charged and neutral forms of few cases in which the method has been these spin probes. Because the equilibrium applied to desiccation-tolerant systems. The between charged and neutral amines and pathways of bulk water penetration into wheat acids depends on pH, the intracellular kernels during imbibition have been studied EPR signals of these spin probes can be using the nitroxide radical TEMPOL (4- used to calculate the intracellular pH. hydroxy-2,2,6,6-tetramethyl-1-piperidinyloxy) Unfortunately, such an approach can only (Smirnov et al., 1988; Golovina et al., 1991). be applied in the presence of a solution of To avoid the problem of dielectric losses, the a broadening agent. To study the changes kernels were placed in liquid nitrogen. in pH in a sample during drying, specific Using perdeuterated 15N nitroxides as imag- pH-sensitive spin probes can be applied. ing substances, tens of micrometre resolu- The reversible effect of pH on EPR spectra tion can be achieved. Such resolution was is associated with proton exchange in the sufficient to obtain EPRI of viable cells in radicals. Protonated and non-protonated hydrated lettuce seeds in the presence of fer- forms have different EPR parameters. The ricyanide (Walczak et al., 1987). The image protonable group in the radical structure enabled contrast between embryo and stor- has to be close to the unpaired electron. age tissue to be observed. The EPRI of the Iminonitroxides are the most promising in penetration and distribution of natural spin this respect (Khramtsov and Weiner, 1988). probes (humic substances) in wheat kernels has also been demonstrated (Smirnov et al., Spin trapping. The free radicals that are 1991). produced in anhydrobiotic organisms during water loss (Section 4.3.4) cannot be detected by EPR because of their short half- POTENTIAL APPROACHES FOR STUDYING ANHYDRO- life resulting in low steady-state concentra- BIOSIS. Additional EPR methods have been tion, or the short relaxation times leading to designed to study several biochemical and very broad lines. These radicals can be biophysical aspects of biology. However, trapped specifically by spin traps and these methods have not yet been applied detected by EPR in organic extracts or in specifically to seeds or other types of anhy- vivo (Knecht and Mason, 1993). Four differ- drobiotic tissues. ent traps are commonly used in biological

Intracellular O2 concentration. Intracellular systems: 2-nitrosopropane (MNP), phenyl- O2 affects the shape of EPR spectra of spin N-tert-butylnitrone (PBN), -(4-pyridyl-1- probes because, as a paramagnetic mole- oxide)-N-tert-butylnitrone (POBN), and cule, it causes line broadening. Broadening 5,5-dimethyl-1-pyrroline-N-oxide (DMPO).

is proportional to O2 concentration, thereby The primary free radicals interact with the allowing the intracellular concentration to double bond of diamagnetic spin-trap mole- be calculated (Swartz, 1987). However, the cules and form radical adducts that are application of this method may not be much more stable than the primary free straightforward for drying biological mater- radicals. The radical adducts of these spin ial because the rise of viscosity also intro- traps are nitroxide radicals. The primary duces changes in line broadening. Another free radical can be identiﬁed either from the

approach is to use the effects of O2 on the spectra of radical adducts, or after puriﬁca- microwave power saturation: the presence tion of radical adducts and further identiﬁ-

of O2 diminishes power saturation, and cation by mass spectroscopy. Various Dessication - Chap 04 18/3/02 1:55 pm Page 127

Methods for Quantifying Desiccation Phenomena 127

shortcomings complicate the application of NMR signals can be characterized by spin trapping in vivo in drying organisms: intensity, frequency, line shape and relax- oxidation of the spin traps and reduction of ation times. All these characteristics are the radical adducts, and amphiphilic affected by the physical and chemical envi- behaviour which may relocate spin traps ronment of the magnetic nucleus and can into the membranes (Knecht and Mason, be used to obtain information of biological 1993). interest such as the state of water, intracellular pH and membrane dynamics. Signal intensity is related to the number of mole- 4.4.2. Nuclear magnetic resonance cules that produce the signal. In relaxation (see also Chapter 2) experiments, the intensity depends on the time of signal registration and on the rate 4.4.2.1. General description of magnetization decay. For quantitative estimation of peaks in NMR spectra, inte- Analogous to EPR spectroscopy, NMR spec- gration of the lines should be used because troscopy is based on the resonance absorp- of the different relaxation times of the sig- tion of electromagnetic radiation by the nals. The limits of integration are deter- system during the transition between two mined by the signal-to-noise ratio of the discrete energy states. The energy differ- signal and the overlapping with other sig- ences studied in NMR spectroscopy are due nals in a spectrum. to the interaction of nuclear magnetic Local fields originating from the local moments with the magnetic field (Zeeman electron density modify the external field splitting for nuclei). The energy differences imposed on magnetic nuclei. As a result, the are smaller than those in EPR because of resonance frequency of a nucleus depends the smaller magnetic moment of nuclei. on its chemical environment, which is This explains why electromagnetic radia- called chemical shift. Magnetization relaxes tion in the radiofrequency range is required exponentially, and the faster the decay, the to excite the transitions that produce the broader the line in the spectrum. Broad NMR signal, whereas that in the microwave lines have lower amplitudes and overlap range is used in EPR spectroscopy. with other lines, which leads to poorly Because the energy differences in NMR resolved spectra. In living systems, the vari- are small, the differences in number of ations of magnetic susceptibility across the nuclei at different energy levels are also sample cause line broadening, which makes small. As a consequence, the signal it difficult to record high-resolution spectra strength is weak, which makes NMR an from dense heterogeneous tissue, such as inherently insensitive technique. Only seeds, and from tissues containing air- those nuclei that have a non-zero spin spaces (leaves and roots).

quantum number resulting in non-zero The T1 (spin–lattice or longitudinal) and magnetic moment can be used. The split- T2 (spin–spin or transverse) relaxation ting between energy levels depends on the times characterize the magnetization decay strength of the magnetic ﬁeld and the mag- because of the interaction of the nuclear netogyric ratio of the nucleus. The highest magnetic moments with the environment

magnetogyric ratio and the almost 100% (T1) and with each other (T2). Relaxation natural abundance make proton (1H) NMR times are mostly determined by the the most sensitive. The reasonably high motional properties of the nucleus. magnetogyric ratio and 100% natural abun- Measurements of relaxation are particu- dance of 31P nuclei give moderately good larly important in NMR studies of tissue receptivity for in vivo phosphorus NMR. In water when information about the exis- contrast, the 13C nucleus has very low tence of different water fractions in the tis- receptivity because of its low natural abun- sue is required. In practice, the dance, but, as a label, this isotope could be measurements are easier to conduct than to useful (Schneider, 1997; Roberts, 2000). interpret (Ratcliffe, 1994). Dessication - Chap 04 18/3/02 1:55 pm Page 128

128 O. Leprince and E.A. Golovina

In basic NMR experiments, the sample is cal systems because of the generally high placed in a magnetic field, and the NMR sig- water content, the high natural abundance nal is generated by irradiation of the sample and the high magnetogyric ratio of 1H. This with a radio-frequency field, given as pulses allows the use of low-field NMR instead of of different sequences. A single pulse cre- expensive high-field NMR magnets. ates a net magnetization, which is regis- tered. The magnetization decays to zero, MEASUREMENTS OF WATER CONTENT. 1H low-field and the time-dependence of the decay (free NMR allows the non-destructive measure- induction decay) is recorded. In low-field ment of the water content in biological sys- studies, this decay is analysed directly. In tems with high precision. There are two high-field NMR, the decay is converted into types of analytical NMR commonly used in spectra by Fourier transformation. The NMR this respect – continuous wave (CW) NMR spectrum is the plot of intensity against fre- (wide-line) (Pohle and Gregory, 1968) and quency of the radio-frequency field. All pulsed NMR (Martin et al., 1980), the latter NMR applications developed for studying now being generally adopted. In CW-NMR living systems can be divided into four the amount of liquid water is estimated from groups: (i) detection of water signal; (ii) the area under the absorption peak. The sig- NMR imaging; (iii) high-resolution multinu- nal from water strongly ‘bound’ to biopoly- clear NMR spectroscopy; and (iv) solid-state mers is not visible because of broadening. NMR spectroscopy (Ratcliffe, 1994). The signals from liquid water and oil are not To exploit the advantage of a non-inva- resolved, but the contribution of oil to the sive technique, NMR experiments need to signal can be estimated by drying. minimize the physiological perturbation Pulsed NMR can be used to analyse the and maintain the tissue in a physiologi- different water fractions. In pulsed NMR cally controllable state. In this respect, the all protons are excited by a short intense whole plant, cell suspensions and intact radio frequency (RF) pulse resulting in a seeds are the easiest tissues, and excised free induction signal, which decays when tissues the most demanding (Ratcliffe, the pulse is switched off. The initial 1994). Often, it is necessary to submerge a amplitude of free induction decay (FID) is sample in water to avoid differences in proportional to the total number of pro- magnetic susceptibility between air and tons in a sample. The signals due to nuclei cellular material, a practice that is incom- in different physical states decay at different rates: signals due to protons in solid patible with drying organisms. Proper O2 supply and illumination have to be main- state decay faster (microseconds) than tained, especially in densely packed sam- those in liquid phase (from milliseconds ples. In solid-state NMR, when magic angle to seconds). This signal decay can be spinning is applied, it is impossible to con- analysed to reveal the contribution of dif- trol the physiological state because of the ferent proton fractions. To avoid the influ- extreme conditions (more than 1000 rota- ence of inhomogeneity of magnetic field tions per minute) imposed on the sample. and water diffusion on the rate of decay, special sequences of pulses such as spin- echo (SE) or Carr–Purcell–Meiboom–Gill 4.4.2.2. The NMR study of water in living (CPMG) are used (Farrar and Becker, systems 1971). In air-dry samples, the signal decay from water associated with polymers GENERAL REMARKS. The study of water in (mainly starch) can be distinguished easily anhydrobiotes is of particular interest from that of oil protons on the basis of the because with drying and rehydration both considerable differences in spin–spin

water content and water properties change. relaxation time T2. Such an approach is NMR is a powerful tool to study water in widely used for rapid and non-destructive vivo. There is no problem with the sensitiv- determination of moisture and oil content ity of detecting the water signal in biologi- in air-dry seeds (e.g. Tiwari et al., 1974; Dessication - Chap 04 18/3/02 1:55 pm Page 129

Methods for Quantifying Desiccation Phenomena 129

Gambhir and Agarwala, 1985; Brusewitz Compartmentation is the reason why and Stone, 1987; Gambhir, 1992; Rubel, more than one fraction of water is generally 1994; Warmsley, 1998). In hydrated seeds, observed in hydrated living systems. A the-

drying or D2O exchange can be used to ory of transverse relaxation in compart- separate the NMR signal of free water from mented systems has been developed, based that of oil (Ratkovic et al., 1982a). on the chemical exchange and diffusion Because the different water fractions properties of the water (Belton and have the same chemical shift, only pulsed Ratcliffe, 1985). Two to three water frac- NMR can be used to characterize them in tions have been shown in hydrated tissue living tissues. The changes in water frac- originating from different plant cell com- tions with different relaxation characteris- partments (Bacic and Ratkovic, 1984; tics can be followed during the Belton and Ratcliffe, 1985; Snaar and van dehydration or rehydration of anhydrobi- As, 1992). However, it appears that there is otic systems. This gives insight into the no simple relationship between the multi-

role of the different water fractions in bio- exponential character of T2 and the com- logical systems (Seewaldt et al., 1981; partmentation of the water (Ratcliffe, 1994). Ratkovic et al., 1982a; Aksyonov and The heterogeneity in cellular size and com- Golovina, 1986a,b; Ishida et al., 1987, position, subcellular compartmentation, 1988b; Bacic et al., 1992; Golovina and and plasmalemma and tonoplast permeabil- Aksyonov, 1993; Marconi et al., 1993). ity could have inﬂuenced the multi-expo- However, data on different water fractions nential decay curves (Snaar and van As, must be interpreted with extreme caution 1992). The detection of the simultaneous (Ratcliffe, 1994). Different water fractions presence of water of different relaxation with speciﬁc relaxation times can be dis- behaviour in anhydrobiotes with reduced criminated only if there is no fast exchange MC may have been caused by the inhomo- of protons between the fractions in the geneous water distribution within the

NMR time window. In the case of fast organisms. Thus, the water with long T2 (or exchange between protons, only one relax- slow-relaxing water) observed in wheat ker- ation time is observed. The number of pro- nels during the ﬁrst hours of imbibition is tons of different mobility and their thought to be localized around the embryo relaxation times will determine the and in the vascular bundle, whereas the observed effective relaxation time. When fast-relaxing water is thought to be associ- associated with macromolecules, water ated with starchy endosperm (Golovina and protons have shorter relaxation times, Aksyonov, 1993). which will inﬂuence the overall relaxation

time. This is the reason why T1 (spin–lattice WATER SELF-DIFFUSION COEFFICIENT. The behav- relaxation time) and T2 (spin–spin relax- iour of water in living systems can also be ation time) values are lower in cellular characterized by the water self-diffusion water than in bulk water and decrease fur- coefﬁcient. The diffusion coefﬁcient is

ther with water loss. Thus, T2 values can measured by the pulsed (spin-echo) NMR also be used to measure moisture content technique in the presence of a (pulsed) 1 (MC) (Ratkovic, 1987). Below 0.2 g H2O g ﬁeld gradient (Fukushima and Roeder, dry weight, the relationship between relax- 1981). In addition to nuclear magnetic

ation times (T1 and T2) and moisture con- relaxation, the spin-echo amplitude tent is reversed (Clegg et al., 1982; Ratkovic decreases in the presence of a ﬁeld gradient et al., 1982b; Wolk et al., 1989). Because if water changes its position during the

the increase in T1 and T2 at low water con- measurement. Diffusion coefficients as a tents has also been observed in measure of water mobility can be calcu- starch/water systems besides anhydrobiotic lated from the signal decay in the presence organisms, the increase might be attributed of a field gradient. As in the case of relax- to water molecules jumping from one sorp- ation times, self-diffusion coefficients of tion site to another. cellular water are lower than those of bulk Dessication - Chap 04 18/3/02 1:55 pm Page 130

130 O. Leprince and E.A. Golovina

water and decrease with drying (Clegg et 4.4.2.3. NMR imaging al., 1982). This can be caused by the presence of diffusion barriers (membranes or GENERAL REMARKS. NMRI is mainly based on cell walls) or macromolecules. These the detection of the water signal. 1H reso- macromolecules can cause either obstruc- nance frequency is independent of the tion of the diffusion or water binding (Seitz location of the water in a tissue, so that tis- et al., 1981; Back et al., 1991). As a result, sue water signal is averaged across the the diffusion coefficient in hydrated anhy- whole sample. The spatial distribution of drobiotes has been shown to be 2–5 times the water signal can be obtained if a mag- smaller than that of bulk water (Clegg et netic field gradient is applied, which arises al., 1982; Fleischer and Werner, 1992). In from the dependence of the resonance fre- Artemia cysts the diffusion coefficient has quency of NMR signals on the magnetic been measured from 0.02 to 1.49 g water field strength. In spite of the simplicity of g1 dry weight, the minimum value being the principle of NMRI, its practical appli- almost 50 times lower in the dry cysts than cation is rather complicated. Information in the hydrated cysts (Seitz et al., 1981). on the spatial distribution of water or water properties (relaxation times or diffusion MEMBRANE PERMEABILITY. Paramagnetic ions coefficients) can be obtained. Dynamic (Mn2+) cause a decrease in relaxation times information can be obtained from time- due to their interaction with nuclei. Conlon dependent properties of the image. There and Outhred (1972) proposed a method of are two different experimental approaches measuring membrane permeability to water, in NMRI: imaging large objects (roots, based on the change in relaxation time of stems or whole plants) with low spatial intracellular water that is in diffusional resolution, and imaging small samples

exchange with an extracellular MnCl2 solu- (seeds, excised tissues) with high spatial tion. From the estimated water-exchange resolution (NMR microscopy) (Ratcliffe, time and the cell dimension, the diffusion 1994; Ishida et al., 2000).

permeability coefficient Pd can be calcu- Spatial resolution is mainly determined lated (Stout et al., 1977, 1978; Bacic and by the signal/noise ratio, but other factors Ratkovic, 1984). Unfortunately, this such as short relaxation times and the pres- approach cannot be applied to the systems ence of air space cause intensity loss and a that are subjected to drying, because the tis- decrease in spatial resolution. The develop- sue has to be in Mn2+ solution. ment of NMRI has led to a resolution that The pulsed-gradient spin-echo method approaches the dimension of single cells in proposed by Stejskal and Tanner (1965) plant tissues (Connelly et al., 1987). The can be used to study the in situ membrane theoretical limit is considered as 10 10 permeability for water during drying. The 10 µm (Ratcliffe, 1994). While NMR is not method allows the water diffusion to be yet able to compete with optical microscopy measured over the time between two in its resolution of cellular structures, it has pulses of field gradient. The presence of the great advantage of being non-invasive partly permeable barriers causes the and, thus, can be used to monitor function- decrease in the apparent diffusion coeffi- ing plant tissue. The ability to resolve struc- cient for water, so that the permeability of tures depends not only on resolution but membranes for water and the size of water also on the image contrast, which is deter- compartments can be calculated (Tanner, mined by the differences in signal intensity 1978; von Meerwall and Ferguson, 1981). between different regions of the sample. This approach has been applied to follow Knowledge of relaxation properties of the the changes in membrane properties in tissue water is central to the understanding developing barley seeds (Ishida et al., of image contrast. Nitroxide radicals 1995) and to calculate the size of oil bodies (Magin et al., 1986; Swartz et al., 1986) and in rape seeds (Fleischer et al., 1990; paramagnetic ions (Ishida et al., 2000) can Fleischer and Werner, 1992). be used as contrasting agents. Dessication - Chap 04 18/3/02 1:55 pm Page 131

Methods for Quantifying Desiccation Phenomena 131

WATER DISTRIBUTION IN SEEDS DURING MATURATION signal of the externally supplied water AND GERMINATION. It is possible to map sta- (Connelly et al., 1987). The changes in relax- tionary, diffusing and ﬂowing water in ation times of tissue water during seed matu- plant tissue (Ratcliffe, 1994). NMRI enables ration or germination cause changes in the the water distribution inside seeds to be image contrast. Relaxation times of water determined. The brightness of the image is depend on the interaction of water with proportional to the proton density. macromolecules. The synthesis of storage Experiments with seeds of different species substances during maturation and their have shown that the signal/noise ratio in hydrolysis during germination result in an the image is sometimes limited by the short apparent decrease or increase in brightness

relaxation time for tissue water (T2 < 10 of the NMR image (Ishida et al., 1990, 1995; ms) (Connelly et al., 1987). The sensitivity McIntyre et al., 1995), so that solubilized problem can be overcome to some extent parts of the storage tissue can become visi- by signal averaging, since the time scale for ble. The changes in image contrast during detectable structural changes in germinat- precocious germination of Phaseolus vul- ing seeds is long in comparison with the garis seeds after ethylene treatment have time required to obtain an image. In NMR been attributed to changes in the water sta- images of seeds, a clear distinction tus and water redistribution from the cotyle- between axis and storage tissue can be don to the axis (Fountain et al., 1998). obtained (Connelly et al., 1987; Kano et al., 1990; Hou et al., 1996; Fountain et al., THE DISTRIBUTION OF OIL AND SUCROSE IN SEEDS. 1998; Carrier et al., 1999). The changes in The spatial image of other compounds, water distribution during drying and rehy- mainly lipids and carbohydrates that accu- dration have shown the transfer routes for mulate in storage tissue, can be mapped in water (Ruan and Litchfield, 1992; Ruan et vivo using the chemical-shift imaging (CSI) al., 1992; Song et al., 1992; Kovacs and technique (Bottomley et al., 1984). The 1H Nemenyi, 1999). NMR spectra of water, oil and sugars have The water content may be more uni- different chemical shifts, but the peaks are formly distributed in seeds than proton not resolved unless the water peak is sup- NMRI indicates. This discrepancy arises pressed. The CSI technique applied to 1 from the inhomogeneity of the susceptibil- day germinating mung bean seeds has ity of the sample associated with the pres- shown uniformly distributed oil, which ence of cell walls and storage substances allowed the changes in the image with ger- (Back et al., 1991). Eccles et al. (1988) mination to be attributed to the bulk water applied pulsed gradient spin-echo and fraction (Connelly et al., 1987). Oil and steady gradient NMRI to maturing wheat sucrose have been mapped in fresh maize kernels and found the spatial distribution kernels (Koizumi et al., 1995), germinating of the self-diffusion coefficient of water. barley seeds (Ishida et al., 1990) and in The diffusion was slowest in endosperm developing pea seeds (Tse et al., 1996). and highest in the vascular bundle. Back et al. (1991) used the dependence between the self-diffusion coefficient of water and 4.4.2.4. High-resolution multinuclear NMR the relative water content obtained by spectroscopy Callaghan et al. (1979) to correct the proton map for wheat grain and showed the more GENERAL REMARKS. High-resolution multi- uniform distribution of water in the cor- nuclear NMR is used to detect ions and rected image. metabolites of low molecular weight, the For experiments in which germination of intracellular pH, the subcellular compart- seeds has to be followed over many hours in mentation of compounds and the flux the magnet, it is necessary to maintain a con- through metabolic pathways (Ratcliffe, tinuous water supply to the seeds, while at 1994; Schneider, 1997; Roberts, 2000). Low the same time minimizing the spectroscopic concentration of the molecules of interest Dessication - Chap 04 18/3/02 1:55 pm Page 132

132 O. Leprince and E.A. Golovina

and low receptivity of nuclei other than 1H than that of the 1H nucleus, which reduces make this approach rather insensitive. The overlapping in the spectra. Secondly, the sensitivity increases with increasing ﬁeld low natural abundance of 13C opens possi- strength. High-resolution NMR spectrome- bilities for labelling the tissue and monitor- ters are usually equipped with high-ﬁeld ing metabolic pathways. The biological use superconducting magnets in the range of NMR to study metabolism is described in 4.7–14.1 T, corresponding to a 1H fre- Section 4.3.2. 13C NMR has also been used quency of 200–600 MHz. The sensitivity to establish changes in soybean seeds dur- can be increased by multiple scanning and ing maturation and germination. The mois- usually permits the detection of millimolar ture content-dependent disappearance or concentrations of metabolites (Ratcliffe, appearance of narrow peaks associated with 1994). To increase the sensitivity further, sugars in in vivo NMR spectra is indicative the tissue volume within the detector has of the presence of free water in these seeds to be maximized. Cell suspensions and (Ishida et al., 1987, 1988a). The sensitivity excised tissues are more suitable for such of natural abundance 13C NMR can be experiments than whole plants or seeds. enhanced, by applying low-speed magic- angle spinning (Ni and Eads, 1992) or by 1H NMR. Different nuclei can be used for dif- the detection of 13C by protons coupled to ferent purposes. The high sensitivity makes the 13C nucleus (Heidenreich et al., 1998). 1H attractive for metabolite detection. 13C labelling gives opportunities for probing Nevertheless, the need to suppress the water different metabolic pathways, such as lipid signal and the complexity of spectra limit synthesis in soybean ovules (Schaeffer et the possibilities for in vivo 1H NMR. The al., 1975) and the metabolism of dormancy- small differences in chemical shift and con- breaking chemicals in red rice (Footitt et siderable overlapping of broad signals in tis- al., 1995). sues make 1H spectra poorly resolved. For example, in germinating seeds only peaks 31P NMR. In vivo 31P NMR has many applica- from sugars and oil under conditions of par- tions because of the convenient magnetic tial water signal suppression can be properties of the 31P nucleus and the physi- resolved (Koizumi et al., 1995; Ishida et al., ological importance of the information that 1996). 1H NMR spectra of oil in dry seeds can be deduced from the spectra. The mea- can be obtained because the signals from surement of cytoplasmic and vacuolar pH is other nuclei are broadened due to immobi- one of the most important applications of in lization. However, the resolution of lines is vivo 31P NMR, which is based on the depen-

not good because of differences in magnetic dence on pH of the chemical shift of Pi. susceptibility. The magic-angle sample This, together with the slow exchange of Pi spinning (MASS) technique eliminates line across the tonoplast, allows the origin of the

broadening arising from differences in mag- Pi signal – either cytoplasmic or vacuolar – netic susceptibility due to fast mechanical to be determined and, consequently, the rotation about an axis, making a magic angle cytoplasmic and vacuolar pH. A number of (54°55), and resulting in 1H spectra from important phosphorylated metabolites can dry seeds with a good resolution (Rutar, be resolved in 31P spectra. For some of them 1 1989). H NMR is widely used to analyse (Pi, polyphosphates), information on the tissue extracts for the presence of speciﬁc subcellular distribution can also be obtained compounds such as, for example, betaine in because of the pH-dependent chemical shift. wild-type and transformed Arabidopsis 31P NMR has been applied to study the pH thaliana seeds (Alia et al., 1998). of intracellular compartments in germinating seeds of Phacelia tanacetifolia (Espen et 13C NMR. 13C NMR is more attractive for al., 1995). Changes in chemical shifts of the application in vivo for two reasons. First, pH-dependent 31P signal from cytoplasmic the chemical shift scale of the 13C nucleus and vacuolar inorganic phosphate correlate is more than an order of magnitude greater with seed germination. 31P can also be used Dessication - Chap 04 4/4/02 2:19 pm Page 133

Methods for Quantifying Desiccation Phenomena 133

to monitor phosphorus compounds and choline (DPPC). It was shown that the head their changes during maturation and germi- groups are in a rigid state above and below nation of seeds, both in extracts and in vivo. the phase transition for both dry DPPC and Because of line broadening in in vivo exper- a mixture of dry DPPC and trehalose. iments, a lower number of phosphorus com- Tsvetkova et al. (1998) used 31P NMR in a pounds can be resolved (Ishida et al., 1987, comparative study of the interaction of glu- 1988a) in comparison with extracts (Ricardo cose, trehalose and hydroxyethyl starch and Santos, 1990). 31P spectra can be used with dry DPPC. The differential effect of car- for the identification of the appearance or bohydrates on the behaviour of head groups disappearance of vacuoles in seeds during has been related to the role of trehalose in germination and maturation (Ishida et al., membrane protection upon drying. 1990). Phospholipids arranged in bilayers or in an inverted hexagonal phase have different line shapes (31P pattern) (Cullis and de 4.4.2.5. Structure and dynamics of cellular Kruijff, 1979). These differences between membranes bilayer and hexagonal phase spectra arise from the fact that the lipids are restricted in GENERAL REMARKS. NMR provides a rapid, motion to the plane of the membrane in the non-invasive method for investigating the lamellar state. In the case of the hexagonal state of membranes in isolated cellular phase, a rapid motion about the cylinder fractions and in living tissues. The axis averages the chemical shift anisotropy. approach in the study of membrane struc- These differences in 31P pattern can be used ture and dynamics is solid-state NMR, to detect the presence of either phase. because of the anisotropic nature of the For many years researchers have been membranes. The main nuclei used for this interested in the membrane transition upon study are 31P and 2H. Sometimes, labelling drying from the bilayer into the hexagonal with 13C has been used, although the line phase (Simon, 1974). In an attempt to shape is difficult to analyse. detect this membrane transition, Priestley and de Kruijff (1982) applied 31P NMR to 31P NMR The chemical shift of the phospho- several dry biological systems. The in vivo lipids depends on the orientation of the spectra were complicated by the superposi- phosphate groups with respect to the mag- tion of the signals from phospholipids and netic field. In the case of unrestricted phosphorus-containing compounds. Pollen motion, all directions are averaged and the of Typha latifolia was the most suitable for spectrum is isotropic and contains the nar- spectra analysis. At 5.2% MC, the line row symmetrical 31P NMR line (Cullis and shape of the spectrum was broad and not de Kruijff, 1979). In some cases, peaks from suitable for analysis. At MC 8.8%, only different phospholipids can be resolved isotropic signals from phosphorus low- (Smith, 1985). In the case of restricted weight molecules could be identified, but, mobility of phospholipids in membranes, at 10.9% MC, a clear peak from phospho- the spectrum is anisotropic. The shape of lipids organized in bilayers became evi- the anisotropic 31P NMR spectrum depends dent. Thus, no evidence was obtained for on the type and rate of motion of the phos- the presence of a hexagonal phase in the pholipids. Thus, 31P NMR spectra are sensi- pollen on drying to 10.9% MC. tive to the physical state of the phospholipids. From the spectra, the order 2H NMR. The relatively small quadrupole parameter can be calculated (Smith, 1985). moment of deuterium makes it an ideal There are a few examples of the successful probe of membrane lipids (Smith, 1985). application of 31P NMR in the field of desic- Fatty acids labelled with 2H at different cation tolerance. Lee et al. (1986, 1989) positions must be synthesized. The 2H studied the interaction of trehalose with the NMR spectrum of membranes contains phospholipid, dipalmitoylphosphatidyl- three clearly separated lines (‘rabbit ears’), Dessication - Chap 04 18/3/02 1:55 pm Page 134

134 O. Leprince and E.A. Golovina

and the separation relates to the ordering of netic ﬁeld. IR spectroscopy is sensitive to the 2H-labelled segment. Quadrupole split- vibrations that modulate a molecule’s ting, overall pattern and relaxation times dipole moment. The range of frequencies are usually used to characterize 2H spectra. is around 1012–1014 Hz or 400–4000 cm1. Spin–lattice relaxation is sensitive to rela- The main problem of IR spectroscopy is tively rapid motions, whereas spin–spin high water absorption in the IR region.

relaxation is sensitive to slow motions D2O substitution or dry films are often (Smith, 1985). This technique can be used used. In plotting IR spectra, the intensity to study membrane phase transitions, the of absorption (A) against wave number influence of acyl chain saturation on mem- (1/) is used. The main characteristics of brane fluidity and changes in membrane the absorption band are wave number of

ﬂuidity. the maximum absorption (Amax), the width 2H NMR was applied by Lee et al. (1986, of the band determined at half of the

1989) in a study on the effect of interaction height of Amax, the optical density at Amax of trehalose with dry DPPC on the behav- and the shape of the band. Every band can iour of acyl chains. 2H quadrupole spectra be assigned to a certain chemical group of dry DPPC labelled at the 7th position and a certain type of vibration. In the case showed that the disorder of lipid acyl of simple molecules, IR spectra consist of chains is much greater in the case of inter- narrow lines. In the case of macromole- action of DPPC with trehalose above the cules, the spectrum is characterized by rel- phase transition than in hydrated or dry atively broad bands because of the DPPC without trehalose. The new type of overlapping of a great number of individ- liquid-crystalline phase observed in the dry ual lines corresponding to different types mixture of trehalose and DPPC is believed of bonds and different conformations. to play a main role in maintaining membrane stability in dehydrating organisms. 4.4.3.2. Biological applications 13C NMR. 13C-labelled phospholipids can be With the introduction of FTIR spectrome- used to study the particular dynamics of ters in the 1970s, in vivo studies became membranes in the interfacial region. Lee et possible, which was not the case with the al. (1989) used 13C-labelled sn-2-carbonyl grating IR spectrometers because of their of DPPC to study the influence of the inter- low energy throughput. FTIR spectroscopy action of dry DPPC with trehalose on inter- can be used for the analysis of certain com- facial behaviour. No changes in 13C NMR pounds, or to study the interaction between powder spectra were observed during the molecules. In dry organisms, the technique phase transition of a dry mixture of is particularly useful because of the DPPC/trehalose, whereas hydrated DPPC ‘absence’ of water. The absorption of water exhibited pronounced changes during the usually obscures other absorption bands phase transition. and thus complicates the interpretation of spectra. A considerable advantage of in vivo FTIR spectroscopy is that it permits the 4.4.3. Fourier transform infrared (FTIR) analysis of macromolecules in their natural spectroscopy environment as opposed to in a solvent. A disadvantage is that information is obtained 4.4.3.1. General description of infrared on the average vibrational absorption of all spectroscopy molecular groups contributing to the IR- Infrared (IR) spectroscopy deals with the absorption band under study. transition between vibrational energy lev- For analysis of small samples or the loca- els that permanently exist in a system, in tion of certain compounds in specific tis- contrast to NMR and EPR where the transi- sues, an IR microscope fixed to the optical tion occurs between energy levels that bench can be used. Improvement in sensi- arise in a system only in an external mag- tivity has been reached by the application of Dessication - Chap 04 18/3/02 1:55 pm Page 135

Methods for Quantifying Desiccation Phenomena 135

liquid nitrogen-cooled MCT (mercury/cad- tein secondary structure with dehydration mium/telluride) detectors, which allow (Wolkers and Hoekstra, 1995, 1997; pollen, microorganisms or slices of seeds to Golovina et al., 1997c; Wolkers et al., be studied. Peak positions or presence of 1998a,b). Conformational changes of pro- shoulders in the spectra can be analysed by teins can be derived from peak positions computer-assisted derivative analysis (Susi in the amide I (1600–1700 cm1) and II and Byler, 1983) and deconvolution (Byler (around 1550 cm1) regions (Byler and and Susi, 1986) procedures, respectively. Susi, 1986; Surewicz and Mantsch, 1988). Depending on transmittance and scattering The amide I band mainly arises from the characteristics of a sample, a transmission, CO stretching vibration of the peptide reflection or attenuated total reflection groups, and the amide II band from the (ATR) mode can be used. N–H bending vibration of the protein An example of the in vivo analysis of cer- backbone (Susi et al., 1967). The CO tain compounds in seeds is scanning in the stretching frequency is very sensitive to transmission mode along a slice of tissue. changes in the nature of the hydrogen Thus, it has been confirmed that the aleu- bonds arising from the different types of rone layer is enriched in proteins and the secondary structure. This causes a charac- endosperm in starch. In the case of dehy- teristic set of IR-absorption bands for each drating organisms, the change in molecular type of secondary structure (Susi et al., interactions or conformation is of interest. 1967). Curve fitting of the different bands The occurrence of an absorption band allows, to a certain extent, the amounts of around 2850 cm1 originating from the sym- -helix, random coil, turn and -sheet

metric stretching vibration of CH2 can safely structures to be established (Surewicz et be attributed to acyl chains, either from oil al., 1993). In some model enzyme sys- or from membranes. If the organism is low tems, a highly characteristic low wave in oil, it is possible to follow, in vivo, the number band (around 1625 cm1 in the decrease in C–H vibrational freedom in the amide I region (the intermolecular acyl chains of membranes with dehydration extended -sheets) is indicative of the for- (Cameron et al., 1983; Crowe et al., 1989; mation of large protein aggregates with Hoekstra et al., 1992). Restriction of vibra- drying (Prestrelski et al., 1993). These tional freedom by molecular interaction aggregates have also been found in vivo on (van der Waals interactions in the case of gel heat denaturation. The stability of pro- phase formation) leads to shifts of the teins against heat denaturation can be fol- absorption peaks to lower wave number and lowed by scanning over a range of sharpening of these peaks. If the sample temperatures (Wolkers and Hoekstra, holder is temperature-controlled, it is possi- 1997; Wolkers et al., 1998a). In the situa- ble to determine the gel-to-liquid crystal tion where the absorption band of water transition temperature of these membranes (HOH scissoring vibration band at from shifts in the absorption maxima with 1650 cm1) interferes with the proper esti- temperature. The same information can be mation of the different protein secondary

obtained from shifts in other absorption structures, H2O can be replaced by D2O, bands, e.g. the asymmetric CH2 stretch which causes a downward shift in wave around 2920 cm1 and the CO stretch of number. The accessibility of the proteins

the ester bond of the acyl chains around for D2O can help identify the protein sec- 1740 cm1 (Sowa et al., 1991). Although the ondary structure. general melting behaviour of oil in seeds Recently, it was established that the can also be analysed by other techniques glassy state can be studied in vivo by (e.g. differential scanning calorimetry), that inspection of the OH-stretch at around of membranes is difﬁcult with other meth- 3300 cm1 (Wolkers et al., 1998c, 1999). ods because of the small amount involved. The interaction of sugars with proteins or In vivo FTIR spectroscopy has been with polar head groups has been veriﬁed successfully applied in the study of pro- in dry model systems in the 3300 cm1 Dessication - Chap 04 18/3/02 1:55 pm Page 136

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(Wolkers et al., 1998d) and 1240 cm1 thereby limiting the range of moisture regions (Crowe et al., 1996), respectively. content that can be studied (Buitink et al., Such interaction upon desiccation has not 1996; Sacandé et al., 2000). However, the been established with certainty in vivo due future of DSC in studying anhydrobiosis to the possible absorption of other molecu- is questionable since no major difference lar groups in these regions. Although in in the calorimetric properties of water was vivo FTIR spectroscopy has disadvantages found between desiccation-tolerant and in that it is an averaging technique and sensitive organisms (Sun et al., 1994; that it is difficult to establish with cer- Buitink et al., 1996; Fig. 10.2, Chapter 10). tainty from which molecules the spectra originate, it has the considerable advantage that molecules are studied in their 4.5.2. Electron microscopy native environment. The disadvantages can be partly alleviated by parallel in vitro Owing to technical difficulties in studying experiments, also employing other meth- ultrastructural characteristics of organ- ods of analysis. elles in the dry state and upon rehydration, two promising microscopic techniques are worth mentioning because they can be con- 4.5. Additional Techniques to Study sidered as non-invasive techniques: atomic Biochemical and Biophysical Aspects of force microscopy (AFM) and low-tempera- Desiccation Tolerance ture scanning electron microscopy (LTSEM). AFM is particularly suitable for 4.5.1. Differential scanning calorimetry imaging, non-invasively, the surface topog- (DSC) raphy of membranes at a nanometer scale. Furthermore, AFM can be used to obtain DSC is applied to the study of thermal information on the mechanical properties events associated with lipid and water of surfaces (Heinz and Hoh, 1999; phase/state transition. In plant anhydro- Claessens et al., 2000). LTSEM overcomes biotes, it is used for two main purposes: problems linked to aqueous fixation. It (i) to determine the calorimetric proper- allows a fast and direct observation of ties of water present in the system; and freeze–fractured specimens with great reso- (ii) to construct a state–phase diagram in lution without altering the sample water which the glass transition temperature content. Application of LTSEM was found to be powerful for studying ultrastructural (Tg) and the ice formation/melting temperature are plotted as a function of moisture damage resulting from imbibitional injury content (Vertucci, 1990; Leprince and in seeds (Leprince et al., 1998; Nijsse et Vertucci, 1995; Buitink et al., 1996). The al., 1998; Sacandé et al., 2001) and cellu- calorimetric behaviour of the glass transi- lar collapse in lichens (Scheidegger et al., tion can be characterized although DSC 1995). In the near future, new technologi- does not give direct access to the physical cal developments (so-called semi-in-lens) and biological properties of glasses. will improve the resolution, which is cur- Sometimes, the heat released during the rently limited to 100 nm in most commer- glass transition is below the sensitivity of cially available equipment. Non-invasive the equipment. For example, in seed fixation (freeze-substitution) and a new species such as rice and tobacco, Tg can- non-aqueous fixative for immunocyto- not be detected by DSC (O. Leprince and J. chemistry (acrolein) are becoming avail- Buitink, unpublished data). Furthermore, able for transmission electron microscopy in oily seeds such as neem and Impatiens, studies (Grote et al., 1999), allowing the lipid melting transitions often mask microscope observation without disturb- the thermal events associated with water, ing the sample water content. Dessication - Chap 04 18/3/02 1:55 pm Page 137

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4.6. Acknowledgements l’Agriculture et de la Pêche, the Contrat de Plan Etat-Région and INRA; E.A.G. The authors thank Dr F.A. Hoekstra for gratefully acknowledges the ﬁnancial his contribution to the section on spec- support by a grant from the Wageningen troscopy methods and for critically read- Centre for Food Sciences and by NATO ing the manuscript. O.L. acknowledges collaborative linkage grant # LST.CLG the ﬁnancial support of the Ministère de 975082.

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Part III

Biology of Dehydration Dessication - Chap 05 18/3/02 2:07 pm Page 148 Dessication - Chap 05 18/3/02 2:07 pm Page 149

5 Desiccation Sensitivity in Orthodox and Recalcitrant Seeds in Relation to Development

Allison R. Kermode1 and Bill E. Finch-Savage2 1Department of Biological Sciences, Simon Fraser University, Burnaby, BC, V5A 1S6, Canada; 2Horticulture Research International,Wellesbourne, Warwick CV35 9EF, UK

5.1. Introduction 150 5.2. Development and Acquisition of Desiccation Tolerance 151 5.2.1. Changes in water status during development of orthodox seeds 151 5.2.2. Acquisition of desiccation tolerance during development of orthodox seeds 152 5.2.3. Loss of desiccation tolerance following germination of orthodox seeds 153 5.2.4. Effects of the rate and extent of desiccation on the acquisition of tolerance of orthodox seeds 153 5.2.5. Variation in desiccation tolerance across species 155 5.2.5.1. Seed development in recalcitrant species 157 5.2.5.2. Time-dependent effects of storage and drying rate 159 5.2.5.3. Desiccation tolerance differs between seed tissues 160 5.2.6. Mechanisms underlying the acquisition of desiccation tolerance: recent ﬁndings and speculations 161 5.2.6.1. The effects of premature desiccation during the tolerant and intolerant stages of orthodox seed development 161 5.2.6.2. Cellular and metabolic changes during the transition to a desiccation-tolerant state 161 5.2.6.3. Desiccation-tolerance mechanisms in sensitive seeds 170 5.3. Conclusions 174 5.4. References 175

150 A.R. Kermode and B.E. Finch-Savage

5.1. Introduction the embryo passes into a metabolically inactive or quiescent state. The development of most seeds can be The majority of seeds are referred to as divided conveniently into three conﬂuent ‘orthodox’, in which desiccation occurs as stages (Fig. 5.1). During histodifferentiation, a pre-programmed and ﬁnal stage in their the single-celled zygote undergoes exten- development (Fig. 5.1). Seeds of the ortho- sive mitotic division, and the resultant cells dox type and other desiccation-tolerant differentiate to form the basic body plan of structures such as spores and pollen are the embryo (the axis and cotyledons); con- unique in the degree of water loss toler- currently, there is the formation of the ated; as much as 90–95% of the original triploid endosperm or haploid megagameto- water is removed during their develop- phyte. Thereafter, cell division ceases dur- ment. In this dehydrated state, the seed can ing the seed expansion stage and there is survive the vagaries of the environment cell expansion and the deposition of and, unless dormant, will resume full meta- reserves (normally proteins along with bolic activity, growth and development lipids or carbohydrates), primarily in the when conditions conducive to germination storage tissues (i.e. cotyledons, endosperm are provided (Fig. 5.1). This chapter dis- or megagametophyte). Finally, the develop- cusses some of the mechanisms underlying ment of most seeds is terminated by some desiccation tolerance of seeds and recent degree of drying (maturation drying), which approaches to elucidate the precise roles of results in a gradual reduction in metabo- protective molecules and repair processes lism as water is lost from seed tissues and at the cellular and subcellular levels.

Development Germination growth

Histodifferentiation Maturation Desiccation Dry (Expansion)

Cell division Reduced Quiescence Renewed Reserve Cell expansion metabolism (Mature dry metabolism breakdown seed) (respiration, Reserve deposition nucleic acid and protein synthesis)

Dormancy Cell elongation (sometimes) Cell division

Desiccation-intolerant Desiccation-tolerant Desiccation-intolerant

Histodifferentiation Cell expansion Maturation drying Germination and growth Fig. 5.1. Some events associated with seed development, germination and growth. (From Kermode, 1995.) Dessication - Chap 05 18/3/02 2:07 pm Page 151

Desiccation Sensitivity in Relation to Seed Development 151

An important approach to elucidating age upon subsequent rehydration and an the basis of desiccation tolerance in seeds inappropriate proportion or distribution of is comparative analyses between seeds that freezable and non-freezable (bound) water differ in their capacity to withstand water within the seed (Berjak et al., 1992; loss, i.e. seeds of orthodox and recalcitrant reviewed in Bewley and Oliver, 1992; plant species. ‘Orthodox’ seeds can be Vertucci and Farrant, 1995). Recent research stored for long periods under conventional in this area will be discussed brieﬂy but see conditions, i.e. in the dry state and at low also Chapters 6–8 of this volume. temperature. Recalcitrant seeds, on the other hand, do not undergo maturation drying, nor are they capable of withstand- 5.2. Development and Acquisition of ing water loss of the magnitude of that Desiccation Tolerance experienced by orthodox seeds. The seeds are shed at relatively high moisture con- 5.2.1. Changes in water status during tents and are highly susceptible to desicca- development of orthodox seeds tion injury; in order to remain viable, they must not undergo any substantial change The three major phases of seed develop- in moisture. They are not storable under ment characteristic of orthodox seeds conditions suitable for orthodox seeds and, (namely histodifferentiation, expansion even when stored under moist conditions, and maturation drying; Fig. 5.1) are marked their viability is frequently brief and only by distinctive changes in fresh weight, dry rarely exceeds a few months (reviewed in weight and water content (Fig. 5.2). During Chin and Roberts, 1980; Bewley and Black, histodifferentiation and early cell expan- 1994; Smith and Berjak, 1995; Vertucci and sion, there is a rapid increase in whole Farrant, 1995; Berjak and Pammenter, seed fresh weight and water content. 1997; Pammenter and Berjak, 1999). Thus Generally, a period of rapid dry-weight the terms ‘orthodox’ and ‘recalcitrant’ have gain follows (when whole seed fresh been used to describe the storage behaviour weight is relatively stable); this takes place of seeds. A category intermediate between during the later part of the seed expansion orthodox and recalcitrant is now recog- phase of development. Most seeds lose nized (e.g. coffee) in which seeds survive water during this phase as reserves are desiccation but become damaged during deposited primarily within storage tissues, dry storage at low temperatures (0°C and displacing water from the cells. This 20°C) (Ellis et al., 1990, 1991a). It is decline in water content slows as the seed important to note, however, that the situa- approaches its maximum dry weight. Then, tion is more complex and there is a gradual as the seed undergoes maturation drying continuum of desiccation tolerance across and approaches quiescence, there is a orthodox and recalcitrant species. period of fresh weight loss accompanied by The question arises as to whether the a rapid decline in whole seed water con- desiccation sensitivity of recalcitrant seeds tent (Kermode, 1990; Fig. 5.2). is at least partially the result of an insufﬁ- Little is known about the mechanism and cient accumulation of protective proteins, route of water loss from seeds. Some studies or whether other factors (including a lack suggest the existence of a passive mecha- of protective sugars) are more important. nism whereby water is lost primarily by Since desiccation tolerance is arguably a evaporation from the surface of surrounding quantitative feature (Vertucci and Farrant, seed structures (Nechiporenko and 1995), the amount of protective proteins, or Rybalova, 1983; Lee and Atkey, 1984; the rate at which the proteins accumulate, Goncharova et al., 1985). Another suggestion may determine the level of tolerance. is that water moves from the seed to the par- Other features that may be part of the ent plant by a metabolically active process, basis of desiccation sensitivity include an i.e. the plant actually ‘pumps’ the water from inability to repair desiccation-induced dam- the seed (Meredith and Jenkins, 1975). Dessication - Chap 05 18/3/02 2:07 pm Page 152

152 A.R. Kermode and B.E. Finch-Savage

Expansion Maturation Histodifferentiation (reserve deposition) drying

dw Grams

I II III

Days of development Fig. 5.2. A general scheme of changes in whole seed fresh weight (fw), dry weight (dw) and water content (WC) during the histodifferentiation, expansion and maturation drying phases of development of orthodox seeds. Three major periods are noted: I, rapid fresh-weight gain; II, rapid dry-weight gain; III, fresh-weight loss. (From Kermode and Bewley, 1986.)

In soybean and castor bean, the desicca- drying of seeds at a desiccation-tolerant tion period is most probably initiated by stage of their development promotes germi- the severing of the vascular supply to the nation upon subsequent rehydration. Air- seed (funiculus detachment) and senes- dried grains of wheat not only germinate at cence of the pod or capsule (Greenwood an earlier stage of development than non- and Bewley, 1982; Murray and Nooden, dried grains, but at later stages may also 1986). This would suggest that relocation germinate at a faster rate than their non- of water from the seed to the parent plant dried counterparts (Mitchell et al., 1980; is not the means by which water loss Symons et al., 1983). Seeds of Phaseolus occurs. Similarly, pectic substances in the vulgaris (French bean) undergo a transition lumina of xylem elements of the rachis of to a desiccation-tolerant state around 26 wheat and barley (laid down during the DAP (days after pollination) approximately ﬁnal stages of grain maturation) may lead halfway through development (Dasgupta et to the progressive dehydration of the ear by al., 1982). Seeds at 26–32 DAP can be cutting off its water supply (Cochrane, induced to germinate to increasing extents 1985). if ﬁrst dried over silica gel, whereas those dried at 22 DAP fail to germinate when subsequently rehydrated and they eventu- 5.2.2. Acquisition of desiccation tolerance ally deteriorate. The 22 DAP seeds do not during development of orthodox seeds recover their full cellular and metabolic integrity following the premature drying Seeds of orthodox plant species cannot tol- treatment. At later times of development, erate drying at all stages of their develop- however, the seeds acquire a tolerance of ment. During very early development, seeds desiccation and also germinability is are generally intolerant of drying, but they induced. later undergo a transition to a desiccation- A similar situation exists for the castor tolerant state at a particular time (reviewed bean seed (Ricinus communis) (Kermode in Kermode, 1990, 1995). In many cases, and Bewley, 1985). Here, germinability is Dessication - Chap 05 18/3/02 2:07 pm Page 153

Desiccation Sensitivity in Relation to Seed Development 153

not achieved until 50–55 DAP, whereas pre- (brought about by air-drying to 10% water mature drying will promote the germina- content) during the course of germination, tion ability of seeds as young as 25 DAP. At while the cotyledons remain tolerant for a earlier stages of development, drying not considerably longer period (Senaratna and only fails to induce germination ability, but McKersie, 1983). also kills the seed. For both P. vulgaris and As will be discussed below, changes that R. communis, the seeds acquire a tolerance occur on dehydration of the most of desiccation and an ability to be potenti- desiccation-sensitive seeds (e.g. those of the ated to germinate by this treatment around mangrove, Avicennia marina) can be very 25 days after development commences. similar to changes brought about by desic- The transition to a desiccation-tolerant cation of orthodox seeds during the intoler- state approximately midway through ant stage following germination (Farrant et development is also characteristic of other al., 1986). Some recalcitrant seeds initiate seeds, e.g. soybean, maize, barley, germination-related metabolism shortly Agrostemma githago (Adams and Rinne, after shedding (reviewed in Vertucci and 1981; deKlerk, 1984; Bartels et al., 1988; Farrant, 1995) and, in A. marina, 10–15 Bochicchio et al., 1988). Tolerance of desic- days before shedding (Farrant et al., 1993b). cation is gained over only a few days of As germination events progress, the seeds development (e.g. between 20 and 25 DAP become increasingly sensitive to drying and in R. communis); it is achieved well before attempting to store these seeds is akin to the completion of major developmental storage of germinated, orthodox seeds events such as reserve deposition and the (Farrant et al., 1986, 1988). There is no commencement of normal maturation dry- clear-cut event delineating the end of seed ing (Kermode and Bewley, 1985; Kermode development and the start of germination; et al., 1986) (Figs 5.1 and 5.3). during both phases, recalcitrant seeds appear to remain metabolically active, although the axes may undergo a very brief 5.2.3. Loss of desiccation tolerance following period of relative quiescence. germination of orthodox seeds

During germination, seeds initially remain 5.2.4. Effects of the rate and extent of tolerant of reimposed desiccation, but at desiccation on the acquisition of tolerance of some stage after axis elongation this ability orthodox seeds is lost (Fig. 5.1). Germinating soybean seeds are tolerant of drying during the The rate at which drying is imposed during early stages, up to 6 h after commencing early development is critical for the subse- imbibition, but they become increasingly quent expression of germinability, and intolerant after this time. Thus, desiccation thus, when it is stated that a seed acquires at 36 h after the start of imbibition kills the a tolerance of desiccation at a particular seed (Senaratna and McKersie, 1983). The stage during its development, it is neces- plasma membrane appears to be a major sary to deﬁne the rate of water loss to site of damage in seeds during the desicca- which it is subjected (see Chapters 2 and tion intolerant stage of germination, as 3). Whole seeds of several legumes (Adams indicated by ultrastructural studies et al., 1983; Ellis et al., 1987) and R. com- (Crevecoeur et al., 1976) and by the munis (Kermode and Bewley, 1985) are increased solute and electrolyte leakage unable to withstand rapidly imposed dry- upon subsequent rehydration (Senaratna ing (over silica gel or under regimes similar and McKersie, 1983). There appears to be a to ambient laboratory conditions) at early differential sensitivity of different seed tis- stages (i.e. during most of development, sues with respect to the loss of desiccation prior to maturation drying) and exhibit no tolerance. For example, axes of soybean germinability upon subsequent rehydra- rapidly lose their tolerance to desiccation tion. This contrasts with seeds at the same Dessication - Chap 05 18/3/02 2:07 pm Page 154

154 A.R. Kermode and B.E. Finch-Savage + Water stress + Induction of protein LEA synthesis Inhibition of reserve breakdown conditions) (Water-stress ABA d (ABA). Desiccation d (ABA). ctive substances are synthesized. ctive Germination/growth nt vegetative tissues when they are tissues when nt vegetative Germination and post- germinative growth Precocious germination Vivipary + Desiccation Cessation of storage protein synthesis of Termination development Adjustment to drying Preparation for germination Maturation drying – + High osmolarity Water stress + Cell expansion High rate of storage protein synthesis Initiation of protein LEA synthesis Inhibition of germination – ABA + ‘Vascular factors’ from mother plant Histodifferentiation Initiation of storage protein synthesis Inhibition of germination Events during the development and germination/growth of seeds that are affected by desiccation, high osmolarity or abscisic aci of seeds that are affected by and germination/growth during the development Events tolerance is generally acquired by seeds around mid-maturation, when late embryogenesis abundant (LEA) proteins and other prote late embryogenesis when seeds around mid-maturation, acquired by is generally tolerance Fig. 5.3. Fig. Induction of a subset of LEA proteins also occurs following the transition to germination and growth, i.e. in seedlings and pla to germination and growth, the transition Induction of a subset LEA proteins also occurs following 1995.) Kermode, stresses. (From subjected to water-deﬁcit-related Dessication - Chap 05 18/3/02 2:07 pm Page 155

Desiccation Sensitivity in Relation to Seed Development 155

stage of development dried slowly over sat- occurs during the ﬁrst 55 days of develop- urated salt solutions or air-dried while ment of R. communis seeds, although these enclosed in the pod, where full germinabil- seeds can tolerate slow drying at stages as ity is evident. Tolerance of rapid drying early as 25 DAP (Kermode and Bewley, generally occurs only at or near the com- 1985). Seeds (and isolated embryos) of some pletion of reserve deposition (as indicated members of the Gramineae, on the other by the attainment of maximum dry weight) hand, can survive and germinate following a just after the onset of natural drying drastic drying treatment (which reduces (Rogerson and Matthews, 1977; Kermode their water content to around 5%) at rela- and Bewley, 1985; Ellis et al., 1987), tively early stages of development (Bartels et although there are exceptions (see subse- al., 1988; Bochicchio et al., 1988). The rea- quent discussion). sons for the variation between species in the Gradual water loss may allow protective rate of water loss tolerated during their changes to occur and hence increase the development are not known. seed’s resistance to disruption by dehydration. Rapid drying presumably would not allow such protective changes to take place 5.2.5. Variation in desiccation tolerance and may cause considerable disruption to across species cellular membranes and internal structures (see also Chapter 8) (see Section 5.2.6). As a result, the seed requires time for metabolic readjustment A wide range of species growing in differ- (i.e. repair) following rapid drying. This ent habitats produce seeds that cannot sur- cannot take place during drying itself vive drying to the low moisture content because the seed reaches a critical dry (and that enables prolonged storage of orthodox quiescent) state before the repair processes seeds. These species are spread widely can be initiated. Such repair is also through the plant kingdom and updated impeded upon imbibition because of a too- lists continue to be produced (Chin and rapid inﬂux of water, which cannot be Roberts, 1980; Hofmann and Steiner, 1989; accommodated by the weakened or dam- Hong et al., 1996). Since Roberts (1973) aged structural components of the cell. In introduced the terms orthodox and recalci- fact, rapid rates of drying may predispose trant, it has become clear that the degree to seeds to imbibitional injury, as indicated which seeds can survive desiccation varies by increased rates of solute leakage, a greatly both within and between non- symptom of cellular membrane disruption. orthodox species. The moisture contents to However, slowing the rate of hydration which seeds of different species can sur- may prevent a loss of germinability of vive dehydration ranges from just less than rapidly dried seeds by allowing time for that of vegetative tissues to almost com- repair to occur. Germinability of soybean plete tolerance. A recent study has shown a seeds (following an accelerated ageing continuous scale of desiccation tolerance treatment) is increased from 10% to 90% across 64 orthodox and recalcitrant by controlling the rate of imbibition species, with critical water contents for (Tilden and West, 1985). Since seeds survival ranging from 0.1 to > 1.2 g g1 dry become capable of surviving rapid water weight (Sun, 1999). Even in genetically loss during later development, they must related species there is wide variation in acquire a greater cellular and metabolic desiccation tolerance at shedding, for resistance to this event or have an example, within the genera Shorea, Hopea enhanced ability to effect repair during the and Dipterocarpus (Tompsett, 1987), Citrus early stages of imbibition. and Quercus (Sun, 1999) and Coffea The capacity to withstand rapid or slow (Dussert et al., 1999). Species within the desiccation during the tolerant phase of genus Acer (Hong and Ellis, 1990; Dickie et orthodox seed development varies between al., 1991; Fig. 5.4) produce orthodox and species. An intolerance of rapid desiccation recalcitrant seeds. Dessication - Chap 05 18/3/02 2:07 pm Page 156

156 A.R. Kermode and B.E. Finch-Savage

The restrictive categorization of seeds Consideration of variation in the desicca- introduced by Roberts (1973), particularly tion tolerance of seeds across species as a with the introduction of an intermediate continuum of behaviour in response to dry- category (Ellis et al., 1990, 1991a,b), ing is arguably more realistic (Farrant et remains useful in so far as it fulﬁls its origi- al., 1988; Berjak and Pammenter, 1997; nal purpose to describe storage behaviour. Pammenter and Berjak, 1999). Such varia- However, it does not accurately reﬂect tion, while tedious to categorize, provides knowledge of seed response to desiccation. an opportunity to increase our understand- Seed dry weight Norway maple Sycamore Shedding 100

20 Seed moisture content (%) Seed development

100

40 Germination Germination before drying after drying Germination (%) 20

August September October November

Seed development

Fig. 5.4. Seed development on adjacent trees of orthodox Acer platanoides (Norway maple) and recalcitrant Acer pseudoplatanus (sycamore). Adapted from data in Hong and Ellis, 1990 and Dickie et al., 1991. Dessication - Chap 05 18/3/02 2:07 pm Page 157

Desiccation Sensitivity in Relation to Seed Development 157

ing of the basis of desiccation tolerance, known what proportion of this variation is which has only just begun to be exploited. genetic in origin or due to the environ- A number of studies have shown that ments in which seed development, storage desiccation-sensitive seeds do not pass or drying occurred. Temperature of drying through a fully desiccation-tolerant phase can alter tolerance to desiccation (Ellis et during their development, but tolerance al., 1990, 1991a; Berjak et al., 1994). Rate tends to increase to a maximum near the of drying and the extent of storage before time of shedding as moisture content drying are also important time-dependent declines (Figs 5.4 and 5.5; reviewed by factors that could alter the extent of metab- Finch-Savage, 1996; Berjak and olism-induced damage accumulated dur- Pammenter, 1997). Thus, the extent of ing desiccation (Pammenter and Berjak, seed maturity at harvest is one of a num- 1999). Such damage would alter the seed’s ber of factors, discussed below, that deter- inherent capacity for desiccation tolerance mines the degree of desiccation tolerance and may obscure discrete critical water observed in sensitive seeds (e.g. as deter- potentials for survival. A further compli- mined by critical water content experi- cating factor is that the onset of metabo- ments). The increase in desiccation lism leading to the completion of tolerance as water content declines during germination is often observed in recalci- development (Fig. 5.5) and the apparent trant seeds following harvest or natural continuous range of critical moisture con- shedding (Farrant et al., 1988; Pammenter tents observed across species suggests that and Berjak, 1999), and this is known to desiccation tolerance is a quantitative fea- progressively increase sensitivity in toler- ture. However, it is more accurate to ant species (Section 5.2.3). express the degree of desiccation tolerance in terms of water potential as this reflects 5.2.5.1. Seed development in recalcitrant the amount of water available to the cyto- species plasm (see Chapter 2). When data are presented in this way, there is convincing With the exception of A. marina, the phys- evidence that tolerance appears to be iology of seed development following ini- acquired in discrete water potential steps tial histodifferentiation has strong during development (Farrant and Walters, similarities across the recalcitrant species 1998), and the tolerance of species may be so far studied in detail (reviewed by Finch- grouped according to these steps (Walters, Savage, 1996; Berjak and Pammenter, 1999; see Chapter 9), though in other stud- 1997). This general pattern of recalcitrant ies this was thought unlikely (Sun, 1999). seed development is also similar to that of It is argued that these critical water poten- orthodox seeds before they reach maxi- tials, which have different water activities mum dry weight (mass maturity) and and associated metabolic processes rapidly lose water following vascular sepa- (reviewed by Vertucci and Farrant, 1995; ration (Finch-Savage, 1996; Farrant et al., see also Chapters 2 and 9), may be related 1997). For example, when adjacent trees of to specific desiccation stresses and dis- the sympatric species Acer pseudoplatanus crete patterns of gene expression (Walters, (recalcitrant) and Acer platanoides (ortho- 1999). Thus, critical water potentials may dox) are compared, there is a strong tempo- be determined by specific tolerance mech- ral correlation in developmental events anisms or by sufficient accumulation of such as the accumulation of seed reserves desiccation protectants. and the development of germinability (Fig. Within a species, seed tolerance of des- 5.4). However, there are a number of com- iccation can vary according to provenance mon characteristics of recalcitrant seed even to the extent that one species, neem development that contrast with those of (Azadirachta indica), has been described orthodox seeds and which result in seeds as both orthodox and recalcitrant (Berjak adapted for rapid germination and estab- and Pammenter, 1997). However, it is not lishment. In general, recalcitrant seeds at Dessication - Chap 05 18/3/02 2:07 pm Page 158

158 A.R. Kermode and B.E. Finch-Savage

Seed development

70 Reserve accumulation (a)

40 45 50 55 60 65 70 75 80 85 35 (b)

30 Moisture content at 50% viability (%)

45 46 47 48 49 50 51 52 53 54 55 Moisture content at harvest (%)

Fig. 5.5. The relationship between moisture content at harvest and desicccation tolerance (moisture content at 50% viability) (a) during seed development in Quercus robur in 1989, and (b) following shedding in 1989 ( ), 1990 ( ), 1991 (), early in 1993 ( ) and late in 1993 (∆ ). The linear regression in (a) and (b) is ﬁtted to 1989 data (r 2 = 0.937, d.f. 5). (From Finch-Savage and Blake, 1994.)

shedding have had almost no net loss of 1993; A. pseudoplatanus, Hong and Ellis, water; they can still be accumulating dry 1990). In contrast, viviparous germination weight, they retain active metabolism, is a common event in other tropical recal- remain desiccation-sensitive and have no citrant species such as Telfairia occiden- requirement for desiccation to stimulate talis (Akoroda, 1986) and A. marina subsequent germination. Despite these (Farrant et al., 1993b). apparent adaptations for rapid germina- During development, tolerance to desic- tion, a few temperate recalcitrant species cation increases throughout reserve accu- are dormant at shedding (Aesculus hip- mulation as percentage moisture content pocastanum, Tompsett and Pritchard, decreases in most recalcitrant seeds as it Dessication - Chap 05 18/3/02 2:07 pm Page 159

Desiccation Sensitivity in Relation to Seed Development 159

does in orthodox species (Hong and Ellis, opment in recalcitrant species have not sig- 1990; Finch-Savage, 1996; Berjak and nificantly improved their desiccation toler- Pammenter, 1997; Farrant et al., 1997). ance, suggesting inherent limitations to the These changes are concomitant with a development of full tolerance. reduction in vacuolar volume, the appear- A. marina will tolerate little drying and ance of a semi-viscous state and other can be considered to be at the extreme sen- changes associated with desiccation toler- sitive end of the tolerance continuum ance (Farrant and Walters, 1998). However, across species; developmental age has lit- in recalcitrant seeds there appears to be no tle influence on the desiccation sensitivity clear end point to development. For exam- of seeds (Farrant et al., 1993b). In some ple, seeds of Quercus robur are shed at dif- other species, maximum desiccation toler- ferent moisture contents in different years ance is reached at a point before shedding on the same tree and those shed with the (e.g. A. pseudoplatanus, Hong and Ellis, lowest moisture content are most tolerant 1990) and tolerance may then subse- to desiccation (Fig. 5.5; Finch-Savage and quently decline (e.g. Litchi chinensis, Blake, 1994). There is a linear relationship Clausena lansium and Coffea arabica) between moisture content at harvest (pre- (Ellis et al., 1991a; Fu et al., 1994). This mature and at shedding) and the moisture decrease in tolerance may be due to the content at which 50% of seeds remain initiation of germination (Farrant et al., viable during drying. This contrasts with 1988; Hong and Ellis, 1992). A gradual orthodox species, where desiccation toler- decrease in tolerance is then shown as ger- ance continues to increase after the acqui- mination proceeds in desiccation-sensitive sition of maximum seed dry weight, during seeds (Farrant et al., 1988) as it does in maturation drying, which results in a qui- orthodox seeds (Hong and Ellis, 1992). escent seed (Sanhewe et al., 1996). In Q. robur, development is indeterminate, but 5.2.5.2. Time-dependent effects of storage consistent in several respects with that of and drying rate orthodox seeds shed early before mass maturity and therefore before full desicca- Many recalcitrant seeds are characteristi- tion tolerance is acquired (Finch-Savage cally large and consequently dry slowly. and Blake, 1994). It is therefore tempting to But even when the seeds are of similar size suggest that the level of desiccation toler- to comparable orthodox seeds, such as in ance may depend upon how far seeds of a A. pseudoplatanus (recalcitrant) and A. species progress through development, platanoides (orthodox), the recalcitrant possibly an evolutionary consequence of Acer takes 12 times as long to reach 20% the environmental and selection pressures moisture content under the same drying that were exerted on them in the past. conditions (Greggains et al., 2000a). A fur- These ideas are taken further in a compari- ther characteristic of recalcitrant seeds is son of development in orthodox P. vulgaris that even in the absence of desiccation they seeds, and the recalcitrant seeds of A. hip- deteriorate rapidly and are therefore short- pocastanum and A. marina (Farrant et al., lived. Differences in the reported critical 1997). One possibility in temperate cli- water contents for recalcitrant seeds can be mates is that the onset of winter truncates greatly influenced by these factors, and the seed development so that full tolerance threshold water potentials for a number of does not develop. However, seeds of Q. other physiological processes can be highly robur produced on trees grown outside the dependent on the postharvest history of the temperate climate zone in the apparently seed (Tompsett and Pritchard, 1998). A non-limiting environment and season common observation is that if seeds are length of South Africa also failed to pro- stored before drying they become more duce desiccation-tolerant seeds (Finch- desiccation-sensitive (Finch-Savage et al., Savage and Blake, 1994). In addition, 1996). This may be because of damage experimental manipulations of seed devel- accumulated during storage (Greggains et Dessication - Chap 05 18/3/02 2:07 pm Page 160

160 A.R. Kermode and B.E. Finch-Savage

al., 2000b) or because storage has allowed (Vertucci et al., 1991). It appears from this the initiation of germination (Farrant et al., and similar published work that desicca- 1985; Berjak et al., 1989). However, in tion sensitivity in recalcitrant seeds and some other species, particularly in seeds excised embryonic axes can occur at a min- shed early, development may continue so imum of two levels, which are influenced there is a considerable delay before the by the rate of drying: initiation of germination (Berjak and 1. The removal of freezable water is toler- Pammenter, 1997). ated and minimum survivable moisture Drying rate can affect the apparent toler- content coincides with the quantity of non- ance of whole seeds (Farrant et al., 1985; freezable (matrix-bound) water in the tis- Pritchard, 1991; Pammenter et al., 1998, sue. Farrant et al. (1988) suggested that 1999; Chapter 3), although this is not recalcitrant seeds, unlike orthodox seeds, always the case (Finch-Savage, 1992). In require this bound water for the mainte- embryonic axes, as discussed below, rapid nance of membrane integrity. drying consistently improves their survival 2. Seed viability is lost as freezable (free) to lower moisture contents, perhaps water is removed. because there is less time for damage to accumulate during drying. However, The former situation usually occurs in Pammenter and Berjak (1999) pointed out rapidly dried embryonic axes (Berjak et that rapid drying does not confer improved al., 1992), but has also been reported in desiccation tolerance because axes that relatively desiccation-tolerant recalcitrant have been rapidly dried to lower moisture seeds (Finch-Savage, 1992; Finch-Savage contents do not survive long under ambi- et al., 1993). The second situation occurs ent conditions. in more sensitive recalcitrant species. A third level of sensitivity, which is unaffected by drying rate, is thought to occur 5.2.5.3. Desiccation tolerance differs in the most sensitive seed: mechanical between seed tissues damage resulting from a reduction in cell In general, when isolated, the embryonic volume in the early stages of drying axis of desiccation-sensitive species is (Pammenter and Berjak, 1999). In all more tolerant than when it is dried in the cases, solute leakage precedes viability whole seed (Berjak et al., 1990; Pammenter loss during desiccation, suggesting that et al., 1991; Leprince et al., 1999), which significant membrane damage has may result from its more rapid drying occurred. These differences in critical when excised than when in situ (Berjak et moisture levels are consistent with the al., 1990). It may also relate to a signifi- concept of discrete water potential steps cantly smaller proportion of ‘matrix-bound’ associated with desiccation tolerance (Finch-Savage, 1992; Finch-Savage et al., (Farrant and Walters, 1998; Walters, 1999) 1993) or non-freezable water (water that is and may be related to the different desic- bound or structure-associated; Berjak et al., cation stresses encountered. In species 1992) in the axis compared to storage tis- where loss of viability appears to coincide sues. Desiccation tolerance of Landolphia with removal of non-freezable water kirkii axes is also affected by developmen- (Berjak et al., 1992; Finch-Savage, 1992), tal status and, in contrast to the whole it may be that these seeds lack mecha- seed, immature axes are more tolerant than nisms required to stabilize membranes as mature ones (Berjak et al., 1992) as a result water is removed. Where viability is lost of a lower content of non-freezable water in as freezable water is removed, other the immature axes. Despite their greater mechanisms may be limiting, such as the desiccation tolerance, the immature axes, provision of adequate protection against unlike mature axes, do not survive expo- free-radical attack. The provision of puta- sure to very low temperatures and are tive protective mechanisms in seeds is therefore unsuitable for cryopreservation reviewed in the following section. Dessication - Chap 05 18/3/02 2:07 pm Page 161

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5.2.6. Mechanisms underlying the ment), but the physical nature of such acquisition of desiccation tolerance: recent changes is enigmatic. While drastic ﬁndings and speculations changes to membranes have been observed upon their drying in vitro (Crowe et al., 5.2.6.1. The effects of premature desiccation 1992), the evidence suggests that mem- during the tolerant and intolerant stages of branes (in the desiccation-tolerant state) orthodox seed development appear to be protected against certain major alterations (e.g. transformation of the Premature desiccation during the early bilayer arrangement to hexagonal-type developmental stages of P. vulgaris (up to arrangements). The importance of a preser- 22 DAP, i.e. during the desiccation-sensi- vation of basic membrane composition dur- tive stage) drastically reduces the metabolic ing drying is obvious, for cells would and cellular integrity of the axis upon sub- surely perish without the prompt re-estab- sequent rehydration. Particularly evident is lishment of functioning membranes upon a loss in the capacity to recover polyribo- rehydration when they are challenged by a some levels and to resume protein synthe- swiftly changing hydration environment. sis (Dasgupta et al., 1982). Considerable Fourier transform infrared microspec- damage is inﬂicted upon cellular organelles troscopy has been useful for elucidating (including protein bodies and mitochon- some of the changes to membranes and dria) and upon the nuclear membrane. In other cellular constituents (e.g. proteins) fol- contrast, such severe perturbations do not lowing desiccation at the tolerant and sensi- occur following desiccation at a tolerant tive stages of seed development (Wolkers et stage, e.g. at 32 DAP. Moreover, the limited al., 1998b, 1999; see Chapter 4). Isolated damage that is sustained during drying at immature maize embryos acquire a toler- this stage is rapidly reversed following ance to rapid drying between 22 and 25 rehydration; cells regain their normal DAP, but can tolerate slow drying from 18 appearance within a very short time. DAP onwards. Rapid drying at the tolerant Studies on the effects of desiccation stages is associated with lower membrane during the sensitive stages of seed develop- permeability upon rehydration in contrast ment (or germination) suffer the limitation to embryos rapidly dried at a sensitive stage, of not distinguishing between the causes of in which there is an almost complete loss of desiccation intolerance and changes during membrane integrity. In addition, there is a the death of cells as a consequence of greater proportion of -helical protein struc- undergoing desiccation. Nevertheless, a tures in embryos rapidly dried at a tolerant few of these studies (particularly those that versus an intolerant stage (Wolkers et al., have compared the effects of drying at the 1998b). The proportion of -helical protein sensitive and tolerant stages) have pro- structures increases in the axes of embryos vided some useful information on the cel- during slow drying of 20 and 25 DAP seeds lular sites and/or metabolic processes that (as compared with that within fresh devel- are most susceptible to damage during des- oping seeds at these stages), and this factor iccation/rehydration, and hence require coincides with the acquisition of additional protection for retention of viability (see tolerance of desiccation. Chapters 9 and 12). As implied earlier, the integrity of membranes in seeds is of crucial importance to the maintenance of via- 5.2.6.2. Cellular and metabolic changes bility; any undue disruption of the during the transition to a desiccation-tolerant membrane systems during drying is likely state to be of immediate consequence once the seed imbibes. It is probable that some DEHYDRINS AND LATE EMBRYOGENESIS ABUNDANT changes in membrane structure are pro- PROTEINS ARE PRODUCED AS PART OF THE DEVELOP- voked as a consequence of desicccation MENTAL PROGRAMME. As noted earlier, (even during the tolerant stages of develop- orthodox seeds are not capable of with- Dessication - Chap 05 18/3/02 2:07 pm Page 162

162 A.R. Kermode and B.E. Finch-Savage

standing desiccation at all stages during glycine content and a high hydrophilicity their development, but their potential index) also accumulate in Escherichia coli acquisition of tolerance is usually sub- and in the yeast Saccharomyces cerevisiae stantially earlier than the onset of the nat- as an adaptive response to hyperosmotic ural drying event itself. A highly conditions; the authors suggest that most abundant set of hydrophilic proteins LEA proteins are part of a more wide- exhibiting temporal regulation during spread group that they term ‘hydrophilins’ seed development (i.e. the late embryoge- (Garay-Arroyo et al., 2000). nesis abundant (LEA) proteins first A subset of the LEA proteins (including described in cotton) has been implicated the LEA D-11 family and some denoted in desiccation tolerance (Dure, 1993; see RAB (responsive to abscisic acid (ABA)) in also Chapters 1, 10 and 11). The genes rice) have been termed dehydrins; they encoding these proteins arise as highly exhibit some common features in their coordinately regulated sets, which on this structure that may be important for their basis comprise two distinct classes in cot- putative protective function (Close, 1996). ton (Hughes and Galau, 1989); the mRNAs Dehydrin genes exhibit a flexible expres- that correspond to the two classes peak sion repertoire, being responsive to both just prior to, or during, desiccation developmental and environmental cues (Hughes and Galau, 1989). LEA protein (reviewed by Thomas et al., 1991). synthesis constitutes a large proportion of Transcription of these genes is also the translational activity of the cotton induced in virtually all seedling tissues embryo during late maturation (up to subjected to water stress (i.e. non-lethal 25%), regulated at the level of transcrip- desiccation). Thus, the protective role of tion, i.e. by the abundance of lea mRNAs dehydrins in the survival of water loss is (Hughes and Galau, 1987). In mature cot- purported to be dual: during maturation ton embryos they comprise about 2% of drying of the developing seed and follow- the total soluble protein (Dure, 1993) or ing germination/growth of the mature seed about 30% of the non-storage protein moi- (i.e. in seedlings or plant vegetative tissues ety (Hughes and Galau, 1987). undergoing mild water stress) (Fig. 5.3). Since their description in cotton, mes- Precocious appearance of the proteins and sages homologous to the lea cDNAs of cot- their mRNAs can be induced in cultured ton (representing at least five conserved immature embryos by exogenous ABA families of corresponding proteins) have treatment. It has been hypothesized that, been found in abundance in mature dry during normal development, high levels of embryos and storage organs of many ABA induce the accumulation of these diverse plant species including polypeptides and hence prepare the Arabidopsis thaliana, several crop species embryo for desiccation or possible cellular and gymnosperms. Protein families related disruption upon subsequent rehydration (reviewed by Kermode, 1990, 1995; Bray, to some of the LEA proteins are induced 1991, 1993; Bewley and Oliver, 1992; during drying of xerophytic species, e.g. Chandler and Robertson, 1994; Ingram and the desiccation-tolerant resurrection plant Bartels, 1996). (Craterostigma plantagineum), which is capable of surviving in the desiccated state REGULATION OF DEHYDRIN AND LEA GENE EXPRESSION for long periods and resumes full physio- BY ABA. Since ABA has been implicated as a logical activity within several hours of mediator of stress responses, especially rehydration (Bartels et al., 1990; where water stress is concerned, its poten- Piatkowski et al., 1990; reviewed in tial role as the primary regulator of lea Ingram and Bartels, 1996; see Chapter 11). genes in vegetative tissues has been inves- The desiccation-related proteins accumu- tigated (reviewed by Bray, 1991; Chandler late in leaves; some are also present within and Robertson, 1994; Kermode, 1995; roots and in seeds. Proteins sharing fea- Ingram and Bartels, 1996; Plant and Bray, tures with plant LEA proteins (e.g. a high 1999). In some cases (but not all) ABA Dessication - Chap 05 18/3/02 2:07 pm Page 163

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application stimulates the accumulation of of maize has been undertaken in order to the mRNAs in the absence of water stress, elucidate the possible regulatory role of and there is some evidence that endoge- ABA and to address whether discrete paral- nous ABA plays a regulatory role in their lel ABA and stress response pathways exist expression in seedling tissues (Fig. 5.3). in developing maize embryos (Finkelstein, For example, there is excellent correlation 1993). However, substantially different (e.g. in barley and maize) between the results have been obtained depending on amounts of mRNA and ABA in shoots, the type of LEA protein under study and roots and aleurone layers from either well- more systematic and detailed investigation watered, dehydrated or dehydrated/rehy- is needed. Several 23- to 25-kDa proteins drated seedlings (Chandler et al., 1988; corresponding to RAB17 are expressed nor- Gomez et al., 1988). Other stresses that mally in the ABA-deficient mutants of lead to increased endogenous ABA (e.g. maize (vp-2 and vp-5) in contrast to the salt, cold and wounding) are often also dependency on applied ABA for their capable of eliciting expression of these expression in vegetative tissues of the genes. The most convincing evidence for mutant seedlings (Pla et al., 1989). Likewise, the role of ABA in dehydrin gene expres- the regulation of the Rab28 gene (a homo- sion comes from studies of ABA-deficient logue of the cotton lea D34 gene) in excised mutants of maize (Pla et al., 1989, 1991). young embryos of the ABA-deficient vp-2 When exposed to dehydration stress, mutant closely resembles that found in non- seedlings homozygous for mutations lead- mutant excised young embryos (Pla et al., ing to vivipary (e.g. vp2 and vp5) fail to 1991). In contrast, embryos of the ABA- elevate ABA levels and show a correspond- insensitive mutant of maize (vp-1) do not ing inability to produce dehydrins. Similar accumulate Rab28 transcripts to significant results have been found in an ABA-defi- amounts during development; surprisingly, cient mutant of tomato (Cohen and Bray, induction of Rab28 mRNA can be achieved 1990; reviewed by Bray, 1991). in these young vp-1 embryos by ABA treat- What is the evidence that ABA plays a ment (Pla et al., 1991). Expression of the central regulatory role in the expression of maize Em gene (a group 1 lea gene) may be lea genes within the developing seed? dependent on both the presence of ABA Generally, the mRNAs encoding LEA pro- within embryos and its perception via a teins are detected in embryos around mid- functional Vp-1 gene product; it is barely development; highest levels of expression detectable in the ABA-deficient mutant occur either at incipient desiccation or embryos and is undetectable in the ABA- during maturation drying itself (Gomez et insensitive vp-1 embryos. Nevertheless, vp-1 al., 1988; Mundy and Chua, 1988; Close et embryos do exhibit a response to both ABA al., 1989). The mRNAs are preserved in and osmotica at the molecular level, since the mature dry seed but are rapidly they accumulate specific gene products (22- degraded upon imbibition, although, in and 30-kDa polypeptides) differentially some cases, certain proteins persist follow- upon imposition of osmotic stress or exoge- ing imbibition (Han et al., 1996). nous ABA (Butler and Cumming, 1993). Precocious appearance of the proteins and The Vp-1 gene is thought to encode a their mRNAs can be induced in cultured novel type of transcription activator, which immature embryos by exogenous ABA. plays a role in the expression of ABA- Thus, high levels of ABA during mid- responsive genes during seed development development are thought to induce the (e.g. maize globulin and Em genes), similar accumulation of these polypeptides and to the Abi-3 gene product in Arabidopsis hence prepare the embryo for desiccation and other species (reviewed by Giraudat et or possible cellular disruption upon subse- al., 1994; Hattori et al., 1995; McCarty, quent rehydration (Fig. 5.3). 1995). The promoter elements of the rice A comparative analysis of wild-type, Osem gene (an Em-type gene) required for ABA-deficient and ABA-insensitive mutants regulation by VP-1 have been identified Dessication - Chap 05 18/3/02 2:07 pm Page 164

164 A.R. Kermode and B.E. Finch-Savage

(Hattori et al., 1995). These include 1 plantlets subjected to drought stress. TACGTGTC (an ABA-responsive element or Thus, there may be two regulation path- ABRE), a small sequence located just down- ways that mediate dehydrin transcript stream of the ABRE and a quantitiative ele- accumulation in seeds and stressed vegeta- ment (the sph box/RY repeat), conserved in tive tissues – an ABA-dependent pathway many seed-specific gene promoters. and an ABA-independent pathway; Accumulation of group 3 LEA proteins together, these pathways may have cumula- in maturing maize embryos may be depen- tive effects (Giordani et al., 1999). dent upon ABA but appears to have no Ectopic expression of the Abi-3 gene specific requirement for the Vp-1 gene product (Giraudat et al., 1992) allows the product (Thomann et al., 1992). ABA-mediated activation of lea genes in Interestingly, when an ABA-deficient, vegetative tissues of A. thaliana (Parcy et viviparous mutant of maize (vp-5) is al., 1994). Seed viability is not altered in manipulated either genetically or via ABA-deficient (aba) and ABA-insensitive biosynthesis inhibitors to induce gib- (abi-3) mutants of A. thaliana, yet seeds of berellin (GA) deficiency during early seed double mutants exhibiting these two traits development, vivipary is suppressed in do not undergo desiccation on the parent developing kernels and the seeds acquire plant, are intolerant of artificial desiccation desiccation tolerance and storage longevity and fail to produce some of the late abun- (White et al., 2000). Major accumulation of dant proteins (Koornneef et al., 1989;

GA1 and GA3 occurs in wild-type maize Meurs et al., 1992). These double-mutant kernels, just prior to a peak in ABA content seeds accumulate only low amounts of the during development. It is speculated that major storage proteins and are deficient in these GAs induce a developmental pro- several low-molecular-weight polypep- gramme that leads to vivipary in the tides, both soluble and bound, some of absence of normal amounts of ABA, and which are heat-soluble. During develop- that a reduction of GAs re-establishes an ment (14–20 DAP), the low amounts of var- ABA/GA ratio appropriate for suppression ious maturation-specific proteins are of germination and induction of matura- degraded and proteins characteristic of ger- tion. Induction of GA deficiency does not mination are induced, in the absence of suppress vivipary in vp-1 mutant kernels, germination. Here, the seed developmental suggesting that VP-1 acts downstream of programme is not completed, and there is a both GA and ABA in programming seed premature (yet incomplete) switching to a development (White et al., 2000). germination programme in the absence of Two ABA-deficient mutants of sun- substances presumed to be protective flower have been isolated – nd-1, an albino, against desiccation. Seeds become desicca- non-dormant and lethal mutant exhibiting tion-tolerant when the plants are watered a very low ABA content and no accumula- with an ABA analogue (LAB 173711) or by tion of ABA in response to stress, and w-1, incubating isolated immature seeds (11–15 a wilty mutant, with reduced ABA accu- DAP) with ABA and sucrose. Whereas mulation during embryo and plantlet sucrose may protect desiccation-sensitive development and drought stress (Giordani structures from damage, ABA inhibits pre- et al., 1999). The w-1 mutant exhibits a cocious germination and may be required reduction of dehydrin transcripts in the for, or is accompanied by, completion of early stages of embryo development as the seed developmental programme and compared with wild-type embryos, indicat- associated acquisition of desiccation toler- ing that ABA affects dehydrin accumula- ance (Meurs et al., 1992). In another study, tion; however, the amount of dehydrin expression of a specific lea gene (a group 1, transcripts appears to be independent of D19/Em homologue) was found to be ABA content during late embryogenesis. slightly reduced in seeds of the Accumulation of dehydrin transcripts Arabidopsis aba mutant, but was reduced occurs in the leaflets and cotyledons of nd- by approximately tenfold in the abi-3 Dessication - Chap 05 18/3/02 2:07 pm Page 165

Desiccation Sensitivity in Relation to Seed Development 165

mutant; expression in the double mutant OTHER PROTECTIVE PROTEINS IMPLICATED IN DESICCA- was not studied (Finkelstein, 1993). A dif- TION TOLERANCE. Specific small heat-shock ferent Arabidopsis abi-3 mutant (abi-3-3, proteins (HSPs) of the cytosolic classes (I isolated by screening for mutants that ger- and II) accumulate in seeds of several plant minate in the presence of the GA biosyn- species. These proteins appear to be homo- thetic inhibitor, Uniconazol) showed geneously distributed in all tissues of the abnormal seed development, remaining seed and a role in the acquisition of desic- green until maturity, had dramatically cation tolerance has been suggested (Coca reduced amounts of storage proteins, was et al., 1994; Wehmeyer et al., 1996; see desiccation-sensitive, and lacked dor- Chapters 1 and 10). In Arabidopsis and mancy, indicative of a possible role for the other species, class I small HSPs are first Abi-3-3 gene in the control of the synthesis detected during mid-maturation and of seed storage proteins and desiccation become most abundant in dry seeds protectants (Nambara et al., 1992). ABI5, a (Wehmeyer et al., 1996; Carranco et al., member of the family of basic leucine zip- 1999). In some seeds (e.g. Arabidopsis), the per transcription factors, regulates a subset proteins decline rapidly during germination of lea genes during seed development and (Wehmeyer et al., 1996); in others (e.g. sun- in vegetative tissues in the presence of flower), they persist (Coca et al., 1994). The ABA (Finkelstein and Lynch, 2000). Abi-3 gene product may activate expression Further studies to clarify the role of of genes encoding specific small HSPs dur- ABA and other components of the signal ing seed development. Transcriptional acti- transduction pathway leading to lea gene vator mutants of Arabidopsis (abi-3-6, expression and other late maturation fus3-3 and lec1-2) that are desiccation-sen- events in developing seeds will be awaited sitive have undetectable amounts of HSP17.4 with interest. Recessive mutants of (abi-3-6) or highly reduced amounts of the Arabidopis with lesions at the Fusca3 protein (fus3-3 and lec1-2), i.e. less than 2% (fus3) and Leafy Cotyledon (lec1) gene loci of that in wild-type seeds (Wehmeyer and lead to various abnomalities during mid- Vierling, 2000). Interestingly, a chimeric embryogenesis and late embryogenesis, gene consisting of the small HSP gene pro- including loss of dormancy and failure to moter linked to -glucuronidase (GUS) acquire desiccation tolerance (Kirik et al., shows strong expression in mutant seeds 1998). FUS3 and LEC1 modulate the abun- that are heat-stressed, indicating that the dance of ABI3 protein in seeds and syner- genes are under distinct developmental gistic interactions between the three and stress regulation. proteins (ABI3, FUS3 and LEC1) are Polypeptides produced in sunflower thought to control various key events, seeds (e.g. HSP17.6 and HSP17.9, belong- including accumulation of chlorophyll and ing to different families of cytoplasmic anthocyanins, sensitivity to ABA and small HSPs) are indistinguishable from expression of individual members of the low-molecular-weight HSPs expressed in 12S storage protein gene family (Parcy et vegetative tissues in response to water al., 1997). Interestingly, part of FUS3 (a deficit, but they are different from homolo- continuous stretch of 100 amino acids) gous proteins expressed in response to shows significant similarity to the B3 thermal stress (Coca et al., 1994; Carranco domain of the ABI3 and VP-1 proteins et al., 1997, 1999). Proteins immunologi- (Luerssen et al., 1998), a domain which cally related to two sunflower small HSPs interacts with the RY cis promoter motif of are detected in unstressed vegetative tis- several seed proteins. Thus, both FUS3 and sues of the desiccation-tolerant resurrec- ABI3 may be essential components of a reg- tion plant C. plantagineum and are ulatory network acting in concert through induced to higher levels in these tissues the RY-promoter element to control gene by water stress and heat shock. In desicca- expression during late embryogenesis and tion-sensitive Craterostigma callus tissue, seed development (Reidt et al., 2000). there are no detectable small HSP-related Dessication - Chap 05 18/3/02 2:07 pm Page 166

166 A.R. Kermode and B.E. Finch-Savage

polypeptides, but their expression, and the However, it is possible that the attainment concurrent acquisition of desiccation tol- of a critical level of reserves is required erance is induced by exogenous ABA before the seed can withstand desiccation (Alamillo et al., 1995). (Kermode, 1997). Highly vacuolated cells Small HSPs, immunologically related to (hence containing little reserve material) a 20-kDa HSP from desiccation-sensitive may undergo severe mechanical disrup- chestnut (Castanea sativa) seeds have been tion during water loss, and tearing or detected in orthodox and recalcitrant seeds shearing of membranes (or other cellular of 13 woody species; hence additional pro- components) could lead to irreversible teins or mechanisms are likely to be changes in their internal morphology. The involved in desiccation tolerance (Collada presence of a critical level of cellular et al., 1997) (see later discussion). reserves would limit such changes (Table Major intrinsic proteins (MIPs) are a 5.1). The quantity of reserves may also family of channel proteins that are mainly merit consideration in relation to the loss represented by aquaporins in plants. They of tolerance during seed germination. As are generally divided into TIPs (tonoplast noted earlier, while soybean seed axes intrinsic proteins) and PIPs (plasma mem- rapidly lose their tolerance to desiccation brane intrinsic proteins) according to their during the course of germination, the subcellular localization (reviewed by cotyledons remain tolerant for a consider- Maurel et al., 1997). The vacuolar mem- ably longer period (Senaratna and brane protein, -TIP (a water-channel pro- McKersie, 1983). The major breakdown of tein), accumulates during seed maturation reserves within the cotyledons is a post- in the parenchyma cells of seed storage germinative event; however, catabolism of organs. Synthesis of this integral membrane reserves within the axes occurs relatively protein does not appear to be related (in a early (i.e. during germination) to provide a quantitative manner) to storage protein source of nutrients. The decline of deposition and a role in seed desiccation, reserves below a critical level within the cytoplasmic osmoregulation and/or seed axes may contribute to a loss of desicca- rehydration has been suggested (Johnson et tion tolerance within this germinating tis- al., 1989). The water-channel activity of the sue. Interestingly, certain seed storage protein can be regulated by phosphoryla- proteins are suggested to play a more tion and the protein assembles as a 60 Å direct role in desiccation tolerance. One 60 Å square in which each subunit is member of the vicilin superfamily in pea formed by a heart-shaped ring comprised of (psp54) is expressed during seed desicca- -helices. This structure is remarkably sim- tion and is not detected prior to this stage; ilar to that of mammalian PIPs, suggesting the mRNA encoding the protein declines that the molecular design of functionally soon after imbibition, but can be detected analogous and genetically homologous in vegetative tissues in response to water- aquaporins is maintained between the plant deﬁcit-related stresses and ABA (Castillo and animal kingdoms (Daniels et al., 1999). et al., 2000). A lower-molecular-weight In the desiccation-tolerant resurrection protein (p1), which corresponds to the C- plant C. plantagineum, homologues to PIPs terminal third of p54, shares some proper- and TIPs are regulated by dehydration and ties with dehydrins and is suggested to ABA, with members of a subset of PIPs protect chromatin structure during desic- (PIPa) being regulated by ABA-dependent cation (Castillo et al., 2000). Seed storage and ABA-independent pathways (Mariaux globulins of spermatophytes are thought to et al., 1998). have evolved from a group of ancient In many seeds, the acquisition of desic- single-domain proteins of prokaryotes cation tolerance during the seed expansion and fungi functional in cellular desicca- stage of development occurs well before tion/hydration processes (Baumlein et al., the completion of reserve deposition. 1995; Shutov et al., 1998). Dessication - Chap 05 18/3/02 2:07 pm Page 167

Desiccation Sensitivity in Relation to Seed Development 167 s number and strength of sorption sites; drying, preventing loss of its protective potential damaging effects of increasing ionic strengths damaging effects cytosol during drying (Based on Kermode (1990). With permission from a transition from liquid crystalline to gel stachyose) inhibits sucrose crystallization during acid accumulation Prevent loss of tightly bound (‘vital’) waternecessary for structural and functional capacity of cells enhanced with Water-binding integrity of biomolecules increased number of sorption sites plus raffinose and/orplus raffinose stachyose due to lateral phase separation of hydrophilic (polar) end groups of membrane phospholipids in the bilayer and phase and/or phospholipids; oligosaccharide (raffinose chaperones, some LEAs degradation of damaged or denatured proteins Lipid-soluble antioxidants(e.g. tocopherols) As above; prevent de-esterification oflipids, proteins Scavenging activity increases resistance to membrane phospholipid and free fattyHydrophilic, denaturation-resistant proteins (e.g. LEAs, free-radical-mediated desiccation injury other desiccation-inducible As above disruption of cellular componentspolypeptides)‘Repair’ proteins, proteases,ubiquitin and extension confers mechanical strength to whole cell protein, HSP/molecular Rapid re-establishment of structural and metabolic integrity following imbibition repair of membranes and other cellular Efficient capacity of cells enhanced with Water-binding components restores normal functioning; aid increased native conformation of protective molecules maintained proteins in recovering their native conformation; throughout drying; bind ions and thereby counteract Possible components of desication tolerance in seeds and their protective action. Refer to review by Kermode (1990) and Ingram Bartels (1996) references therein. Table 5.1. Table CRC Press, Inc.) Site or process affectedMembranes Protective component Carbohydrates: sucrose Prevent changes in selective permeability Structure/metabolism Protective action Hydroxyl groups of sucrose replace water on Reserves: carbohydrates, Prevent ‘whole scale’ mechanical Critical level of reserves in vacuoles/storage bodie Possible mode of protection a Dessication - Chap 05 18/3/02 2:07 pm Page 168

168 A.R. Kermode and B.E. Finch-Savage

ROLE OF SUGARS (see also Chapters 1, 10 and humidity (Blackman et al., 1992). However, 11). It is likely that the underlying basis an increase in the amount of raffinose is of desiccation tolerance is diverse and is not correlated with the acquisition of des- not simply restricted to the synthesis of iccation tolerance of wheat embryos (Black specific proteins. The ability to withstand et al., 1999). Accumulation of fagopyritol desiccation may also depend upon B1 (a galactopinitol) in buckwheat seeds is increased amounts (or a heightened capac- temporally associated with the acquisition ity to synthesize) molecules which stabi- of desiccation tolerance during develop- lize membranes. Carbohydrates such as ment (Horbowicz et al., 1998). This major trehalose (a non-reducing disaccharide of soluble carbohydrate, which comprises glucose) are effective in preserving the 40% of the carbohydrate of the mature structural and functional integrity of mem- buckwheat embryo, declines with the loss branes in vitro at low water contents of desiccation tolerance following germina- (reviewed by Crowe et al., 1992). Drying tion. Temperature conditions during seed and rehydration of the model membrane development that have a favourable effect on sarcoplasmic reticulum usually results in vigour and storability of buckwheat seeds the fusion of vesicles and loss of the ability result in seeds having a lower sucrose-to- to transport calcium. However, when disac- fagopyritol ratio as compared with those charides such as trehalose are present in that develop under non-optimal tempera- concentrations equivalent to those in desic- ture conditions (Horbowicz et al., 1998). cation-tolerant organisms, functional vesi- Although trehalose is not abundant in cles are preserved. Membrane fusion vascular plants, it has been identified as a during desiccation is thought to be pre- major carbohydrate in more than 70 vented as a result of the sugars’ hydroxyl species of desiccation-tolerant lower plants groups interacting (i.e. forming hydrogen (reviewed by Muller et al., 1995). There are bonds) with the polar head groups of phos- recent reports of trehalose in relatively pholipids and functional groups of pro- high amount in two desiccation-tolerant teins (Crowe et al., 1992). Thus, the sugars angiosperms (reviewed by Muller et al., are thought to alter physical properties of 1995; see references therein). One is dry membranes so that they resemble those Myrothammus flabellifolia, a dicotyledo- of fully hydrated biomolecules (Crowe et nous plant living in arid, rocky regions in al., 1992) (Table 5.1). southern Africa, the leaves of which con- The occurrence of trehalose in high contain about 3% trehalose on a dry weight centrations in anhydrobiotic organisms basis. The other is the grass Sporobolus such as yeast and nematodes (up to 20% of stapfianus, in which trehalose comprises their dry weight) suggests that this sub- 2–5% of the total soluble carbohydrates. stance may be involved in their desiccation Interestingly, even though most higher tolerance (Crowe et al., 1992). A role for plants contain low amounts of trehalose, sucrose and raffinose (carbohydrates found high activities of trehalase, an enzyme in much greater abundance in seed tissues which degrades trehalose, have been found than trehalose) in the preservation of mem- (reviewed by Muller et al., 1995). A. branes during drying has been suggested by thaliana possesses genes for at least one of Leopold and Vertucci (1986) and several the enzymes required for trehalose synthe- others. Orthodox seeds accumulate consid- sis, trehalose-6-phosphate phosphatase erable amounts of soluble proteins and sug- (Vogel et al., 1998). ars throughout maturation, and these As noted above, the protective effect of collectively may be important in the acqui- sugars probably extends to preventing irre- sition of a desiccation-tolerant state (Amuti versible changes to proteins. For example, and Pollard, 1977). Stachyose accumulates phosphofructokinase is a tetrameric in immature soybean seeds subjected to enzyme, which is irreversibly denatured slow drying, but does not increase signifi- during desiccation, dissociating into inac- cantly when seeds are maintained at high tive dimers. However, the disaccharides Dessication - Chap 05 18/3/02 2:07 pm Page 169

Desiccation Sensitivity in Relation to Seed Development 169

sucrose, maltose and trehalose stabilize the dried embryos cannot account for their activity of the enzyme (in vitro) during dry- enhanced viability as compared with ing (Carpenter et al., 1987). Although the rapidly dried embryos; however, enhanced evidence from these experiments carried synthesis of LEA proteins embedded in the out in vitro is convincing, the role of sugars glassy matrix may be a contributing factor (in vivo) in protecting cells during water (Wolkers et al., 1999). deficit and during desiccation of orthodox The proteins of maturation-defective seeds remains to be elucidated. mutants of Arabidopsis (e.g. abi-3 and lec One way sugars may protect the cell mutants) appear to be more susceptible to during severe desiccation is by glass forma- denaturation during heating (Wolkers et tion (see Chapter 10); in the presence of al., 1998a). Proteins in dry wild-type seeds sugars a supersaturated liquid is produced do not denature at temperatures up to with the mechanical properties of a solid 150°C; those of dry desiccation-sensitive (Koster, 1991). Only sugar mixtures equiva- seeds (lec1-1, lec1-3 and abi3-5) denature lent in concentration and composition to at 68, 89 and 87°C, respectively. In con- those of desiccation-tolerant embryos are trast, in desiccation-tolerant seeds (abi3-7 able to form glass at ambient temperature and abi3-1), denaturation commenced (Koster, 1991) and this ability has been above 120 and 135°C, respectively. The dif- associated with retention of viability of ferential sensitivity of the seed proteins of maize embryos (Williams and Leopold, the mutants to denaturation has been 1989). Glass formation has been suggested attributed in part to differences in molecu- to prevent cellular collapse during desicca- lar packing density, which is higher in dry tion and to promote a state of metabolic desiccation-tolerant seeds than in dry des- quiescence by restricting diffusion of sub- iccation-sensitive seeds (Wolkers et al., strates and products within cells (Koster, 1998a). 1991). Carrot somatic embryos, when pre- treated with ABA, are able to tolerate slow OTHER MECHANISMS UNDERLYING DESICCATION TOLER- drying, but are still intolerant of rapid dry- ANCE. The loss of desiccation tolerance during. This appears to be due in part to the ing germination of soybean seeds is not extent of protein denaturation, which is associated with any compositional changes greater after rapid drying (Wolkers et al., in fatty acids, but is correlated with a 1999). In contrast to slowly dried embryos, decline in the quantity of lipid-soluble which form a glassy state at room tempera- antioxidants in the membrane (Senaratna et ture, no clearly defined glassy matrix is al., 1985a,b). These antioxidants may con- formed in rapidly dried embryos. The aver- tribute to the desiccation tolerance of axes age strength of hydrogen bonding is less in during the early stages of germination by rapidly dried versus slowly dried embryos, preventing changes in membrane fluidity which may be indicative of less extensive caused by free-radical attack on phospho- ‘molecular packing’ in the former. Sucrose lipids in response to drying (Senaratna et accumulates following rapid drying of al., 1985a,b). The transition to desiccation embryos; following slow drying, the trisac- sensitivity following germination of pea and charide umbelliferose is accumulated at cucumber seeds is accompanied by a desic- the expense of sucrose. In phospholipid cation-induced imbalance of metabolism

model systems, both carbohydrates are able (i.e. increased emission of CO2 and fermen- to form a stable glass with drying; they tation products such as acetaldehyde) in the depress the transition temperature of dry radicle, which precedes loss of membrane liposomal membranes to an equal extent as integrity (Leprince et al., 2000). Imbalanced well as preventing leakage from dry lipo- metabolism is signiﬁcantly reduced when

somes upon subsequent rehydration. sensitive axes are dried in 50% O2 instead Likewise, both exhibit an equal capacity to of air and it is suggested that a balance

protect a desiccation-sensitive protein. between down-regulated metabolism and O2 Thus, increased umbelliferose in slowly availability is associated with desiccation Dessication - Chap 05 18/3/02 2:07 pm Page 170

170 A.R. Kermode and B.E. Finch-Savage

tolerance. Products resulting from imbal- by facilitating the conversion of abnormal anced metabolism (e.g. acetaldehyde) dis- L-isoaspartyl residues to normal L-aspartyl turb the phase behaviour of phospholipid residues (Mudgett and Clarke, 1994). A vesicles and thus may aggravate membrane summary of some of the possible compo- damage induced by dehydration (Leprince nents of desiccation tolerance in seeds is et al., 2000). presented in Table 5.1. Within the developing seed, the thiol- requiring (1-cysteine) peroxiredoxin family DIFFICULTIES IN ASSESSING THE ROLE OF PROTECTANTS of antioxidants may protect tissues (e.g. the IN DESICCATION TOLERANCE. As indicated embryo and aleurone layer of cereals) from above, a wealth of information has been reactive oxygen species during desiccation derived from the study of desiccation-toler- and early imbibition (Haslekas et al., 1998; ant systems, such as seeds and resurrection Stacy et al., 1999). PER1, a protein belong- plants, and from the various molecular and ing to this family, is maintained in imbibed biochemical analyses that have contributed dormant barley seeds, but declines in the to our understanding of gene and protein non-dormant seeds. In immature embryos function. In orthodox seeds, the metabolic and aleurone layers, the protein resides in changes that occur either prior to or during the nucleus and is most abundant within maturation drying (including the accumu- the nucleolus (Stacy et al., 1999). In con- lation of oligosaccharides, sugars and LEA trast, in mature imbibed dormant seeds, an proteins) may have functional significance equivalent amount of protein is present in in protecting them against the rigours of the cytosol. In Arabidopsis, the expression desiccation and/or subsequent rehydration. of AtPer1, a gene encoding a protein with However, some of the changes in metabo- similarity to barley PER1, is reduced in lism of orthodox seeds during the time of seeds of the ABA-insensitive mutant, acquisition of desiccation tolerance may abi3-1, but is unaltered in an ABA-defi- not directly contribute to the ability to cient mutant of Arabidopis (aba-1) withstand water loss, but rather may be (Haslekas et al., 1998). pre-programmed changes that are part of Serotonin accumulation in walnut other seed developmental programmes ulti- cotyledons is thought to protect seeds from mately important for seedling survival. toxic ammonia concentrations following What are other research strategies that may seed desiccation (Schroder et al., 1999). contribute to our understanding of the In conclusion, the basis of desiccation underlying basis of desiccation tolerance? tolerance of developing seeds is still a One strategy is the transfer of genes encod- poorly understood phenomenon. What ing putative desiccation protectants into emerges from the evidence available at pre- transgenic host plants with the ultimate sent is a complex process involving various goal of testing protein function and metabolic and/or structural adjustments, enhancing stress tolerance (see Chapter which allow cells to undergo extensive 11). Another approach is the comparative water loss with a minimum of damage analysis of LEA- and dehydrin-related pro- (Table 5.1). However, while maturation teins and other putative desiccation protec- drying may inflict limited damage on cells tants in recalcitrant versus orthodox seeds of orthodox seeds, the capacity to reverse (discussed below). Both approaches have such changes (i.e. to effect repair) upon limitations. subsequent rehydration is probably an integral feature of desiccation tolerance 5.2.6.3. Desiccation-tolerance mechanisms (Bewley and Oliver, 1992; Ingram and in sensitive seeds Bartels, 1996; O’Mahony and Oliver, 1999a,b). An L-isoaspartyl protein methyl- In the previous sections we have shown transferase that accumulates in wheat that development of orthodox seeds fol- seeds during the late stages of caryopsis lows a largely predetermined sequence of development may repair damaged proteins events that leads to desiccation and then Dessication - Chap 05 18/3/02 2:07 pm Page 171

Desiccation Sensitivity in Relation to Seed Development 171

shedding of the seed in a quiescent (and sidered to be particularly sensitive to des- sometimes dormant) state. In evolutionary iccation. The pattern of ABA concentration terms, it is not known whether the ability in seeds of the tropical wetland species, A. to develop full desiccation tolerance has marina, during seed expansion differs from been lost in species with recalcitrant seeds, that of other recalcitrant species; low and was never gained, or is just not fully decreasing concentrations of ABA are pre- expressed. The progress of evolution is also sent in the axis during reserve accumula- likely to have differed among taxa (von tion (Farrant et al., 1993a). Moreover, ABA Teichman and van Wyk, 1994; see Chapter concentration does not increase with dry- 8). Despite increasing interest in recalci- ing and dehydrin proteins are not detected trant seeds, it is clear from recent reviews (Farrant et al., 1996). In general, the pres- (Berjak and Pammenter, 1997; Pammenter ence of dehydrins in recalcitrant seeds is and Berjak, 1999) that the cause of their associated with those species that have desiccation sensitivity is still far from high ABA concentrations and are most understood. However, comparison of likely to be exposed to moisture stress desiccation-sensitive seeds with tolerant (Farrant et al., 1996). In addition, an earlier orthodox seeds can clarify our understand- peak in ABA concentration of recalcitrant ing of desiccation-tolerance mechanisms. Q. robur seeds is associated with greater In the following section, the occurrence of desiccation tolerance at shedding (Finch- these putative mechanisms in desiccation- Savage and Farrant, 1997). However, in T. sensitive seeds is reported. cacao embryos in vitro, ABA is associated with maturation events as it is in orthodox ABSCISIC ACID AND PROTEINS. As noted in the seeds, but does not influence desiccation previous sections, ABA may play a role in tolerance (Pence, 1992). the regulation of dehydrin and lea gene In orthodox cotton, lea mRNAs that expression in orthodox seeds. In the recal- have protein homology with dehydrins citrant seeds of Theobroma cacao (Pence, accumulate relatively slowly during the 1991) and Q. robur (Finch-Savage et al., period of cotyledon expansion, but then 1992; Finch-Savage and Blake, 1994), there increase rapidly at the point of vascular is a clear pattern of ABA accumulation separation (Galau et al., 1991). Recalcitrant during seed expansion, similar to that seeds are often shed at a time when dry reported in orthodox species, such as P. weight is still increasing and may therefore vulgaris (Prevost and Le Page-Degivry, lack the phase of rapid increase in lea 1985a,b). In both the axis and cotyledons, mRNAs that occurs at the end of orthodox ABA increases to a maximum and then seed development. Desiccation sensitivity decreases before shedding. However, the may therefore be due in part to an inability decline in ABA concentration prior to to accumulate a sufficient quantity of dehy- shedding in recalcitrant seeds is limited drins or other LEA proteins. and consistent with a continuing role for Small HSPs have also been associated ABA in preventing precocious germination with desiccation tolerance in orthodox (Finch-Savage and Farrant, 1997). seeds (see earlier discussion; DeRocher and Dehydrin proteins accumulate during seed Vierling, 1994; Wehmeyer et al., 1996), and development, and in response to seed dry- have been shown, like dehydrins, to accu- ing, in a number of recalcitrant species mulate to significant levels in recalcitrant (Finch-Savage et al., 1994; Gee et al., 1994; C. sativa seeds during development and to Farrant et al., 1996; Han et al., 1997; be present in seeds of other recalcitrant Greggains et al., 2000a). However, dehy- species (Collada et al., 1997). As discussed drin proteins are not detected in mature earlier, the presence of dehydrin and small undried axes of a range of recalcitrant trop- HSPs in recalcitrant seeds supports the ical wetland species (Farrant et al., 1996). view that they alone are not sufficient to These species would not normally be confer desiccation tolerance. However, exposed to significant drying and are con- Farrant et al. (1996) suggested that the con- Dessication - Chap 05 18/3/02 2:07 pm Page 172

172 A.R. Kermode and B.E. Finch-Savage

verse might be true, i.e. their absence may sue in recalcitrant seeds tends to have a imply an inability to tolerate desiccation, much lower oligosaccharide:sucrose ratio and the absence of a specific protective than that generally present in orthodox protein cannot be ruled out as the cause of seeds (Steadman et al., 1996). This ratio is desiccation sensitivity. For example, the therefore a potential indicator of seed membranes surrounding oil bodies of seeds behaviour; however, seeds of cocoa and A. contain integral proteins, called oleosins, marina are exceptions (Steadman et al., which may maintain the integrity of these 1996). Pammenter and Berjak (1999) organelles during desiccation and subse- pointed out that the proposed mechanisms quent imbibition (Murphy et al., 1995; for the involvement of sugars in desicca- Leprince et al., 1998). Oleosins are pre- tion tolerance operate at moisture contents sent in the membranes of oil bodies in below those at which most recalcitrant desiccation-tolerant seeds, but are absent, or seeds can survive. It is therefore perhaps their amount is diminished, in desiccation- not surprising that there is no clear rela- sensitive seeds (Leprince et al., 1998). tionship between the degree of desiccation Thus, a lack of oleosins may be an impor- tolerance in recalcitrant seeds and sugar tant factor in the desiccation sensitivity of accumulation. oil-storing recalcitrant seeds. High monosaccharide levels have been linked with desiccation sensitivity and the SUGARS. Studies with recalcitrant seeds potential for damage resulting from the show that there is no clear link between Maillard reaction (Koster and Leopold, the presence of sugars and the level of des- 1988). In the later stages of development in iccation tolerance in seeds. For example, orthodox seeds, monosaccharide levels are large amounts of sugars including sucrose reduced and this also occurs in some and stachyose accumulate during develop- (Farrant and Walters, 1998), but not all, ment in the highly desiccation-sensitive species with recalcitrant seeds (Farrant et seeds of A. marina (Farrant et al., 1993b). al., 1992, 1993b; Finch-Savage et al., 1993). In the more tolerant Q. robur, sucrose and Monosaccharide levels were generally low raffinose accumulate in the cotyledons and in most of the 18 species studied by axes during the later stages of reserve accu- Steadman et al. (1996), including the recal- mulation (Finch-Savage et al., 1993; Finch- citrant ones. Savage and Blake, 1994) and, in mature axes of Quercus rubra, desiccation sensitiv- IS DESICCATION SENSITIVITY DUE TO RETENTION OF ity is not caused simply by the absence of METABOLIC ACTIVITY AT SHEDDING? Recalcitrant non-reducing sugars (Sun et al., 1994). In a seeds, perhaps because they remain moist, more comprehensive study, Steadman et maintain active metabolism throughout al. (1996) determined the sugar composi- development to the time of shedding. For tion of a range of recalcitrant, intermediate example, respiration of A. marina seeds, and orthodox species and combined this after a small decline at the start of reserve with published data for additional species. accumulation, remains relatively constant They found no simple relationship between until abscission (Farrant et al., 1992). High seed type and total sugar content or sucrose respiration rates have also been recorded in level; however, the content of raffinose and the seeds of other recalcitrant species at stachyose was generally lower in recalci- shedding (Farrant et al., 1992, 1997; trant than in orthodox seeds. These Poulsen and Eriksen, 1992; Finch-Savage oligosaccharides were also found to be and Blake, 1994; Salmen Espindola et al., lower in seeds of recalcitrant A. pseudopla- 1994; Leprince et al., 1999). The absence of tanus than in seeds of the orthodox A. pla- substantial developmental arrest as seeds tanoides (Greggains et al., 2000a). In approach shedding is confirmed by ultra- general, there are large variations in the structural and biochemical studies (Dodd content of sugars between tissues of desic- et al., 1989; Berjak et al. 1992; Farrant et cation-sensitive seeds, but at least one tis- al., 1992; Farrant and Walters, 1998). Dessication - Chap 05 18/3/02 2:07 pm Page 173

Desiccation Sensitivity in Relation to Seed Development 173

Indeed, in A. marina the limited de-differ- like that thought to occur in orthodox entiation of subcellular components species. towards the end of development allows In most cases, the viability of recalci- changes indicative of germination to begin trant seeds is lost during drying in region immediately upon shedding (Farrant et al., three of the ﬁve hydration levels summa- 1992). Interestingly, in contrast to this and rized in Vertucci and Farrant (1995). In this other recalcitrant species studied, respira- region (c. 3 to 11 MPa), seeds are meta- tion in the dormant seeds of A. pseudopla- bolically active, respiration is measurable tanus declines to a rate similar to that of and presumably membranes are still the orthodox A. platanoides at shedding hydrated. However, at this level of hydra- (Greggains et al., 2000a). tion, it is thought that metabolism becomes There are differences in the activities of ‘unregulated’, repair processes become respiratory enzymes between the recalci- inoperative and catabolic activities con- trant Guilfoylia monostylis and the ortho- tinue unabated, but the processes utilizing dox Erythrina caffra (Nkang and Chandler, the high-energy intermediates are impaired 1986). These differences may be indicative (Vertucci and Farrant, 1995). As Q. robur of the seeds’ different germination strate- seeds are dried, increasing quantities of gies; the recalcitrant seeds maintained a several harmful volatiles are produced, balance of enzymes suitable for immediate including ethanol and acetaldehyde germination, whereas, in the orthodox (Finch-Savage et al., 1993). These volatiles seed, biosynthetic processes were drasti- are indicative of ‘unregulated’ respiration cally reduced (Nkang and Chandler, 1986). and are a potential source of the free radi- In contrast to recalcitrant seeds, the meta- cals that accumulate around the time of bolic activity of orthodox seeds is thought viability loss in Q. robur (Hendry et al., to decline in a programmed way before or 1992). These free radicals could alter the during the early stages of maturation dry- physical/chemical properties of mem- ing, so that seeds are shed in a quiescent branes, causing them to lose liquid/crys- state (Rogerson and Matthews, 1977; Miller talline structure (McKersie and Leshem, et al., 1983; Farrant et al., 1997). This orga- 1994). Thus, it is reasonable to speculate nized decline in metabolic activity, which that membrane damage and viability loss presumably has a role in protection against during drying of recalcitrant seeds may desiccation damage (reviewed by Vertucci result from unregulated metabolism and Farrant 1995), does not occur in enhanced by inadequate protection by free- species with the most sensitive seeds and radical scavengers. is not completed in those species with more tolerant recalcitrant seeds (Berjak and ANTIOXIDANT SYSTEMS. Lipid peroxidation and Pammenter, 1997; Farrant et al., 1997). free-radical activity have been associated Recent studies on seeds of a number of with seed viability loss in several recalci- more tolerant temperate recalcitrant trant species during desiccation (Hendry et species suggest that, although high at shed- al., 1992; Chaitanya and Naithani, 1994; ding, respiration rates decline like those of Finch-Savage et al., 1996; Li and Sun, orthodox seeds during desiccation (V. 1999). So far, it is difﬁcult to tell whether Greggains and W.E. Finch-Savage, unpub- the reported accumulation of free radicals lished data). However, in the more sensi- is a cause or a consequence of viability tive Araucaria angustifolia, respiration is loss. In either case, adequate protective only reduced by levels of desiccation that systems to limit free-radical damage are cause viability loss (Côme and Corbineau, likely to be essential for the maintenance of 1996). In temperate Castanea sativa seeds, viability (Côme and Corbineau, 1996) and a disruption of the electron transport chain range of antioxidant systems is present in occurs during drying (Leprince et al., recalcitrant seeds (Hendry et al., 1992; 1999), suggesting that the decline in respi- Chaitanya and Naithani, 1994; Li and Sun, ration due to drying is not programmed, 1999; Tommasi et al., 1999). In Q. robur, Dessication - Chap 05 18/3/02 2:07 pm Page 174

174 A.R. Kermode and B.E. Finch-Savage

there appear to be different protective sys- differentiation that occurs in orthodox seeds tems in the embryonic axis and cotyledons during maturation drying (reviewed by (Hendry et al., 1992). In the axis, protec- Vertucci and Farrant, 1995). tion occurs predominantly through the It is essential that there is effective antioxidants ascorbic acid and -toco- maintenance of the integrity of DNA during pherol, whereas in the cotyledons protec- desiccation and that any damage is tion is largely enzymatic, with relatively repaired on rehydration for seed viability high and increasing activities of superox- to be maintained (see Chapter 12). In A. ide dismutase and glutathione reductase. marina, DNA repair processes are Decreased levels of protection from lipid markedly compromised after limited dry- peroxidation during desiccation may con- ing and DNA replication does not fully tribute to the loss of seed viability (Hendry recover after only 8% water loss (Boubriak et al., 1992). Increased lipid peroxidation et al., 2000). The arrest of cell cycle activ- during desiccation, which precedes viability at the stage where DNA per nucleus is ity loss in Shorea robusta (Chaitanya and lowest may render embryos more resistant Naithani, 1994) and T. cacao (Li and to stress conditions (Deltour, 1985). Sun, 1999) seeds, was associated with Desiccation tolerance may therefore be decreased activities of free-radical-scaveng- related to the stage of cell cycle activity at ing enzymes. In contrast to these findings, which desiccation occurs (Bino et al., in a comparison of orthodox and recalci- 1992). However, Sacandé et al. (1997) have trant Acer species, it was concluded that presented data suggesting this is not true, the limitation to desiccation tolerance does and further convincing evidence against not result from inadequate free-radical the possibility comes from a comparison of scavenging (Greggains et al., 2000a). the orthodox species A. platanoides and However, A. pseudoplatanus used in the the recalcitrant species A. pseudoplatanus study can be placed at the most tolerant (Finch-Savage et al., 1998). Both species end of the continuum of desiccation sensi- produce seeds with stable high levels of 4C tivity among recalcitrant species. Its respi- DNA during the later stages of develop- ration rate at shedding is similar to that of ment, and both contain nuclei arrested at the orthodox Acer species, and there is no the 2C and 4C levels at maturity. evidence of increased lipid peroxidation as So far there has been little emphasis on viability is lost. the study of post-desiccation repair mechanisms in seeds and few studies have been OTHER FACTORS. Differences in cellular struc- published on this topic in recalcitrant ture could influence desiccation tolerance seeds; this may well be a limiting factor in (reviewed by Ruhl, 1996) such that the ini- desiccation-sensitive seed tissues. tial loss of water and consequent reduction in cell volume in very sensitive seeds causes mechanical damage (reviewed by 5.3. Conclusions Berjak and Pammenter, 1997; Pammenter and Berjak, 1999). For example, the seeds of Essential components of desiccation toler- A. marina are highly vacuolated and this ance of seeds include the accumulation of has been connected to their level of desicca- protective substances, which limit the tion sensitivity compared with other species amount of damage that otherwise would be (Farrant et al., 1997). However, Vertucci and induced by water loss, and the ability to Farrant (1995) showed that the evidence for repair cellular components upon subse- this is conflicting. Considerable disruption quent rehydration. Sugars (disaccharides, of the cytoskeleton has also been observed such as sucrose, and oligosaccharides, such during drying of Q. robur axes, and it is not as raffinose and stachyose) have been sug- reassembled during rehydration (Mycock et gested to play a key protective role by al., 1999), which may contrast with the accumulating under water deficit condi- apparently organized intracellular de- tions and functioning to replace water, thus Dessication - Chap 05 18/3/02 2:07 pm Page 175

Desiccation Sensitivity in Relation to Seed Development 175

stabilizing membranes and other sensitive 1998) for examining protein–protein inter- systems. Another protective mechanism actions and the use of differential display may involve dehydrins (Table 5.1). reverse-transcription PCR (Rodriguez-Uribe Desiccation tolerance is acquired during et al., 2000), gene and enhancer trap tag- development of orthodox seeds; tolerance to ging (Rojas-Pierce et al., 2000) and micro- full desiccation is generally lost after germi- arrays (Nevarez et al., 2000) to identify nation. Recalcitrant seeds, unlike orthodox water-deﬁcit-regulated genes. Proteomics seeds, are sensitive to desiccation when approaches could yield invaluable infor- shed from the parent plant, and thus pro- mation concerning post-translational con- vide a system to study temporal and stress- trols over desiccation-induced gene induced changes in dehydrins and other expression. These and similar research putative desiccation protectants. Although avenues may yield more decisive results studies on recalcitrant seeds provide indi- than the traditional approaches. rect evidence in relation to elucidating the Finally, it is noteworthy that desicca- roles of putative desiccation protectants, tion tolerance of seeds is a complex and more work needs to be done in this area. multifaceted property involving a multi- It is currently an exciting time to under- tude of genes whose expression ultimately take the challenges of understanding the leads to mechanisms of both cellular pro- biochemical and genetic components of tection, to sustain limited damage during desiccation tolerance of seeds. Several drying itself, and cellular repair, to novel approaches are now available, includ- reverse any desiccation-induced changes ing the yeast one- and two-hybrid systems when the appropriate hydrated conditions (Ingram and Bartels, 1996; Frank et al., are re-established.

5.4. References

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The effect of temperature and CO2 on seed quality development in wheat (Triticum aestivum L.). Journal of Experimental Botany 47, 631–637. Schroder, P., Abele, C., Gohr, P., Stuhlfauth-Roisch, U. and Grosse, W. (1999) Latest on enzymology of serotonin biosynthesis in walnut seeds. Advances in Experimental Medical Biology 467, 637–644. Senaratna, T. and McKersie, B.D. (1983) Characterization of solute efflux from dehydration injured soybean (Glycine max L. Merr) seeds. Plant Physiology 72, 911–914. Senaratna, T., McKersie, B.D. and Stinson, R.H. (1985a) Simulation of dehydration injury to membranes from soybean axes by free radicals. Plant Physiology 77, 472–474. Senaratna, T., McKersie, B.D. and Stinson, R.H. (1985b) Antioxidant levels in germinating soybean seed axes in relation to free radical and dehydration tolerance. Plant Physiology 78, 168–171. Shutov, A.D., Braun, H., Chesnolov, Y.V. and Baumlein, H. (1998) A gene encoding a vicilin-like protein is specifically expressed in fern spores. Evolutionary pathway of seed storage globulins. European Journal of Biochemistry 252, 79–89. Smith, M.T. and Berjak, P. (1995) Deteriorative changes associated with the loss of viability of stored desiccation-tolerant and desiccation-sensitive seeds. In: Galili, G. and Kigel, J. (eds) Seed Development and Germination. Marcel Dekker, New York, pp. 237–271. Stacy, R.A., Nordeng, T.W., Culianez-Macia, F.A. and Aalen, R.B. (1999) The dormancy-related peroxiredoxin anti-oxidant, PER1, is localized to the nucleus of barley embryo and aleurone cells. The Plant Journal 19, 1–8. Steadman, K.J., Pritchard, H.W. and Dey, P.M. (1996) Tissue-specific soluble sugars in seeds as indicators of storage category. Annals of Botany 77, 667–674. Sun, W.Q. (1999) Desiccation sensitivity of recalcitrant seeds and germinated orthodox seeds: can germinated orthodox seeds serve as a model system for studies of recalcitrance? In: Marzalina, M., Khoo, K.C., Jayanthi, N., Tsan, F.Y. and Krishnapillay, B. (eds) Recalcitrant Seeds, Proceedings of IUFRO Seed Symposium 1998. Forest Research Institute, Kuala Lumpur, Malaysia, pp. 29–42. Sun, W.Q., Irving, T.C. and Leopold, A.C. (1994) The role of sugar, vitrification and membrane phase transition in seed desiccation tolerance. Physiologia Plantarum 90, 621–628. Symons, S.J., Angold, R.E., Black, M. and Chapman, J.M. (1983) Changes in the growth capacity of the developing wheat embryo. I. The influences of the enveloping tissues and premature drying. Journal of Experimental Botany 34, 1541–1550. Thomann, E.B., Sollinger, J., White, C. and Rivin, C.J. (1992) Accumulation of group 3 late embryogenesis abundant proteins in Zea mays embryos. Roles of abscisic acid and the viviparous-1 gene product. Plant Physiology 99, 607–614. Thomas, T.L., Vivekananda, J. and Bogue, M.A. (1991) ABA regulation of gene expression in embryos and mature plants. In: Davies, W.J. and Jones, H.G. (eds) Abscisic Acid Physiology and Biochemistry. BIOS, Oxford, UK, pp. 125–135. Tilden, R.L. and West, S.H. (1985) Reversal of the effects of aging in soybean seeds. Plant Physiology 77, 584–586. Tommasi, F., Paciolla, C. and Arrigoni, O. (1999) The ascorbate system in recalcitrant and orthodox seeds. Physiologia Plantarum 105, 193–198. Tompsett, P.B. (1987) A review of the literature on storage of dipterocarp seeds. In: Kamra, S.K. and Ayling, R.D. (eds) Proceedings of the International Symposium on Forest Seed Problems in Africa. Harare, Zimbabwe, pp. 348–365. Tompsett, P.B. and Pritchard, H.W. (1993) Water status changes during development in relation to the germination and desiccation tolerance of Aesculus hippocastanum L. seeds. Annals of Botany 71, 107–116. Tompsett, P.B. and Pritchard, H.W. (1998) The effect of chilling and moisture status on the germination, desiccation tolerance and longevity of Aesculus hippocastanum L. seed. Annals of Botany 82, 249–261. Vertucci, C.W. and Farrant, J.M. (1995) Acquisition and loss of desiccation tolerance. In: Galili, G. and Kigel, J. (eds) Seed Development and Germination. Marcel Dekker, New York, pp. 701–746. Vertucci, C.W., Berjak, P., Pammenter, N.W. and Crane, J. (1991) Cryopreservation of embryonic axes of an homeohydrous (recalcitrant) seed in relation to calorimetric properties of tissue water. Cryo-Letters 12, 339–350. Dessication - Chap 05 18/3/02 2:07 pm Page 184

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Vogel, G., Aeschbacher, R.A., Muller, J., Boller, T. and Wiemken, A. (1998) Trehalose-6-phosphate phosphatases from Arabidopsis thaliana: identiﬁcation by functional complementation of the yeast tps2 mutant. The Plant Journal 13, 673–683. von Teichman, I. and van Wyk, A.E. (1994) Structural aspects and trends in the evolution of recalcitrant seeds in dicotyledons. Seed Science Research 4, 225–240. Walters, C. (1999) Levels of recalcitrance in seeds. In: Marzalina, M., Khoo, K.C., Jayanthi, N., Tsan, F.Y. and Krishnapillay, B. (eds) Recalcitrant Seeds. Proceedings of IUFRO Seed Symposium 1998. Forest Research Institute, Kuala Lumpur, Malaysia, pp. 1–13. Wehmeyer, N. and Vierling, E. (2000) The expression of small heat shock proteins in seeds responds to discrete developmental signals and suggests a general protective role in desiccation tolerance. Plant Physiology 122, 1099–1108. Wehmeyer, N., Hernandez, L.D., Finkelstein, R.R. and Vierling, E. (1996) Synthesis of small heat- shock proteins is part of the developmental program of late seed maturation. Plant Physiology 112, 747–757. White, C.N., Proebsting, W.M., Hedden, P. and Rivin, C.J. (2000) Gibberellins and seed development in maize. I. Evidence that gibberellin/abscisic acid balance governs germination versus maturation pathways. Plant Physiology 122, 1081–1088. Williams, R.J. and Leopold, A.C. (1989) The glassy state in maize embryos. Plant Physiology 89, 977–981. Wolkers, W.F., Alberda, M., Koornneef, M., Leon-Kloosterziel, K.M. and Hoekstra, F.A. (1998a) Properties of proteins and the glassy matrix in maturation-defective mutant seeds of Arabidopsis thaliana. The Plant Journal 16, 133–143. Wolkers, W.F., Bochicchio, A., Selvaggi, G. and Hoekstra, F.A. (1998b) Fourier transform infrared microspectroscopy detects changes in protein secondary structure associated with desiccation tolerance in developing maize embryos. Plant Physiology 116, 1169–1177. Wolkers, W.F., Tetteroo, F.A., Alberda, M. and Hoekstra, F.A. (1999) Changed properties of the dried carrot somatic embryos. An in situ Fourier transform infrared microspectroscopy study. Plant Physiology 120, 153–164. Desiccation - Chap 06 18/3/02 1:56 pm Page 185

6 Pollen and Spores: Desiccation Tolerance in Pollen and the Spores of Lower Plants and Fungi

Folkert A. Hoekstra Laboratory of Plant Physiology, Department of Plant Sciences, University of Wageningen, Arboretumlaan 4, 6703 BD Wageningen, The Netherlands

6.1. Introduction 186 6.2. Pollen 187 6.2.1. Desiccation tolerance 187 6.2.2. Characteristics 188 6.2.3. Longevity 190 6.2.4. Imbibitional stress 191 6.2.5. (Cryo)preservation 191 6.3. Fern Spores 192 6.3.1. Desiccation tolerance 192 6.3.2. Characteristics 192 6.3.3. Longevity 193 6.3.4. Cryopreservation 193 6.4. Moss Spores 193 6.4.1. Desiccation tolerance, longevity and imbibitional stress 194 6.4.2. Characteristics 194 6.4.3. Cryopreservation 194 6.5. Spores of Horsetails, Lycopodia and Selaginella 194 6.6. Fungal Spores 195 6.6.1. Desiccation tolerance 195 6.6.2. Characteristics 195 6.6.3. Longevity 197 6.6.4. Imbibitional stress 197 6.6.5. Cryopreservation 197 6.7. Conclusion 197 6.8. Acknowledgement 197 6.9. References 199

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6.1 Introduction other propagules is linked with considerable differences in physiology. Pollen grains of seed plants and spores of Pollen is dispersed mostly through the lower plants and fungi have diverse devel- air by wind, insects or vertebrates to meet a opmental histories. However, they are simi- female receptive structure, where it is pro- lar in size, usually below 200 µm, which voked to germinate immediately after rehy- promotes their effective dispersal via the air. dration, sometimes depending on The propagules are exposed to often hostile recognition and/or the presence of self- environmental conditions, without the abil- incompatibility. The rapid start of pollen ity to actively control their own hydration tube growth might be linked with the com- status. Under similar environmental condi- petition of the grains for the available tions, microscopic propagules dry out much ovules (Mulcahy, 1979), as discussed in faster than, for example, the much larger Section 6.2. Those pollen grains that fail to seeds. It is, therefore, not surprising that land on the proper site are lost, because pollen and spores are endowed with mecha- they have very little subsequent chance to nisms that allow them to withstand a cer- meet the appropriate vehicle for transport. tain degree of water loss and to survive the After dehiscence, rainwater is generally period from their release from the produc- detrimental to pollen, as it causes loss of tion site to the target site and, often, beyond viability, bursting or germination at inap- that. The seeds of about 15% of all plant propriate sites (Lidforss, 1896). This species are supposed to have problems as a extreme sensitivity to rainwater is associ- result of water loss (Hong et al., 1996). ated with the absence of dormancy in Drought tolerance is the term used to pollen. Only in some gymnosperms has it indicate tolerance of moderate dehydration, been found that the pollen remains viable for example, not below the moisture con- after several hours of soaking of dry pollen tent (MC) at which bulk water has disap- in rainwater followed by redrying peared (approx. 20–25%, on a fresh weight (Hoekstra, 1983). 1 basis, or 0.25–0.33 g H2O g dry weight). Fungal, fern and moss spores are also Desiccation tolerance refers to further dehy- often transported via the air after the dration and is understood to include not release from the mother plant upon slight only the ability of cells to become air-dry drying of the spore-bearing structures. without loss of viability, but also to success- They will dehydrate to some extent, fully rehydrate. The period of anhydrobio- depending on the niche in which the par- sis in between these two events is referred ent organism grows. In these propagules, to as longevity or life span in the dried dormancy mechanisms occur, which sup- state. This chapter focuses on the possible press germ tube emergence in the presence tolerance of spores of eukaryotic plants to of sufﬁcient water, when the environmen- reduced levels of hydration. tal conditions are unfavourable for sup- Pollen, the male gametophyte of porting growth. This also implies the higher plants, is designed to deliver its activity of dormancy-breaking mechanisms haploid sperm cells to the ovules in at some time when the conditions improve. order to bring about fertilization. There As a consequence of the difference in tar- is a speciﬁc and highly specialized target get, pollen does not survive in the hydrated for the pollen to land on – the stigma in state for a long time, whereas the other the case of angiosperms, or a pollination propagules usually do. droplet or pollen-collecting apparatus in A considerable amount of physiological, the case of gymnosperms. In contrast, the biochemical and biophysical research in propagules of the spore-forming plants relation to desiccation tolerance has been and fungi are less restricted as to their performed on pollen and yeast, but com- initial target site, which is usually the paratively little on the other propagules. soil or a host organism. This difference For each particular propagule described in in specialization between pollen and the this chapter, special emphasis is placed on Desiccation - Chap 06 18/3/02 1:56 pm Page 187

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how widely spread the phenomenon of Bruinsma, 1975; Linskens et al., 1989), or desiccation tolerance is (as far as is pollen may form germ tubes inside the known), including possible problems with anthers (Pacini and Franchi, 1982). When the rehydration of the dried specimens. In viable pollen rehydrates on the appropriate tests for desiccation tolerance, improper female receptive structure, it germinates, rehydration may kill otherwise viable dry producing a ﬁliform structure (pollen specimens, leading to false negatives. tube), which grows through the style Longevity in the dried state is surveyed, towards the ovules by a process much like because the span may be indicative of the a parasitic invasive action. depth of desiccation tolerance. The occurrence of compounds generally associated with anhydrobiosis is listed, particularly 6.2.1. Desiccation tolerance sugars, compatible solutes and dehydration proteins. The feasibility of (cryo)preserva- The pollens of a large number of plant tion is highlighted as a practical guideline species can withstand air-drying (Stanley for those interested in preserving the and Linskens, 1974; Towill, 1985; Hoekstra, propagules. 1986, 1995). Studies by the author of the pollen literature have revealed that the pollens of plants belonging to some genera of 6.2. Pollen about half of all plant families have been tested for their tolerance to various levels of The male gametophytes of higher plants dehydration. While desiccation tolerance develop in the anthers (Stanley and appears to be widespread among pollen of Linskens, 1974). When the ﬂower opens, most plant families studied, there are a few the anthers become exposed to the environ- exceptions. It is expected that more such ment and dehydration then triggers a recalcitrant pollen types will be found in mechanism in the anthers for the release of very humid climates and niches not yet the pollen. In the process, pollen also investigated, particularly in the hot, humid dehydrates. Continuous rain may prevent tropics. anthers from opening. Once pollen has From the results of in vitro and in vivo passed physiological maturity, ageing may germination experiments, it is clear that the start within the anthers under such condi- pollen of genera in Araceae, Cucurbitaceae, tions. Ultimately, pollen with reduced via- Gramineae and Zingiberaceae experience bility and vigour is released (Hoekstra and problems during drying (see Table 6.1 for

Table 6.1. List of plant families and their genera in which desiccation-sensitive pollens have been reported. Family Genus Reference Araceae Dieffenbachia Henny, 1980a,b Aglaonema Henny, 1985 Cucurbitaceae Cucurbita Wang and Robinson, 1983; Gay et al., 1987 Momordica Dubey and Gaur, 1989 Gramineae Avena Wallace and Karbassi, 1968 Hordeum Anthony and Harlan, 1920; Firbas, 1922 Saccharum Sartoris, 1942; Moore, 1976 Secale Chaudhury and Shivanna, 1987; Shi and Tian, 1989a Sorghum Sanchez and Smeltzer, 1965 Triticum Firbas, 1922; Goss, 1968 Zea Barnabas, 1985; Digonnet-Kerhoas and Gay, 1990 Pennisetum Aken’ova and Chheda, 1970 Zingiberaceae Zingiber Adaniya and Higa, 1988; Adaniya, 2002 Desiccation - Chap 06 18/3/02 1:56 pm Page 188

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details). They can be kept alive only under more subtle rehydration requirements and 100% relative humidity (RH) are often difficult to germinate on such (Zingiberaceae) or at slightly lower RH, media (Bar-Shalom and Mattsson, 1977). albeit for short periods of time. Although During the development of the male Gramineae pollens are generally sensitive angiosperm gametophyte, two nuclear divi- to air-drying, they usually have a consider- sions take place. The first one leads to one able tolerance to drought. Thus, pollen of vegetative and one generative cell that Secale cereale is tolerant to MCs as low as gives rise to two sperm cells in the subse- 11.5% (fresh weight basis) (Shi and Tian, quent division. Depending on whether 1989a), and that of Zea mays to 13% MC dehiscence occurs after the first or the sec- (Digonnet-Kerhoas and Gay, 1990). In the ond mitosis, mature pollen is bicellular or genus Pennisetum, tolerance (Pennisetum tricellular. Bicellular pollen grains have to typhoides (Pennisetum americanum), perform the second mitosis during tube Chaudhury and Shivanna, 1986, 1987; growth. Thus, tricellular pollen is ontoge- Hoekstra et al., 1989) as well as intoler- netically advanced. This characteristic ance (Pennisetum purpureum, Aken’ova occurs in about 30% of angiosperm families and Chheda, 1970) to air-drying have been that are considered as being evolutionarily observed. Some orchid pollens rapidly advanced (Brewbaker, 1959, 1967; Sporne, lose viability on storage above silica gel, 1969). Typical families with tricellular which cannot be restored by a day of pre- pollen are the Asteraceae, Caryophyllaceae, hydration in humid air prior to the in vitro Chenopodiaceae, Cruciferae, Gramineae, test (Pritchard and Prendergast, 1989). Juncaceae and Umbelliferae. Tricellular This may point to reduced desiccation tol- pollen tends to be associated with dry stig- erance, but may also indicate that the mas and is not readily germinated in vitro dried pollen is sensitive to the humid air (Heslop-Harrison and Shivanna, 1977). treatment (see Section 6.2.4). The time from contact with the germination medium or appropriate stigmatic surface to emergence of a pollen tube is 6.2.2. Characteristics generally short. It ranges from a few minutes for Gramineae and Compositae pol- On dehydration, pollen changes in shape lens to a few hours for some other pollens in different ways, dependent on the (Hoekstra and Bruinsma, 1978; Hoekstra, species. While indentation is observed in 1986). Only in the case of gymnosperm some pollens, e.g. those of Gramineae, in pollens does it take a few days before others the major to minor axis ratio is emergence occurs (e.g. Pettitt, 1985). A increased as the shape changes from rapid start of tube emergence is generally round into ellipsoid along special fur- followed by extremely fast tube growth of rows, as in the pollens of Solanaceae and up to 1–2 cm h1 (Hoekstra and Bruinsma, Papaveraceae (for low-temperature scan- 1978). It has been argued that recurrent ning electron micrographs, see Fig. 6.1). competition for the fertilization of avail- The pattern of size reduction with dehy- able ovules has led to such short lag peri- dration also depends on the distribution ods and fast growth, which may have and number of germ pores. made mature pollen extremely sensitive to Germination ability of pollen is assayed stress. This problem may have been suc- in vivo by the analysis of tube growth in cessfully evaded by the regular production compatible stigmas or styles, or in vitro on of fresh pollen during the flowering artificial germination media (see Hoekstra, period. Some characteristics of fast-grow- 1995, for a review). Those pollens that are ing pollens that are often tricellular are the adapted to plants having wet stigmatic sur- high proportion of polyunsaturated fatty faces easily germinate in liquid or solidified acids (linolenic acid) in the phospholipids germination media. In contrast, pollen grains and neutral lipids, the high rates of metab- from plants with dry stigmatic surfaces have olism associated with well-developed Desiccation - Chap 06 18/3/02 1:56 pm Page 189

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mitochondria and, in the case of short tional and desiccation-tolerant at approxi- styles, even the absence of protein synthe- mately 1–2 days prior to anthesis, coinci- sis during tube growth (see Hoekstra, dent with the degradation of starch and a 1986, for a review). The high level of doubling of the amount of sucrose. polyunsaturated acyl chains that are par- Precocious drying leads to loss of viability ticularly sensitive to peroxidation might and damaged plasma membranes. When contribute to the intrinsically short life immature pollen is liberated mechanically span. from the anthers and allowed to mature in Mature pollen is generally endowed humid air, starch degrades and sucrose with high contents of sucrose, ranging from content nearly doubles, and the grains 7 to 23% of the pollen dry weight, but become largely functional and dehydration- oligosaccharides have not been encoun- tolerant. Apparently, maturation during the tered (Hoekstra et al., 1992). Sucrose may last 3 days of development is independent play an essential role in the acquired toler- of the parent plant, and sucrose may play ance of severe dehydration, as illustrated an essential role in the acquired desicca- by the following examples. Developing tion tolerance (Hoekstra and van Roekel, pollen of Papaver dubium becomes func- 1988). During the slow dehydration of

Fig. 6.1. Low-temperature scanning electron micrographs of partly dehydrated (A,C) and hydrated pollens (B,D). Maize pollen, (A) partly dehydrated and (B) hydrated (fresh); poppy (Papaver rhoeas) pollen, which was liberated from an anther in a partly dehydrated state (C) and upon rehydration in water (D). Upon dehydration, maize pollen becomes indented, whereas poppy pollen becomes elongated. Bars are 10 µm. (Micrograph, courtesy of Adriaan van Aelst, Laboratory of Experimental Plant Morphology and Cell Biology, Wageningen University.) Desiccation - Chap 06 18/3/02 1:56 pm Page 190

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fresh maize pollen, the sucrose content to biopolymers under conditions when increases from 5 to 12% of the dry weight. there is still bulk water present during When fresh pollen is dried in the cold dehydration, is discussed in Chapter 10. (2°C) or at a high rate, the increase in As in seeds, heat-stable proteins with sucrose content is curtailed, and the toler- late embryogenesis abundant (LEA) ance of dehydration is affected, from which (Wolkers et al., 2001) and dehydrin (Wang it has been concluded that survival of et al., 1996) characteristics have been iso- dehydration is correlated with the presence lated from pollen. There are further indica- of sucrose (Hoekstra et al., 1989). The tions that, as a result of osmotic stress, mode of action of sugars in dry plant mate- dehydration and ABA, a number of pro- rial is discussed in detail in Chapter 10. teins are produced in pollen with homolo- Free amino acids are abundant in gies to stress proteins in seeds. The mature pollen grains. On the basis of an possible mode of action of these proteins is analysis of the pollen of a large number of discussed in Chapter 10. plants (about 200 species from 63 families), it has been shown that pollen differs from other organs in having an unusually high 6.2.3. Longevity content of free proline, exceeding 1.5% per crude weight in many species (Britikov and Longevity of pollen varies considerably Musatova, 1964). A high content of free among species and is dependent on water proline has repeatedly been confirmed for content and temperature (see Hoekstra, other pollens, even up to 3% of the dry 1986, for a review). Tricellular pollen tends weight. Instead of proline, free arginine to be shorter-lived than bicellular pollen. and glutamate may also occur. There are However, short storage life and extremely indications that the free proline content of rapid tube emergence and growth have also pollen is positively related to viability been found in bicellular pollen, e.g. (Palfi and Köves, 1984; Palfi and Mihalik, in Balsaminaceae, Cucurbitaceae and 1985; Lansac et al., 1996). It is remarkable Commelinaceae. The average survival peri- to note that the desiccation-sensitive ods at 20–25°C and equilibrium RHs of pollen of Cucurbita has low proline con- approximately 40% range from a few hours tents (Gulyas and Palfi, 1986). On the other in desiccation-sensitive pollens to several hand, dehydration-sensitive maize pollen, months in the most stable, desiccation-tol- which can withstand dehydration to erant pollens (Hoekstra, 1995). Only for approximately 13% MC, does contain con- gymnosperm pollens have longevities of siderable amounts of proline (Linskens and over 1 year under these conditions been Pfahler, 1973; Palfi and Köves, 1984). The reported. The survival of pollen in storage accumulation of proline is preceded by a conforms to a cumulative negative normal peak in free abscisic acid (ABA) (Lipp, distribution (van Bilsen and Hoekstra, 1991; Chibi et al., 1995). Whereas the ABA 1993; Buitink et al., 1998b). Based on the content decreases towards maturity (added data of Buitink et al. (1998b), an empirical ABA inhibits germination), the proline model for the storage behaviour of pollen content further increases to a maximum, has been constructed, which can predict coinciding with the stage of anther desicca- the viability of a pollen lot over time at a tion (Zhang et al., 1985). Proline added to broad range of different water contents and the germination medium makes pollen storage temperatures (Hong et al., 1999a). more resistant to heat (Zhang and Croes, There is a negative logarithmic relation 1983), which suggests that the high between longevity and pollen MC and a endogenous amounts in pollen may have a curvilinear semi-logarithmic relation role in conferring resistance to between longevity and temperature. unfavourable temperatures. The role of free It has long been known that there is a proline and some amino acids as compati- lower MC limit, below which further reduc- ble solutes, providing structural protection tion in MC does not increase pollen Desiccation - Chap 06 18/3/02 1:56 pm Page 191

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longevity, but, instead, decreases it (Pfundt, is predicted to be considerably above 10% 1910; Hoekstra, 1986). At high MC, pollen MC at cryogenic temperatures. This may be longevity is not extended as in dormant the reason for the successful cryogenic spores and seeds, but the pollen germi- storage of desiccation-sensitive Gramineae nates, or engages in inappropriate synthe- pollen, as mentioned below. ses, such as callose deposition (Hoekstra, 1986). As mentioned earlier, dormancy mechanisms are absent in pollen. An 6.2.4. Imbibitional stress exception to the rule that fully hydrated pollen does not survive for a long time is Sensitivity of pollen to imbibitional stress orchid pollen that is enclosed in pollinia in has been known for many years (see long-lasting ﬂowers. On agar, survival times Hoekstra, 1986, for a review). The imbibi- of a few weeks at 2°C have been reported tion of dry pollen in germination medium, (Pritchard and Prendergast, 1989). particularly at chilling temperatures, can Recently, it has been established that lead to loss of endogenous solutes and pollen is in a glassy state (see Chapter 10) delay of tube emergence, or even a com- at below approximately 10% MC at room plete failure of the grains to become turges- temperature. In a glass, molecular mobility cent and form a pollen tube. The problem is considerably reduced. It has been found is widespread among pollens and can be that the molecular mobility of a small guest circumvented by prehydration in humid (spin-probe) molecule in the cytoplasm is air or by warm imbibition, or a combina- inversely correlated with storage longevity tion of both (Hoekstra, 1984; Hoekstra and (Buitink et al., 1998a). Thus, the slower the van der Wal, 1988). Some caution has to be molecular mobility, the greater is the life exercised with prehydration in humid air, span. Elevated water contents and tempera- as this treatment may lead to a rapid loss of tures increase molecular mobility and, viability in some cases. The extensive leak- thus, decrease the life span. For long-term age as a result of imbibitional stress is survival it is therefore important that the indicative of problems at the level of the pollen cytoplasm is in the glassy state. plasma membrane. Few pollens have been From the linear relationship between the found that are resistant to this stress. They logarithms of molecular mobility and are characterized by highly polyunsatu- longevity, Buitink et al. (2000a) have been rated acyl chains in their membranes able to predict storage longevities under (reviewed by Hoekstra and Golovina, varying conditions of temperature and MC. 1999), which may increase membrane ﬂu- Thus, survival times at low temperatures, idity and thus more easily accommodate for which experimental determination is the expanding protoplast on rehydration. practically impossible, can be estimated. This approach on the basis of molecular mobility probably gives more accurate esti- 6.2.5. (Cryo)preservation mates of survival times for sub-zero temperatures than does the empirical model Desiccation-tolerant pollen that is air- proposed by Hong et al. (1999a), which is dried to approximately 7% MC can be more conservative. stored for extended periods of time at A possible cause of the critical lower sub-zero temperatures. The experience of MC limit may be encompassed in the phe- the author with air-dried cattail (Typha nomenon that, during the removal of water, latifolia) pollen is that a half-life of about molecular mobility reaches a minimum, 25 years is possible at 18°C in a deep- but increases again on further drying to freezer. However, for long-term storage of very low water contents (Buitink et al., many decades or even longer, cryogenic 2000b). In addition, the MC at which this storage is essential. Some excellent minimum molecular mobility occurs reviews of this subject have been pub- increases with decreasing temperature, and lished (Binder et al., 1974; Stanley and Desiccation - Chap 06 18/3/02 1:56 pm Page 192

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Linskens, 1974; Franklin, 1981; Shivanna 6.3.1. Desiccation tolerance and Johri, 1985; Towill, 1985). The storage of desiccation-sensitive Because the release of spores from the spo- pollen is limited to several days or at most rangia requires a certain degree of dehydra- a few weeks under conditions of high RH tion, some tolerance to dehydration in the at 0–2°C (see Hoekstra, 1995). Desiccation- spores is likely. This has, indeed, been sensitive Gramineae pollen can usually established in the spores of a number of withstand drying to 20–25% MC. This is ferns, e.g. Adiantum capillus-veneris the MC at which bulk water has disap- (Uchida et al., 1998), Dryopteris filix-mas peared and ice crystals do not rapidly form (Haas and Scheuerlein, 1991), Dryopteris at sub-zero temperatures. When pollen, paleacea (Scheuerlein et al., 1988; Haupt carefully dried to just below this water and Psaras, 1989), Anemia phyllitidis content, is quenched in liquid nitrogen, a (Grill, 1988), Lygodium japonicum glassy state is rapidly formed, and viability (Manabe et al., 1987) and Onoclea sensi- is maintained because detrimental ice crys- bilis (Raghavan, 1992), but precise informa- tals cannot grow. In this way, successful tion on longevity is scarce. storage at 196°C is possible with maize pollen (Barnabas and Rajki, 1976; Barnabas et al., 1988; Shi and Tian, 1989b; Barnabas, 6.3.2. Characteristics 1994 (even for more than 10 years)) and rye pollen (Shi and Tian, 1989a). After pollina- To detect spore survival by germination tion with the thawed pollen, excellent seed tests is laborious and slow. Fern spores ger- set can be obtained. minate in soil or on aseptic agar-containing It has been reported that storage in vac- or liquid nutrient media after a dormancy- uum ampoules at 5°C after vacuum-drying releasing treatment. Criteria of viability are allows maize pollen to survive for more swelling of the cell, coat splitting, greening than 1 year (Jensen, 1964). Also freeze-dry- and rhizoid formation, which usually ing followed by storage at 0–5°C gives require weeks after induction for their some survival up to 5 months (Nath and expression. A chlorophyll fluorescence Anderson, 1975). Short lyophilization, fol- technique has been applied, which has the lowed by vacuum storage at 40°C pro- advantage that it can be used to quantify longs the life span of pollen of the grass the germination capacity of non-green Dactylis glomerata for more than 2 years spores (D. paleacea) just 2 days after (Cauneau-Pigot, 1991). The beneficial photoinduction (Scheuerlein et al., 1988). effect of the vacuum during dehydration There is ample evidence for the occur- and storage might be associated with the rence of dormancy in fern spores. Spore exclusion of oxygen: oxidative stress germination in hybrid Azolla increases might be an important factor in the desic- progressively to attain a maximum after 5 cation sensitivity and storability of months of storage and declines after 7 Gramineae pollen. months to reach a minimum after 11 months (Bhattacharyya and Kushari, 1999). It is clear that fern spores do not 6.3. Fern Spores simply germinate when they come into contact with water. They are stimulated by Fern spores are formed by meiosis in spo- red light and reversibly inhibited by far rangia, often underneath the leaves and red light. The red-light-induced germina- above ground. They are released when tion is irreversibly inhibited by ultraviolet dehydration causes rupture of the sporan- (275 nm) and blue (440 nm) light (Sugai gia. They are dispersed and can germinate, and Furuya, 1985). The induction of spore giving rise to prothalli, where eventually germination is mediated by phytochrome, fertilization takes place followed by out- and the effect of a red pulse irradiation growth of the sporophytic plant. can be enhanced by nitrate added to the Desiccation - Chap 06 18/3/02 1:56 pm Page 193

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culture medium (Haas and Scheuerlein, collected in the ﬁeld (Janes, 1998). From 1991). The application of gibberellic acid the scanty data in the literature, one cannot

(GA3) also allows spores to germinate, but get an idea of how widespread the occur- this hormone strongly inhibits further rence of desiccation tolerance is among gametophyte development (Fernandez et fern spores. al., 1997). The red-light-induced spore ger- Some fern species (e.g. A. filix-femina, mination is inhibited by AMO 1618, an Gymnocarpium dryopteris, Phegopteris inhibitor of gibberellin biosynthesis, from connectilis) form spore banks, which are which it has been suggested that red light soil reservoirs of viable spores that remain induces the biosynthesis of gibberellin via dormant while buried, but germinate in the phytochrome system and that gib- light if brought to the surface (Dyer, 1994). berellin induces spore germination Fern spores remain stable in the soil for (Kagawa and Sugai, 1991). Added ABA more than a year (Dyer and Lindsay, 1992). significantly inhibits spore germination (Singh et al., 1996). Spores accumulate reserve lipids 6.3.4. Cryopreservation (Gemmrich, 1977) and storage proteins that are genetically similar to seed storage pro- It has been found that spores of Cyathea teins (Templeman et al., 1988). The spores of spinulosa survive storage in liquid nitro- Osmunda japonica contain large amounts of gen (196°C) followed by slow thawing free proline and arginine (Wada et al., 1998). (Agrawal et al., 1993). In this case, the water content of the spores was uncon- trolled. Prior dehydration to at least below 6.3.3. Longevity the level of the non-frozen water content 1 (approximately 0.25–0.33 g H2O g dry Storage of spores at 20°C in the air-dried weight) is expected to provide possibilities state and under hydrated conditions has for long-term survival under conditions of been compared for four species (Athyrium cryogenic storage, if the spores survive filix-femina, Blechnum spicant, Polystichum dehydration to this low water content. setiferum and Phyllitis scolopendrium) (Lindsay et al., 1992). The viability of their hydrated, non-green spores remained un- 6.4. Moss Spores changed after 2 years of storage, while that of the air-dried spores decreased with time. Although the release of moss spores from In addition, the time required for germina- the sporophyte capsules upon drying tion of the air-dried spores increased dur- would suggest that desiccation tolerance in ing storage. The chlorophyllous spores of these spores could be common, little is Todea barbara, which cannot be stored for known about how widely distributed this long periods by conventional methods, also phenomenon really is among species. In lost viability during storage much less the soil, the unicellular haploid spore ger- quickly when hydrated than when air- minates to form a small green protonema. dried. From this, Lindsay et al. (1992) have Later on, plants grow from these protone- concluded that wet storage of fern spores is mata. Beside the spores that arise from the far more effective than dry storage. inner tissue of the capsule by meiotic divi- However, prior dehydration allows spores sion, a variety of gametophytic cells and (Cyathea delgadii) to be stored at sub-zero fragments, called ‘diaspores’, can be pro- temperatures, which extends survival duced from the gametophytic plant and times considerably in comparison with protonemata by different cellular separa- other storage methods (Simabukuro et al., tion mechanisms (Duckett and Ligrone, 1998). Sporocarps of Azolla filiculoides 1992). Here, diaspores will also be consid- are able to survive storage in water for 3 ered in relation to their possible desicca- years and to germinate from mud samples tion tolerance. Desiccation - Chap 06 18/3/02 1:56 pm Page 194

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6.4.1. Desiccation tolerance, longevity and present. These authors have found that on imbibitional stress agar the germination of Archidium alternifolium spores continues to increase over a When comparing the survival of spores period of several months. Percentage ger- from ﬁve species (Schistidium rivulare, mination was consistently less than 65% in Racomitrium aciculare, Dicranoweisia freshly collected material, and increased crispula, Oligotrichum hercynicum and with the age of the spores up to 4 years of Ceratodon purpureus) in water and after storage. There is no information about the desiccation, Dalen and Soderstrom (1999) major constituents of spores. For compari- found that the spores generally survived son, sucrose is the main sugar in the dried better in the dried state than in water. gametophyte (Smirnoff, 1992). However, the survival times were limited, i.e. not exceeding 6 months. In these ﬁve species, fragments (diaspores) have been 6.4.3. Cryopreservation found to survive equally well in water as in the dried state. The (dia)spore bank of Spores can be expected to survive drying, bryophytes that are soil reservoirs of viable and also to survive well in the dried state spores appears to play a role similar to that at sub-zero temperatures. However, cryo- of the soil seed bank in seed plants. preservation of (hydrated) cultures solves Redifferentiation of moss protonemata problems associated with long-term cul- into spherical, thick-walled brood cells ture, particularly when spores are short- (brachycytes) is a widespread phenome- lived or not produced. Improvements in non, which occurs when protonemal survival after cryopreservation have been colonies are cultured for long periods of made by preconditioning cultures in ABA time, allowed to dry out or are treated with and proline (Christianson, 1998). There is ABA. Brood cells in some species retain information that suggests that moss spores viability for long periods even in a desic- buried in the Siberian permafrost have sur- cated state and germinate rapidly in new vived 40,000 years, as they could still form medium lacking ABA (Duckett et al., 1993; protonemata upon culture (www.science. Schnepf and Reinhard, 1997). It is likely nasa.gov/newhome/headlines/ast27jul99_ that ABA is the natural compound that 1.htm). triggers brood cell development and induces tolerance to desiccation (Goode et al., 1993), including the synthesis of 6.5. Spores of Horsetails, Lycopodia and extremely heat-resistant soluble proteins Selaginella (Werner and Bopp, 1992). It has been concluded that ABA has the same function in The chlorophyllous spores of the sole bryophytes as in higher plants, acting as a horsetail genus, Equisetum, survive desic- mediator in stress conditions (Bopp and cation, yet do not live for more than Werner, 1993). Dry intact moss gameto- approximately 2 weeks when desiccated at phytes are sensitive to imbibitional stress 2% RH and 25°C (Lebkuecher, 1997). (Schonbeck and Bewley, 1981). Whether Under these conditions, disseminated dry spores and diaspores are similarly sen- spores of Equisetum hyemale have an sitive has not been studied. extremely short life span, possibly due to the inability to recover losses of water oxidation and photosystem II core function. 6.4.2. Characteristics Storing ripe cones of E. hyemale at 70°C extends the viability of the spores for more Although no special studies have been con- than a year (Whittier, 1996). ducted as to possible dormancy mecha- Spores of Equisetum arvense, when cul- nisms in spores, the study of Miles and tured in Murashige and Skoog liquid Longton (1992) suggests that dormancy is medium, germinate 2–3 days after sowing Desiccation - Chap 06 18/3/02 1:56 pm Page 195

Desiccation Tolerance in Pollen and Spores 195

(Kuriyama and Maeda, 1999). Large amounts duce a powdered formulation of skimmed of free proline and arginine have been milk/P. bilaji spore particles that could be detected in these spores (Wada et al., 1998). kept alive under refrigeration for at least 3 The spores of the Lycopodiaceae, some months. Drying techniques for spores and of them chlorophyllous, germinate rela- yeast cells that have been applied include tively slowly (months/years). There air-drying, freeze-drying, spray-drying and appears to be no information on desicca- ﬂuid bed-drying by contact-sorption. tion tolerance of the spores, including those of Selaginella. 6.6.2. Characteristics

6.6. Fungal Spores Spores change their shape as a result of dehydration, with size reductions to Spores of terrestrial fungi will be consid- 70–80% of the hydrated size. The critical ered here and not those of aquatic fungi. At RH, above which spores are expanded and spore maturity, ejection mechanisms occur below which they are collapsed, ranges that ensure the dispersal of spores into the between 80 and 90% RH, as established for air, but sometimes spores develop in an a number of urediniospores (Littlefield and aqueous environment. Schimming, 1989). Desiccation tolerance of the spores is enhanced when the fungi are grown in spe- 6.6.1. Desiccation tolerance cial media, e.g. enriched with glycerol, glucose and casamino acids, or when spores Desiccation tolerance has been established are harvested at mature rather than early in the spores of a wide variety of fungi (see stages of development. It has been found Table 6.2). Particularly well studied are the that the life span of Metarhizium desiccation-tolerant spores of economically flavoviride conidia is greater after slow important fungi, including yeast. Among rather than rapid drying (Hong et al., them are the entomopathogenic fungi of the 2000). This suggests that there is a certain genera Beauveria, Metarhizium and time required for protective mechanisms to Paecilomyces, the phytopathogenic fungi of be expressed during the stress, which is the genera Alternaria, Helminthosporium, also observed in other anhydrobiotic Pseudopezicula, Puccinia, Sclerotinia, propagules, e.g. seeds and pollen. Venturia, Uromyces and Ustilago, and the A compound that typically accumulates bioherbicidal fungus, Stagonospora. The in fungal spores is trehalose (see Feofilova, main issues in the research were the explo- 1992, for a review). Additional stresses, ration of how long spores can survive under such as heat shock or stationary phase natural conditions, which is important in (stress because of crowding) (Pedreschi and relation to plant pathogens, and the estab- Aguilera, 1997; Pedreschi et al., 1997) and lishment of culture conditions and drying high osmolality (Hallsworth and Magan, protocols that give adequate shelf stability 1994; Eleutherio et al., 1997), are particu- of spores in the case of commercial biologi- larly effective at increasing the content of cal products for agricultural application. trehalose, and enhancing viability and des- Desiccation-sensitive spores have been iccation tolerance, such as in yeast cells. found as a spin-off in the search for toler- Apart from its effect as an osmoprotectant, ance. For example, the conidia of trehalose causes a depression of the gel-to- Penicillium bilaji appear to be desiccation- liquid crystalline transition temperature of sensitive, because they could be kept alive membranes in dried yeast (Leslie et al., only under conditions of 100% RH 1994; see Chapter 10), alleviates the effects (Cunningham et al., 1990). However, using a of ethanol stress (Mansure et al., 1994) and fluidized bed drier and drying system, can be involved in the formation of a glassy Tadayyon et al. (1997) have managed to pro- state in the dried organism (Wolkers et al., Desiccation - Chap 06 18/3/02 1:56 pm Page 196

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Table 6.2. List of spore types of different fungal species in which desiccation tolerance has been established. Species Type of spore Reference Alternaria porri Conidia Hong et al., 1997 Aspergillus japonicus Conidia Gornova et al., 1992 Beauveria bassiana Conidia Hallsworth and Magan, 1994; Pfirter et al., 1999 Beauveria brongniarti Conidia Hong et al., 1997 Colletotrichum gloeosporioides Conidia Cunningham et al., 1990 Helminthosporium oryzae Conidia Hong et al., 1997 Metarhizium anisopliae Conidia Hong et al., 1997 Metarhizium flavoviride Conidia Moore et al., 1997; Hong et al., 2000 Neosartorya fischeri Ascospores Beuchat, 1992 Paecilomyces farinosus Conidia Hallsworth and Magan, 1994 Paecilomyces fumosoroseus Blastospores Cliquet and Jackson, 1997; Jackson et al., 1997 Conidia Stephan and Zimmermann, 1998 Phycomyces blakesleeanus Zygospores Rivero and Cerda Olmedo, 1994 Pseudopezicula tracheiphila Ascospores Pearson et al., 1991 Puccinia graminis Urediniospores Eversmeyer and Kramer, 1994 Puccinia recondita Urediniospores Eversmeyer and Kramer, 1994 Saccharomyces cerevisiae Cells van Steveninck and Ledeboer, 1974 Saccharomyces uvarum Cells Eleutherio et al., 1997 Sclerotinia sclerotiorum Ascospores Hong et al., 1997 Sordaria macrospora Ascospores Read and Lord, 1991 Stagonospora convolvuli Conidia Pfirter et al., 1999 Talaromyces flavus Ascospores Beuchat, 1992 Trichoderma harzianum Conidia Jin et al., 1996; Pedreschi and Aguilera, 1997 Uromyces appendiculatus Urediniospores Hong et al., 1997 Ustilago scitaminea Teliospores Hoy et al., 1993 Venturia inaequalis Conidia Becker and Burr, 1994

1998). Saccharomyces cerevisiae mutants, the percentage germination increases with in which proline and the charged amino spore age (Perry and Fleming, 1989; Ben acids such as glutamate, arginine and lysine Ze’ev et al., 1990; Gazey et al., 1993) or after are increased, have a marked cryoprotective chemical treatment (Rivero and Cerda activity, nearly equivalent to that of glycerol Olmedo, 1994). Usually, exposure of the or trehalose, both known as major cryopro- spores to low temperatures for some time, or tectants in this yeast (Takagi et al., 1997). to heat, certain chemicals or light, increases Other typical products are heat-shock pro- the germination percentage. The presence of teins (HSPs), which, for example, in yeast a thick wall may play an important role in are produced in the stationary phase of fer- the constitutive dormancy of some spores mentation (Praekelt and Meacock, 1990; (Ulanowski and Ludlow, 1989). In the Sanchez et al., 1992). They are thought to be spores of ectomycorrhizal and several sapro- crucial for the naturally high thermo-toler- trophic fungi, fluorescein diacetate (FDA) ance of these cell types and for their long- staining has been found to be unreliable as term viability at low temperatures. A small an indicator of viability, but a good predic- HSP in yeast may function in the protection tor of dormancy (Miller et al., 1993). As the of the plasma membrane against desiccation spores age, the percentage of fluorescent and ethanol-induced stress (Sales et al., spores increases. It is not known whether 2000). Further, high superoxide dismutase this increase is the result of a better penetra- (SOD) activity is essential for stationary tion of FDA through the spore wall, or of the phase survival in yeast (Longo et al., 1996). synthesis or activation of esterase capable of Evidence for the existence of dormancy cleaving the fluorogenic ester to form the in spores comes from the observation that fluorescent dye, fluorescein. Desiccation - Chap 06 18/3/02 1:56 pm Page 197

Desiccation Tolerance in Pollen and Spores 197

6.6.3. Longevity dry intervals (Becker and Burr, 1994). Spores can also survive for considerable periods of Longevity of spores varies considerably time when stored fully hydrated. Septoria among species. Thus, the viability of nodorum spores have been shown still to be teliospores of Ustilago scitaminea in air-dried infectious after 24 months of storage in water soils does not begin to decrease until after 18 (Wilkinson and Murphy, 1990). weeks, but is lost after maintenance, free of soil, for 23 weeks at ambient RH (Hoy et al., 1993). Air-dried spores of Stagonospora con- 6.6.4. Imbibitional stress volvuli remain viable at 3°C for 180 days (Pfirter et al., 1999). Particularly long-lived As in pollen, there is evidence in fungi and are the heat-resistant ascospores of yeast that dried cells suffer from being Neosartorya fischeri and Talaromyces flavus, plunged into liquid medium, particularly at which have been shown to survive dry stor- low temperatures. For example, dry conidia age in fruit powders (RH = 23%; 25°C) for up of M. flavoviride (4–5% MC) display to 30 months (Beuchat, 1992). It has been reduced viability when rapidly rehydrated reported that some Talaromyces spores sur- in free water. Prehydration in an atmosphere vive in coating material of dry seeds for as of high humidity allows dry conidia to long as 17 years at room temperature absorb sufficient moisture to avoid imbibi- (Nagtzaam and Bollen, 1994). tional damage (Moore et al., 1997). A similar The survival of conidia conforms to a avoidance of damage has been demonstrated cumulative negative normal distribution. As when dried yeast was allowed to imbibe at in seeds and pollen, longevity of conidia is elevated temperatures of 39–42°C (van determined by MC and temperature, beside Steveninck and Ledeboer, 1974). The mecha- endogenous factors (Hong et al., 1997, 1998). nism of imbibitional damage is dealt with in These authors have constructed an empirical Chapters 10 and 12. model for the storage behaviour of conidia, which can predict the viability of a spore lot over time at a broad range of different water 6.6.5. Cryopreservation contents and storage temperatures. A negative logarithmic relation has been observed Spores of vesicular–arbuscular (VA) myocor- between longevity and conidia MC. The MC rhizal fungi are usually stored in soil just of the conidia is dependent on the sorption above 0°C, but some of them may be stored properties of the endogenous compounds frozen. When cryoprotectants such as and the RH in which the conidia are equili- dimethylsulphoxide (DMSO), glycerol, man- brated. The relation between longevity and nitol or sucrose are ineffective, incubation of equilibrium RH can be described by a nega- freeze-sensitive spores for 2 days in tive semi-logarithmic relation. There is a 0.75–1.0 M trehalose confers a measure of lower MC limit, below which value further freeze-damage protection. However, it has reduction in MC does not increase conidia been found that slow drying of spores in situ longevity, and an upper MC limit, above (in soil) prior to freezing gives satisfactory which longevity no longer decreases. survival of the VA spores (Douds and Analysis of the effect of different tempera- Schenck, 1990). In the case of full desiccation

tures has shown that the Q10 for the loss in tolerance, sub-zero temperature storage of the conidia viability increases, the warmer the dried spores will generally be successful. temperature (Hong et al., 1999b). With ﬂuc- tuating day/night temperatures, the warmer temperature mainly determines conidia 6.7. Conclusion longevity. Viability of ungerminated desiccation-tolerant conidia is not affected by expo- In the introduction, it was suggested that sure to wet and dry intervals, but germinated pollen and spores are likely to have at conidia (germlings) are generally sensitive to least a certain level of tolerance to dehy- Desiccation - Chap 06 18/3/02 1:56 pm Page 198

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dration. This expectation appears to be which makes them more attractive to generally correct. study. The economic importance of pollen Most pollens are tolerant to MCs and fungal spores has also encouraged below 10%. However, pollens of some considerable research, particularly into the plant families have an elevated lower MC fundamental aspects of stress tolerance. limit (>10% MC), below which viability The occurrence of imbibitional damage in is lost. Such a reduced tolerance is com- dry pollen and fungal spores/yeast, and parable with that of the so-called ‘inter- probably also in the other propagules, may mediate’ seeds. It seems that real have reduced the enthusiasm for the appli- desiccation sensitivity (recalcitrance), cation of dry storage: the probably still i.e. sensitivity already below 40% MC, as viable propagules may have been killed on is frequently observed in seeds, is rehydration. Considering the generally uncommon among pollens. This might be limited longevity of the pollen/spores at explained by the usually larger size of room temperature, sub-zero temperature the seeds: under the same humid condi- storage has to be applied for successful tions, the comparatively large seeds do long-term storage. not dry out as fast as the microscopic The life span in the dry state at room pollens. Thus, desiccation tolerance is temperature generally ranges from a few less imperative for seeds that are dis- days to several months for both desicca- persed in humid climates. tion-tolerant pollen and spores. This is in The phenomenon of desiccation toler- sharp contrast to plant seeds, which often ance has been extensively studied in survive a number of years (see Priestley, pollen. Some of the compatible solutes, 1986, for a review). Pollen, for example, such as sucrose and proline, that are typi- is not typically designed to overcome cally associated with drought and desicca- long periods of adverse conditions, as is tion tolerance are abundantly present. The common for seeds. Although the cause of heat-stable dehydration proteins, known the difference in longevity between from seeds, also occur in pollen. pollen and spores, on the one hand, and A similar picture emerges for fungal seeds, on the other hand, is a matter of spores and yeasts. However, the situation speculation, it may be envisaged that the is less clear for fern and moss spores, comparatively large size of seeds gives mainly because considerably less work has them an extra protection against molecu- been done on this material. Desiccation- lar oxygen and peroxidative degradation. tolerant fern and moss spores have been The exceptionally long life span, as found found, nevertheless, and drought tolerance in some thick-walled fungal spores, might might at least be common. Only for the depend on a possible impermeability of spores of the fern Osmunda has analysis these walls to oxygen. shown that free proline and arginine The major difference between pollen abound. Little is known about horsetail, and the other propagules is the lack of dor- Lycopodium and Selaginella spores, mancy in the former. In hydrated pollen, except that dehydrating horsetail spores metabolism seems unrestricted, which leads have a very short life span and contain to a rapid loss of viability. Thus, while sur- free amino acids. The problem with vival in the hydrated state is a common screening for desiccation tolerance in strategy in spores, it is not in pollen. these propagules (not pollen and fungal spores) is that germ tube emergence is comparatively slow, taking at least a week 6.8. Acknowledgement and often much longer. Until rapid assay methods for viability are developed, it will The author is indebted to Dr Elena A. not be attractive to study anhydrobiosis in Golovina from the Timiryazev Institute of these spores. In contrast, germ tube emer- Plant Physiology, Russian Academy of gence is generally a matter of hours in fun- Sciences, Moscow, for critically reading gal spores and even minutes in pollen, the manuscript. Desiccation - Chap 06 18/3/02 1:56 pm Page 199

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6.9. References

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Wilkinson, C.A. and Murphy, J.P. (1990) A comparison of preservation methods for Septoria nodorum isolates. Cereal Research Communications 18, 33–40. Wolkers, W.F., Oldenhof, H., Alberda, M. and Hoekstra, F.A. (1998) A Fourier transform infrared microspectroscopy study of sugar glasses: application to anhydrobiotic higher plant cells. Biochimica et Biophysica Acta 1379, 83–96. Wolkers, W.F., McCready, S., Brandt, W., Lindsey, G.G. and Hoekstra, F.A. (2001) Isolation and characterization of a D-7 LEA protein from pollen that stabilizes glasses in vitro. Biochimica et Biophysica Acta 1544, 196–206. Zhang, H.Q. and Croes, A.F. (1983) Protection of pollen germination from adverse temperatures: a possible role for proline. Plant, Cell and Environment 6, 471–476. Zhang, H.Q., Croes, A.F. and Linskens, H.F. (1985) Proline accumulation during anther development in Petunia hybrida. Acta Botanica Neerlandica 34, 213–222. Desiccation - Chap 06 18/3/02 1:56 pm Page 206 Dessication 07 18/3/02 1:56 pm Page 207

7 Vegetative Tissues: Bryophytes, Vascular Resurrection Plants and Vegetative Propagules

Michael C.F. Proctor1 and Valerie C. Pence2 1School of Biological Sciences, University of Exeter, Washington Singer Laboratories, Perry Road, Exeter EX4 4QG, UK; 2CREW, Cincinnati Zoo and Botanical Garden, 3400 Vine Street, Cincinnati, OH 45220, USA

7.1. Introduction 207 7.2. Bryophytes 208 7.2.1. Desiccation time and recovery time: the typical pattern of desiccation response 210 7.2.2. The effect of intensity of desiccation 211 7.2.3. Effects of temperature 213 7.2.4. Events on rehydration 213 7.2.5. Drying rate and drought hardening 215 7.2.6. Constitutive and induced tolerance 216 7.2.7. How long is needed for complete recovery? Processes and criteria of recovery; long-term survival 216 7.3. Vascular Plants 217 7.3.1. Ecological and morphological adaptations 224 7.3.2. The effect of the intensity of desiccation 225 7.3.3. Effect of the rate of desiccation 226 7.3.4. Morphological and cytological changes that occur with drying 226 7.3.5. Rehydration and recovery 227 7.3.6. Vegetative propagules: bulbils, corms, tubers and plant fragments 228 7.4. Concluding Comments 228 7.5. References 230

7.1. Introduction iccation tolerance in vegetative parts of plants. However, desiccation tolerance is a We take for granted the desiccation toler- widespread phenomenon (Bewley and ance of seeds and pollen, but see it as a Krochko, 1982; Stewart, 1989; Crowe et al., matter for remark when we encounter des- 1992; Oliver and Bewley, 1997). It is found © CAB International 2002. Desiccation and Survival in Plants: Drying Without Dying (eds M. Black and H.W. Pritchard) 207 Dessication 07 18/3/02 1:56 pm Page 208

208 M.C.F. Proctor and V.C. Pence

in many microorganisms, in animals such There are probably some features and as tardigrades, rotifers and nematodes and mechanisms common to all desiccation- in larval cysts of crustacea of seasonal tolerant cells, but there are also some major water bodies, and in plants it is a frequent differences. In general, the more tolerant and characteristic feature of the vegetative bryophytes are what have been character- cells of terrestrial algae, lichens and ized as ‘fully desiccation-tolerant’ (Bewley bryophytes. In vascular plants it is the and Oliver, 1992; Oliver, 1996; Oliver and norm in spores, pollen and seeds, but Bewley, 1997; Oliver et al., 2000); their tol- uncommon in vegetative tissues. erance is essentially constitutive and little Desiccation tolerance implies the ability affected by the rate of drying. By contrast, of an organism to dry to equilibrium with many desiccation-tolerant vascular plants the ambient air, and to recover and return to show little tolerance if they are dried fast, normal metabolism on remoistening. It is a but tolerance is induced in the course of qualitatively different phenomenon from slow drying, which, because of their vascu- drought tolerance. Drought-tolerant plants lar system and relatively large size, is gen- can maintain more or less normal metabo- erally what happens in nature. Some of the lism in the presence of soil water potentials less tolerant bryophytes probably behave that may often be low enough to wilt the similarly. These have been referred to as leaves, but are killed if the relative water ‘modified desiccation-tolerant’ plants content (RWC) of the tissues falls below (Oliver and Bewley, 1997). Desiccation-tol- ~ 0.2–0.3, i.e. 20–30% of full turgor, corre- erant plants vary greatly in the length and sponding to a tissue water potential of frequency of the wet and dry periods to perhaps 5 to 10 MPa. Many desiccation- which they are adapted. Small bryophytes tolerant plants withstand drying to water of sun-exposed dry habitats may achieve a potentials of 100 MPa or lower, at which positive carbon balance from moist periods no liquid phase remains in the cells and of an hour or less and can benefit from metabolism is at a standstill. Desiccation tol- brief showers, or even dewfall following erance must have evolved early in the colo- clear nights. Vascular plants that retain nization of land by microorganisms their chlorophyll when desiccated may including cyanobacteria and simple algae. typically require around a day for recovery. Amongst more complex photosynthetic land In poikilochlorophyllous species, which organisms, the bryophytes are a numerous lose their chlorophyll on drying, regreen- group, growing in diverse habitats from the ing and return to normal metabolism takes tropics to the polar regions, in which a several days and these plants generally degree of desiccation tolerance seems to be a occupy situations with relatively long alter- fundamental part of their life strategy, and is nating wet and dry periods. presumably primitive. Much the same may be said of the symbiotic associations between fungi and the photosynthetic organ- 7.2. Bryophytes (see also Chapter 1) isms that we call lichens (Kershaw, 1985; Nash, 1996). On the other hand, the vascular Bryophytes are the second largest group of plants that dominate the world’s flora gener- photosynthetic land plants, with some ally depend on maintenance of a high inter- 25,000–30,000 species occupying diverse nal water content by conduction of water habitats from the tropics (Pócs, 1982; from the soil, with varying degrees of control Richards, 1984) to polar regions (Longton, of water loss by the stomata (Raven, 1977). 1988). By contrast with the vascular plants, Desiccation-tolerant vascular plants are the they may be seen as having followed an rare exception. They are relatively few in alternative strategy of adaptation to the number and taxonomically scattered and irregular and intermittent availability of isolated – products of independent selection water on land. Conduction of water is typi- for intermittently or seasonally desiccated cally external and water is freely gained habitats, mostly warm-temperate to tropical. and lost over much or all of the surface of Dessication 07 4/4/02 2:20 pm Page 209

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the plant, and dry periods are passed in a early investigators were well aware state of suspended metabolism until wet (Irmscher, 1912; Höfler, 1946; Clausen, conditions return. Clearly, bryophytes are 1952; Abel, 1956; Hosokawa and Kubota, in some respects limited by this mode of 1957), though appreciation of their com- life; they cannot compete with vascular plexity was constrained by limited tech- plants in size or in productivity on fertile niques. The earlier studies generally used water-retentive soils. On the other hand, plasmolysis as an indicator of cell survival. they are freed from some constraints: they This necessarily gave a somewhat static can occupy hard substrates that are impene- picture, but it allowed exploration of the trable to roots and so untenable to vascular effect of different times and intensities of plants, and they are well adapted to nutri- desiccation, and demonstration of drought- ent capture in N- and P-deficient situations. hardening (Höfler, 1946; Abel, 1956); The majority of bryophytes will with- Höfler, Clausen, Abel, Hosokawa and stand at least modest levels of desiccation Kubota and others laid a foundation of (to equilibrium with, for example 20 to knowledge which is still valuable as back- 40 MPa) for at least a few days, but some ground for more analytical research. Ried are much more tolerant than that. Breuil- (1960a,b), in his work on lichens, measured Sée (1993) observed regrowth following photosynthesis and respiration by classical remoistening of gametophytes of the liver- gas-analysis techniques, which, though wort Riccia macrocarpa kept as dried laborious, gave newly dynamic insights herbarium specimens for over 23 years. into the process and timing of recovery. Maheu (1922) observed regeneration of pro- Various authors in the 1970s and early tonema from leaves of ‘Barbula’ (= Tortula, 1980s (Hinshiri and Proctor, 1971; Dilks Syntrichia) ruralis that had been dry for 14 and Proctor, 1974; Schonbeck and Bewley, years. Keever (1957) found that most speci- 1981a,b) measured gas exchange manomet- mens of Grimmia laevigata were still viable rically, but by the mid-1980s that had been after 3 years’ dry storage, but only 20% largely superseded by infrared gas analysis remained viable after 10 years. Of taxo- (IRGA). This allowed continuous measure- nomic orders within the bryophytes, those ment and, as IRGA systems developed, pro- that stand out as particularly desiccation- gressively better stability, sensitivity and tolerant include the Andreaeales (Andreaea: time resolution (Sˇesták et al., 1971; Dilks ‘granite mosses’), Encalyptales (Encalypta), and Proctor, 1976; Kershaw, 1985; Lange, Pottiales (Tortula, Syntrichia, Tortella, etc.), 1988; Long and Hällgren, 1993; Tuba et al., Orthotrichales (Orthotrichum, Ulota, 1996). Since the 1990s, chlorophyll fluores- Zygodon), Hedwigiales (Hedwigia) and cence has provided a powerful new Grimmiales (Grimmia, Racomitrium, etc.), method for rapid in vivo assessment of but a number of other orders include at some aspects of photosynthetic function to least some highly tolerant species, e.g. complement gas-exchange measurements Polytrichales (Polytrichum piliferum, etc.), (Seel et al., 1992b; Deltoro et al., 1998a,b; Dicranales (Dicranoweisia cirrata, Csintalan et al., 1999; Marschall and Cheilothela chloropus, etc.), Hypnales (e.g. Proctor, 1999). Eurhynchium pulchellum, Scleropodium Alongside this strand of research centred tourretii, Hypnum spp., Leucodon sci- on gas exchange and carbon balance, uroides, Anomodon viticulosus). Some Bewley, Oliver and their co-workers have other groups, such as the Hookeriales concentrated particularly on effects of desic- among the mosses and many genera of leafy cation and rehydration on protein synthesis. and thalloid liverworts, are relatively sensi- Their very extensive work, reviewed by tive and characteristically confined to shel- Bewley (1979), Bewley and Krochko (1982), tered humid places. Most bryophytes fall Bewley and Oliver (1992), Oliver (1996) and somewhere between these two extremes. Oliver and Bewley (1997), leads naturally The responses of bryophytes to desicca- into modern molecular biological tech- tion are multifaceted, a fact of which the niques (Wood et al., 1999) and to the possi- Dessication 07 18/3/02 1:56 pm Page 210

210 M.C.F. Proctor and V.C. Pence

bility of genetic engineering for increased viticulosus (measured as net O2 evolution), drought tolerance in arid-zone crops. as a function of desiccation time (at c. 94 MPa) on one axis and rehydration time on the other (Hinshiri and Proctor, 1971). For 7.2.1. Desiccation time and recovery time: periods of up to about 10–15 days desicca- the typical pattern of desiccation response tion, recovery is quick and essentially complete within 3–4 h. This may be seen as a Figure 7.1 shows the photosynthetic per- period of ‘pure’ desiccation tolerance. formance of a rehydrated moss, Anomodon From this point to about 40–45 days’ desic-

20 Hours rehydration

22 28

35 42

+500 Oxygen output 49 l g–1 h–1 56

0 67

–500 Days desiccation

Fig. 7.1. The course of the net photosynthesis rate of Anomodon viticulosus following moistening after various periods of desiccation at 50% RH, 20°C. Curves are interpolated for net photosynthesis rates attained 2.5, 5, 10, 15 and 20 h after moistening. The shaded area indicates the part of the graph over which net C assimilation

is negative. Warburg manometer measurements of oxygen evolution or uptake; CO2 concentration maintained by carbonate–bicarbonate buffer in centre wells; with a few exceptions points are means of two readings. Measurements up to 28 days were made at 20°C, the remainder at 25°C. Somewhat simpliﬁed and in part redrawn from Hinshiri and Proctor (1971). (Reproduced with permission from Proctor, 2001.) Dessication 07 4/4/02 2:21 pm Page 211

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cation, recovery becomes progressively Hookeria lucens 1 slower and less complete. Evidently, one or more progressive damaging processes take –3 MPa 0.8 effect, which can at least to some extent be –9 MPa repaired given sufﬁcient recovery time. 0.6 After prolonged desiccation, remoistening –37 MPa leads to long-persistent oxygen uptake, 0.4 –154 MPa implying corresponding carbon loss. This probably reﬂects major metabolic disrup- 0.2 tion, from which ultimate recovery, if it takes place at all, can only be slow. 0

0 20406080 7.2.2. The effect of intensity of desiccation Rhytidiadelphus loreus 1 In addition to the factors considered in Fig. 7.1, recovery and long-term survival 0.8 depend on the equilibrium water potential to which the bryophyte has been desiccated. 0.6 m F

Dilks and Proctor (1974) gave recovery data / v

for several species following desiccation at F 0.4 different water potentials, which showed that some, like Plagiothecium undulatum, 0.2 are more severely damaged the lower the water potential to which they have equili- 0 brated, while others, such as Racomitrium 0 20406080 lanuginosum, show better long-term sur- Tortula ruralis vival at low than at high water potentials. 1 Experiments using chlorophyll fluorescence as a measure of plant performance show the 0.8 same pattern (Proctor, 2001; Fig. 7.2). Species of more-or-less shaded habitats, 0.6 never exposed in the field to extreme desiccation stress, were most quickly and 0.4 severely damaged at the lowest water potential, at which species of dry, open, sun- 0.2 exposed situations, such as R. lanuginosum and Tortula ruralis, showed best survival of prolonged drying. Given a modest level of 0 daylight illumination (comparable with a 0 20406080 shady woodland floor) all the investigated Desiccation time (days) species survived well for several weeks at Fig. 7.2. The chlorophyll-fluorescence parameter 3 MPa, corresponding to a cell RWC of Fv /Fm (estimating the maximum quantum efficiency about 50%. Over this period, the highly des- of photosystem II), measured 20 min after iccation-tolerant species, R. lanuginosum remoistening, following desiccation at a range of and T. ruralis, performed best at either this water potentials ( 3 to 154 MPa). The curves are smoothed from the original data (n = 3) by a single highest water potential or at the lowest, and pass through a binomial-average smoothing routine. it was only after about 6 weeks that material Collecting sites: Hookeria lucens, Stoke Woods, stored dry outperformed material kept lightly Exeter, Devon, UK, August 1999; Rhytidiadelphus wilted. It was noteworthy that the more des- loreus, White Wood, Holne, Devon, UK, August iccation-tolerant species were damaged most 1999; Tortula ruralis, Fülöpházá, Kiskunság rapidly in the range 9 to 22 MPa. In fact, National Park, Hungary, July 1998. Dessication 07 18/3/02 1:57 pm Page 212

212 M.C.F. Proctor and V.C. Pence 0 Pleurochaete –100 , Clyst St Mary; –200 , Devon, UK, November 1999). UK, November , Devon, tandard deviations. Collecting sites: 24 h 20 min –300 T. ruralis T. Anomodon viticulosus Ulota crispa –400 Grimmia pulvinata 0 –100 ) after remoistening, following desiccation for 60 days at different water desiccation for 60 days ) after remoistening, following , Chudleigh Rocks (all except , Chudleigh Rocks –200 Water potential (MPa) Water –300 Pleurochaete squarrosa ) and 24 h ( –400 Anomodon viticulosus 0 , Felsötárkány, near Eger, Hungary, October 1999; Hungary, near Eger, , Felsötárkány, measured 20 min ( m –100 F / v F Tortula ruralis Tortula –200 , New Bridge, Dartmoor; –300 Ulota crispa , O Brook, Dartmoor; –400 Tortula ruralisTortula Grimmia pulvinata Rac. lanuginosum 0 0 1 1

0.8 0.6 0.2 0.8 0.6 0.2 0.4 0.4

m v F / F , Chudleigh Rocks; , Chudleigh Rocks; The chlorophyll-ﬂuorescence parameter parameter chlorophyll-ﬂuorescence The squarrosa Fig. 7.3. Fig. potentials. Data points are means of three replicate readings; error bars (in some cases hidden within the data symbols) show s potentials. Data points are means of three replicate readings; error bars (in some cases hidden within the data symbols) show Racomitrium lanuginosum Dessication 07 18/3/02 1:57 pm Page 213

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even for the most desiccation-tolerant to less than 0.05 K in liquid helium, and species, there is an optimum range of water Bewley (1973) reported recovery of protein potential for longest survival and most rapid synthesis in T. ruralis shoots that had been recovery, generally around 100 to 300 cooled in the dry state to –196°C in liquid MPa. After 24 h recovery from desiccation nitrogen. This is perhaps no matter for sur- periods of up to 60 days, the most tolerant prise. However, by analogy with seeds in species showed very little difference storage, increased temperature would be between 41, 113, 218 and 412 MPa, expected to shorten survival time, and this the chlorophyll-ﬂuorescence parameter is indeed so. Hearnshaw and Proctor (1982)

Fv/Fm (a measure of the maximum quantum measured mean survival of seven species efﬁciency of photosystem II, widely used as (in terms of chlorophyll content after a a general indicator of plant stress) recover- period of moist recovery) at temperatures ing within 24 h to normal unstressed levels from 20 to 100°C. Five of the bryophytes after all of them (Fig. 7.3). The water content gave good straight-line relationships on an of a bryophyte (as % dry weight (dw)) is ‘Arrhenius plot’ relating the logarithm of closely determined by the water potential half-survival time to the reciprocal of with which it is in equilibrium (Dilks and absolute temperature, as did data for viabil- Proctor, 1979; Schonbeck and Bewley, ity of rice in storage (Roberts, 1975; 1981a). Water contents of 5% dw or less Proctor, 1982). This is the pattern that may be reached by bryophytes in exposed would be expected for the temperature places in the heat of the sun, corresponding response of chemical reactions in general to water potentials below 300 MPa. (Morris, 1974). Two Racomitrium species gave non-linear plots, but for all the bryophytes survival times ranged continu- 7.2.3. Effects of temperature ously from minutes at 100°C to weeks or months at normal ambient temperatures. When fully hydrated, bryophytes show Ambient ‘shade’ temperatures are com- generally similar temperature responses to monly in the range 0–30°C and rarely C3 vascular plants. The lower limit for exceed 40°C, but dry bryophytes in the sun most species is probably set by loss of on hot days can reach 40–60°C, and it is water as the cell contents equilibrate with probably these high temperature episodes the water potential of extracellular ice that drive selection for the apparently (Kallio and Heinonen (1975) found positive extravagant levels of desiccation tolerance net photosynthesis in R. lanuginosum and seen in, e.g. Tortula ruralis, Grimmia Dicranum elongatum down to 8°C); pos- laevigata, R. lanuginosum and Andreaea sible chilling effects in warm-climate rothii. bryophytes at non-freezing temperatures seem not to have been explored. The upper lethal limit for metabolically active 7.2.4. Events on rehydration bryophytes is usually in the range 40–50°C. Bryophytes become much more The preceding sections have principally con- tolerant of extremes of temperature as they sidered responses along the desiccation- dry out (Meyer and Santarius, 1998). time axis of Fig. 7.1. There are also impor- Although there has been a general aware- tant questions to answer on the rewetting ness that temperature must inﬂuence the (recovery) axis. As the time-resolution of survival of dry bryophytes, there has been measurement techniques has improved, it rather little systematic work on the subject has become increasingly apparent that (Irmscher, 1912; Lange, 1955; Nörr, 1974). recovery of normal photosynthetic function Desiccation-tolerant bryophytes, when dry, on remoistening can be very rapid. can survive extremely low temperatures. Manometric and IRGA measurements have Becquerel (1951) reported survival of demonstrated recovery to near-normal rates Barbula and Grimmia leaves after cooling of net photosynthesis within an hour or Dessication 07 18/3/02 1:57 pm Page 214

214 M.C.F. Proctor and V.C. Pence

Tortula ruraliformis: two of remoistening in a number of species 8 days desiccation (Hinshiri and Proctor, 1971; Dilks and 8 (a) Proctor, 1974, 1976; Fig. 7.4). IRGA data show net photosynthesis in T. ruralis to be 6 negative 15 min after remoistening, but 4 almost completely recovered after 30 min (Tuba et al., 1996). Measurements from 2 modulated chlorophyll ﬂuorometers show 0 that photosystem II (PSII) can return to near-normal quantum efﬁciency within a –2 few minutes. Initial recovery of the ratio F /F on remoistening shoots of T. ruralis –4 v m in the dark after 3 days air dry (c. 100 –6 MPa; water content c. 10% dw) approxi- mated closely to an exponential curve with –8 a half-recovery time of c. 20 s. The corre- 0 60 120 180 sponding initial half-recovery time for the pendulous African forest moss Pilotrichella Grimmia pulvinata: 14 days desiccation ampullacea (after 20 h at 37 MPa) was c. 20 40 s (M.C.F. Proctor, unpublished data). (b) This rapid initial phase of recovery is fol- 10 lowed by a much longer phase of slow recovery, which may last an hour or two in 0 the most tolerant species and many hours in the more sensitive. Recovery of –10 (Fm Fm )/Fm ( PSII, a measure of effective

uptake (vpm) quantum yield of PSII) in light is slower 2 –20 than recovery of Fv/Fm. Even so, in tolerant CO Net photosynthesis species recovery of photosynthetic electron –30 ﬂow (which of course may include a pho- Dark respiration torespiratory component as well as carbon –40 ﬁxation) can be largely complete within 15–20 min (Csintalan et al., 1999). 0 60 120 180 240 It is clear that full recovery must involve a Andreaea rothii: diversity of processes. First, membrane 4 days desiccation integrity must be re-established. Probably all 6 (c) bryophytes (indeed, all desiccation- tolerant cells (Crowe et al., 1992)) show 4 detectable leakage of solutes immediately on remoistening (Brown and Buck, 1979; 2 Bewley and Krochko, 1982) but in tolerant species this in only transient and most leaked 0 Fig. 7.4. Rate of net CO2 uptake ( ) and dark –2 respiration () of some desiccation-tolerant mosses following remoistening, after various periods air dry –4 (at ~ 100 MPa), measured by infrared gas analysis (LCA-2, ADC, Hoddesdon, UK); PPFD 500 mol –6 m2 s1; ~ 23°C. (a) Tortula ruralis ssp. ruraliformis, 8 days; Braunton Burrows, Devon, UK; (b) Grimmia 0 306090120 pulvinata, c. 14 days; stone parapet of bridge, Clyst Recovery time (min) St Mary, Devon, UK; (c) Andreaea rothii, 4 days; granite outcrop, Haytor, Devon, UK. Dessication 07 18/3/02 1:57 pm Page 215

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solutes are rapidly reabsorbed; little or no net water stress, but recommences within a leakage of inorganic ions is detectable after a minute or two of remoistening. In T. ruralis few minutes (Deltoro et al., 1998a). Of the the pattern of protein synthesis in the first major metabolic systems, photosynthesis, hours of rehydration is distinctly different dark respiration and protein synthesis from that of hydrated controls, but no novel (Gwózdz et al., 1973; Oliver, 1991) are reiniti- transcripts are made in response to desicca- ated very rapidly (within a minute or two) in tion; the changes appear to be due to alter- desiccation-tolerant species such as R. lanug- ations in translational controls. Oliver inosum, T. ruralis and A. viticulosus. Much (1991) showed that in T. ruralis the synthe- or all of the process of the recovery of all sis of 25 proteins ceased (or substantially three systems seems likely to be essentially decreased) and the synthesis of 74 proteins physical, involving reinstatement of water was initiated (or substantially increased) into macromolecules and re-establishment of during the first 2 h of rehydration. He spatial and conformational relationships. The named these protein groups ‘hydrins’ and evidence indicates that recovery of the photo- ‘rehydrins’, respectively. The synthesis of systems is essentially independent of protein all the hydrins returned to control levels synthesis, but that some protein synthesis is within 2–4 h, but, while some rehydrins needed for full return to predesiccation rates were synthesized only transiently within of photosynthetic carbon fixation (Proctor the first hour or two of rehydration, others and Smirnoff, 2000; Proctor, 2001). were still being synthesized at elevated lev- Respiration recommences immediately els 10–12 h later. Synthesis of all proteins on remoistening; indeed, in lichens there had returned to normal levels within 24 h. are indications of low levels of respiratory The exact nature and function of most of activity even at water potentials as low as these hydrins and rehydrins is not yet 100 MPa (Cowan et al., 1979). The initial known, but some homologies with known rate of respiration on remoistening is proteins are beginning to emerge from mol- always higher, and sometimes very much ecular biological investigations (Wood et higher, than the normal steady rate of dark al., 1999; Chapters 1 and 11). respiration, perhaps reflecting some (proba- Growth of the bryophyte plant requires bly variable) combination of uncoupling of not only a positive carbon balance and the respiration from other metabolic systems ability to synthesize proteins, but also the and the demands of repair processes. In the re-establishment of the cell cycle, and IRGA measurements of Dilks and Proctor translocation of metabolites, mineral nutri- (1976), dark respiration of A. viticulosus ents and growth regulators to the meristem- and Rhytidiadelphus loreus took 5–10 h to atic regions. Cell division and probably also return to steady levels following a few days’ translocation depend on processes mediated desiccation. The data of Tuba et al. (1996) by microtubules, which are broken down on showed dark respiration in T. ruralis and desiccation and must be reconstituted as a the dry-grassland lichens Cladonia convo- part of the recovery process. A degree of luta and Cladonia furcata, following a few repair of DNA will always be necessary, and hours’ desiccation, falling rapidly within 2 the requirement for this is likely to increase h to a rather steady level still greater than with increasing desiccation time. that measured before or during drying. The recovery of protein synthesis has been the subject of extensive research by 7.2.5. Drying rate and drought hardening J.D. Bewley, M.J. Oliver and their co- workers, reviewed by Bewley (1979), Höfler (1946) and Abel (1956) found that the Bewley and Krochko (1982), Bewley and desiccation tolerance of bryophytes was Oliver (1992), Oliver (1996), Oliver and enhanced following a period of less severe Bewley (1997), Oliver et al. (1998) and in drying. Abel surveyed the recovery of a wide Chapters 1 and 11 of this book. Protein syn- range of moss species after 24–48 h desicca- thesis declines and ceases quickly under tion at a range of humidities from 0.08 to Dessication 07 18/3/02 1:57 pm Page 216

216 M.C.F. Proctor and V.C. Pence

96% relative humidity (RH), with and with- 1997; Oliver et al., 1998), R. lanuginosum out a previous light drying at 96% RH for 24 and A. viticulosus. However, in many species h. The predrying treatment led to clearly of more mesic habitats, tolerance is induced greater desiccation tolerance in the majority to varying degrees. The physiology of the of species, sometimes very strikingly so, as in induction of enhanced tolerance in such Bryum capillare, Bryum caespiticium, important groups as the Bryaceae, Mniaceae Mnium marginatum, Plagiomnium rostra- and the common pleurocarpous families in tum, Ceratodon purpureus, Fissidens the Hypnobryales is almost entirely unex- adiantoides, Pohlia elongata, Timmia austri- plored. Abscisic acid (ABA)-induced toler- aca and P. undulatum. In some species (gen- ance (involving synthesis of specific proteins erally the most sensitive and the most during drying) has been demonstrated in pro- tolerant) predrying made little difference, as tonema of Funaria hygrometrica (Werner et in Bryum pseudotriquetrum (bimum), al., 1991; Bopp and Werner, 1993; Schnepf Mielichhoferia elongata, Philonotis seriata and Reinhard, 1997), and short, thick-walled and Rhynchostegium riparioides (Platy- desiccation-tolerant protonemal cells form hypnidium rusciforme) (sensitive), and T. under the influence of desiccation or ABA in ruralis, Encalypta streptocarpa (contorta), Aloina aloides (Goode et al., 1994) and Grimmia pulvinata, Hedwigia ciliata (albi- Diphyscium foliosum (Duckett, 1994). cans), Pleurozium schreiberi and Beckett (1999) showed that partial dehydra- Rhytidiadelphus spp. (tolerant). Schonbeck tion of the moss Atrichum androgynum for 3 and Bewley (1981a) explored the effects of days increased resistance to desiccation- slow and fast drying and rehydration on pho- induced cation leakage, and that treatment tosynthesis by T. ruralis following 2 days’ with ABA produced the same effect. Induced and 7 days’ desiccation at 21 or 208 MPa. or seasonally switched desiccation tolerance Rate of drying made little difference to recov- is probably common in marchantialean liver- ery after desiccation at 21 MPa, but recov- worts of seasonally dry habitats. In Lunularia ery of photosynthesis was severely impaired cruciata, the switch from the desiccation-sen- after rapid drying to 208 MPa. Damaging sitive winter state to the tolerant summer effects of rapid drying to –600 MPa were state appears to be mediated by lunularic largely eliminated if the moss samples were acid (Schwabe and Nachmony-Bascomb, first dried slowly to 21 MPa, and were 1963). Hellewege et al. (1994) showed that greatly reduced if the samples were equili- ABA induces desiccation tolerance in the liv- brated for 5 h in saturated air before remoist- erwort Exormotheca holstii, and Hellewege et ening. Oliver and Bewley (1997) suggested al. (1996) have shown that ABA can bring that the limited ‘hardening’ effect seen in T. about the transition from the aquatic to the ruralis may reflect sequestration of ‘recovery’ land form of Riccia fluitans. The ability to mRNAs during slow drying. Krochko et al. respond to ABA with enhanced desiccation (1978) found very large differences between tolerance thus seems likely to be widespread fast- and slow-dried material of the relatively among bryophytes, but how widely endoge- desiccation-sensitive moss Cratoneuron fil- nous ABA occurs within the bryophytes is icinum. There is clearly much of interest in unknown. ABA is undetectable in T. ruralis, the effects of different rates of drying and in which a high level of desiccation tolerance rehydration, which invites further investiga- is constitutive (Oliver and Bewley, 1997). tion and study of a wider range of species.

7.2.7. How long is needed for complete 7.2.6. Constitutive and induced tolerance recovery? Processes and criteria of recovery; long-term survival Desiccation tolerance apears to be largely constitutive in the highly desiccation-tolerant The recovery processes outlined in Section bryophytes on which most work has been 7.2.4 proceed on different time scales. done, such as T. ruralis (Oliver and Bewley, What should we take as the criteria of over- Dessication 07 18/3/02 1:57 pm Page 217

Desiccation Tolerance of Vegetative Tissues 217

all ‘recovery’? There may be no hard and total, have been documented as being fast answer to this question. Very short desiccation-tolerant in their vegetative parts moist periods will lead to net carbon loss. (Table 7.1; Porembski and Barthlott, 2000). Moist periods long enough for a positive Because the rehydration of some of these net carbon balance may be insufficient for plants gives the appearance of a revitaliza- cell division and growth, but might per- tion of apparently dead tissues, they are haps allow significant DNA repair. This is often referred to as ‘resurrection plants’. conjectural, but indicates the kind of ques- The first scientific report of a resurrection tions on which research is needed. The species was made by Hooker (1837) in a limited available measurements indicate description of Selaginella lepidophylla from that the moist periods experienced by des- the southwestern USA and Mexico. iccation-tolerant bryophytes in the field Subsequent reports were made in the next vary greatly in length (Proctor, 1990; century, the earliest from field observations Proctor and Smith, 1995). Maintenance of a made in dry areas of central Asia and sub- positive net carbon balance must be impor- Saharan Africa. The remarkable ability of tant, even on a rather short time scale, these plants to exhibit the effects of extreme whereas a bryophyte may have an entirely desiccation and yet to remain alive and viable annual life cycle in which growth is revive when rewetted was reported with largely confined to particular seasons, interest and often careful notes (Thoday, while for the rest of the year the plant is 1921; Vassiljev, 1931; Hambler, 1961, 1964; doing no more than maintaining its Hornby and Hornby, 1964). Carex physodes, foothold in the habitat. Whether ‘recovery’ Myrothamnus flabellifolia and Craterostigma is seen as return to a normal rate of carbon plantagineum were among the first desicca- fixation, or as requiring full restoration of tion-tolerant angiosperms described, while all metabolic systems to their optimum early reports confirmed the phenomenon in moist-period activity, is a question of other pteridophytes, such as Polypodium context – indeed, there may be no single polypodioides, Notochlaena marantae, ‘optimum’. Further, bryophyte shoots com- Selaginella njam-njamensis and Platycerium monly show progressive formation of new stemaria (Pessin, 1924; Iljin, 1931; Hambler, leaves and death of old ones; recovery from 1961, 1964). desiccation of the apical parts of the shoot Resurrection species have been docu- may be accompanied by accelerated senes- mented thus far in nine families of pterido- cence and death of the older parts. phytes and ten families of angiosperms (Table 7.1). They are conspicuously lacking in gymnosperms, though foliage of 7.3. Vascular Plants (see also Chapter 1) Welwitschia mirabilis shows some degree of tolerance (Gaff, 1972). In angiosperms, Although often observed in seeds, spores they occur among both monocotyledons and pollen, desiccation tolerance is the and dicotyledons. Although a higher pro- exception in vegetative tissues of vascular portion of pteridophyte taxa than seed plants. The combination of vascular tissue plants are tolerant of desiccation, and intercellular spaces with cuticle and angiosperms often exhibit a higher degree stomata allows these species to maintain a of tolerance than ferns (Gaff, 1977). water potential higher than that of their Desiccation tolerance shows a wide tax- above-ground environment (homoiohydry), onomic scatter, and appears to have avoiding the need to tolerate large fluctua- evolved independently a number of times tions in moisture availability. Nevertheless, as an adaptation to extremes in water avail- in intermittently arid habitats some species ability. Some genera, such as Cheilanthes, have adapted to survive desiccation rather Pellaea, Selaginella and Xerophyta, have than avoid it. However, of the quarter of a large numbers of tolerant species while million or so species of vascular plants, others, such as Boea, have only a single only some 330 species, or < 0.15% of the species that is known to be tolerant. A Dessication 07 18/3/02 1:57 pm Page 218

218 M.C.F. Proctor and V.C. Pence

Table 7.1. Desiccation-tolerant (DT) vascular plants. With a few exceptions, names and authorities are those used by the cited authors. Species of a genus recorded in a single publication from the same geographical area are listed together; otherwise the arrangement is alphabetical within major taxonomic categories.

Species Country Reference

PTERIDOPHYTES LYCOPSIDA (Clubmosses) Isoetaceae Isoetes australis Williams Australia Gaff and Latz (1978) (Only the corms are DT; other terrestrial Isoetes spp. of seasonally desiccated habitats, e.g. in southern Europe, are likely to behave similarly) Selaginellaceae Selaginella caffrorum (Milde) Hieron., South Africa Gaff (1977) S. digitata Spring, S. dregei (C. Presl) Hieron., S. echinata Baker, S. imbricata (Forsk.) Spring ex Decaisne, S. nivea Alston Selaginella convoluta Spring, South AmericaGaff (1987) S. peruviana (Milde) Hieron., S. sellowii Hieron. Selaginella lepidophylla (Hook. and Southwestern USA, Hooker (1837), Gaff (1971), Eickmeier Grev.) Spring Mexico (1979), Iturriaga et al. (2000) Selaginella njam-njamensis Hieron. West Africa Hambler (1961) Selaginella pilifera A. Br. Southwestern US Eickmeier (1980) Selaginella sartorii Hieron. Mexico Iturriaga et al. (2000) PTEROPSIDA (Ferns) Adiantaceae (sensu lato) Actiniopteris dimorpha Pic. Serm. South Africa Gaff (1977) A. radiata (Sw.) Link South Africa, India Gaff (1977), Sharma and Purohit (1986) Adiantum incisum Forsk. South Africa, India Gaff (1977), Sharma and Purohit (1986) Cheilanthes albomarginata India Sharma and Purohit (1986) Cheilanthes bonariensis (Wild) Proctor, Mexico Iturriaga et al. (2000) C. integerrima (Hook.) Mickel., C. myriophylla Desv. Cheilanthes buchtienii (Rosenst.) South AmericaGaff (1987) Capurro, C. glauca (Cav.) Mett., C. marginata H.B.K. Cheilanthes farinosa (Forsk.) Klf. South Africa Gaff (1977), Sharma and Purohit (1986) Cheilanthes capensis (Thunb.) Desv., South Africa Gaff (1977) C. depauperata Bak., C. dinteri Brause, C. eckloniana (Kunze) Mett., C. hirta Sw., C. inaequalis (Kunze) Mett., C. marlothii (Hieron) Mett., C. multiﬁda (Sw.) Sw., C. parviloba Sw. Dessication 07 18/3/02 1:57 pm Page 219

Desiccation Tolerance of Vegetative Tissues 219

Table 7.1. Continued

Species Country Reference

Cheilanthes distans (R. Br.), Australia Gaff and Latz (1978) C. fragillima F. Muell., C. lasiophylla Pic.-Ser., C. paucijuga Benth., C. tenuifolia (Burm.f.) Sw., C. vellea (R. Br.) F. Muell. Cheilanthes lendigera Swartz Wittrock (1891) Cheilanthes pringlei Daven., Southwestern USA, Helvy (1963) C. wrightii Hooker Mexico Cheilanthes sieberi Kunze Australia, Gaff and Latz (1978), Gaff and South Africa McGregor (1979) Doryopteris concolor (Lag. and Risch.) South Africa Gaff (1971, 1977) Kahn Doryopteris kitchingii (Bak.) Bonap. South Africa Gaff (1977) Doryopteris pedata (L.) Fée, D. triphylla South America Gaff (1987) (Lam.) Christ Notholaena marantae R.Br. Europe Iljin (1931) Notholaena parryi D. C. Eat. North America Witham (1972), Nobel (1978) Notholaena R. Br. sp. South America Porembski and Barthlott (2000) Paraceterach muelleri (Hook.) Copel. Northeastern Gaff and Latz (1978) Australia Pellaea atropurpurea (L.) Link Mexico Pickett and Manuel (1926) Pellaea boivinii Hook., P. calomelanos South Africa Gaff (1977) (Sw.) Link, P. hastata (L.f.) Link, P. quadripinnata (Forsk.) Prantl., P. viridis (Forsk.) Prantl. Pellaea falcata (R. Br.) Fée Australia Gaff and Latz (1978) Pellaea glabella Mett. USA Pickett and Manuel (1926) Pellaea longimucronata Hooker Southwestern USA, Helvy (1963) Mexico Pellaea ovata (Desv.) Weatherby (commercial) Iturriaga et al. (2000) Pellaea rotundifolia (Forsk.) Hk. (RBG Kew: Gaff and Latz (1978) origin unknown) Pellaea sagittata (Cav.) Link f. var. Mexico Iturriaga et al. (2000) cordata (Cav.) Tyron. Pellaea ternifolia (Cav.) Link South America Gaff (1987) Aspleniaceae Asplenium aethiopicum (Burm. f.) South Africa Gaff (1977) Bech., A. rutifolium (Berg.) Kunze var. bipinnatum (Forsk.) Schelpe, A. sandersoni H. K. Asplenium bourgaei Mediterranean Greuter et al. (1983) Asplenium pringlei Davenp. Wittrock (1891) Continued Dessication 07 18/3/02 1:57 pm Page 220

220 M.C.F. Proctor and V.C. Pence

Table 7.1. Continued

Species Country Reference

Asplenium ruta-muraria L. Western Kappen (1964) Europe Asplenium septentrionale (L.) Hoffm. Europe Kappen (1964) Asplenium trichomanes L. Europe Wittrock (1891), Kappen (1964) Ceterach cordatum (Thunb.) Desv. South Africa Gaff (1977) Ceterach ofﬁcinarum Lam. et DC Southern and Oppenheimer and Halevy (1962), Western Europe, Schwab et al. (1989), M.C.F. Proctor Mediterranean (unpublished data) Pleurosorus rutifolius (R. Br.) Fée Western Australia Gaff and Latz (1978) Woodsia ilvensis (L.) R.Br. Europe Wittrock (1891) Davalliaceae Arthropteris orientalis (Gmel.) Porth. South Africa Gaff (1977) Grammitidaceae Ctenopteris heterophylla (Labill.) Tindale New Zealand Gaff and Latz (1978) Hymenophyllaceae Hymenophyllum tunbridgense (L.) Southwestern Smith, H. wilsonii Hook. England M.C.F. Proctor (unpublished data) Hymenophyllum sanguinolentum New Zealand J.G. Duckett and M.C. Proctor (Forst. F.) Swartz (unpublished data) Polypodiaceae Platycerium stemaria (P. Beauv.) Desv. West Africa Hambler (1961) Polypodium cambricum L. Southwestern M.C.F. Proctor (unpublished data) England Polypodium polypodioides (L.) North America Pessin (1924), Stuart (1968), Gaff Hitchcock (1977), Iturriaga et al. (2000) Polypodium virginianum L. North America Reynolds and Bewley (1993) Polypodium vulgare L. Wittrock (1891), Kappen (1964) Schizaeaceae Anemia tomentosa (Sav.) Swartz South America Gaff (1987) Mohria caffrorum (L.) Desv. South Africa Gaff (1971, 1977) Schizaea Sm. sp. East Africa, Porembski and Barthlott (2000) Seychelles ANGIOSPERMS MONOCOTYLEDONS Cyperaceae Afrotrilepis pilosa (Boeck.) J. Raynal West Africa Hambler (1961), Owoseye and Sanford (1972) Carex physodes M. Bieb Central Asia Vassiljev (1931) Dessication 07 18/3/02 1:57 pm Page 221

Desiccation Tolerance of Vegetative Tissues 221

Table 7.1. Continued

Species Country Reference

Coleochloa pallidior Nelmes South Africa Gaff and Ellis (1974) Coleochloa setifera (Ridley) Gilly South Africa Gaff (1971), Gaff and Ellis (1974) Cyperus bellis Kunth South Africa Gaff and Ellis (1974) Fimbristylis dichotoma (L.) Vahl Australia Gaff and Latz (1978) Fimbristylis Vahl Tropical Africa Porembski and Barthlott (2000) Kyllinga alba Nees South Africa Gaff and Ellis (1974) Mariscus capensis Schrad. South Africa Gaff and Ellis (1974) Microdracoides squamosa Hua West Africa Porembski and Barthlott (2000) (monotypic) Trilepis Nees South America Porembski and Barthlott (2000) Liliaceae (Anthericaceae) Borya inopinata Australia Forster and Thompson (1997) Borya nitida Labill. Australia Gaff and Churchill (1976) Borya septentrionalis F. Muell. Australia Gaff and Latz (1978) Poaceae Brachyachne patentiﬂora (Stent and South Africa Gaff and Ellis (1974) Rattray) C.E. Hubb. Eragrostiella bifaria (Vahl) Bor. Australia Gaff and Latz (1978) Eragrostiella brachyphylla (Stapf) Bor., India Gaff and Bole (1986) E. nardioides (Trin.) Bor. Eragrostis hispida K. Schum., South Africa Gaff and Ellis (1974) E. nindensis Fic. and Hiern, E. paradoxa Launert Eragrostis invalida Pilger West, East and Gaff (1986), Nugent and Gaff (1989) South Africa Micraira adamsii Australia Gaff (1989) Micraira spinifera Lazar, M. tenuis Lazar Australia Gaff and Sutaryono (1991) Micraira subulifolia F. Muell. Australia Gaff and Latz (1978) Microchloa caffra Nees, M. kunthii Desv. South Africa Gaff and Ellis (1974) Microchloa indica (L.f.) O. Kunze South America, Gaff (1987), Iturriaga et al. (2000) Mexico Oropetium capense Stapf South Africa Gaff (1971), Gaff and Ellis (1974) Oropetium roxburghianum (Steudel) India Gaff and Bole (1986) S. Phillips, O. thomaeum Trin. Poa bulbosa L. Europe Gaff and Latz (1978) Sporobolus atrovirens (Kunth) Kunth Mexico Iturriaga et al. (2000) Sporobolus elongatus R. Br. Australia Gaff and Sutaryono (1991) Sporobolus festivus Hochst. South Africa Gaff and Ellis (1974), Kaiser et al. (1985) Continued Dessication 07 18/3/02 1:57 pm Page 222

222 M.C.F. Proctor and V.C. Pence

Table 7.1. Continued

Species Country Reference

Sporobolus fimbriatus Australia Gaff and Ellis (1974) Sporobolus lampranthus Prig. South Africa Gaff and Ellis (1974) Sporobolus pellucidus Hochst. East Africa Gaff (1986), Nugent and Gaff (1989) Sporobolus stapfianus Gandoger South Africa Gaff and Ellis (1974), Kaiser et al. (1985), Sgherri et al. (1994) Tripogon capillatus Jaub. et Spach, India Gaff and Bole (1986) T. filiformis (Stapf) Nees ex Steud., T. jacquemontii Stapf, T. lisboae Stapf, T. polyanthus Naik. et Patunkar Tripogon curvatus Phillips and Launert Africa Gaff and Sutaryono (1991) Tripogon lolioformis (F. Muell.) Australia Gaff and Latz (1978) C. E. Hubbard Tripogon minimus (A. Rich.) Hochst. South Africa Gaff and Ellis (1974) ex Steud. Tripogon spicatus (Nees) Ekman South America, Gaff (1987), Iturriaga et al. (2000) Mexico Velloziaceae Aylthonia blackii (L.B.Smith) Menezes South America Gaff (1987) Barbacenia flava Martius ex Schultes f., South America Gaff (1987) B. longiflora Martius, B. riedeliana Goethart and Henrare, B. sellovii Goethart and Henrard Barbaceniopsis boliviensis (Baker) South AmericaGaff (1987) L.B. Smith, B. humahuaguensis Noher Nanuza plicata (Speng) L.B. Smith South America Rosetto and Dolder (1996) and Ayensu Pleurostima Raff. South America Porembski and Barthlott (2000) Vellozia Vand. (~ 124 spp., probably all South America Gaff (1987), Porembski and Barthlott desiccation-tolerant) (2000) Xerophyta Juss. (~ 28 spp., probably all Sub-Saharan Gaff (1971, 1977), Owoseye and desiccation-tolerant) Africa, Madagascar Sanford (1972); Gaff and Hallam (1974), Hallam and Gaff (1978), Tuba et al. (1993), Porembski and Barthlott (2000) DICOTYLEDONS Acanthaceae Talbotia elegans Balfour South Africa Gaff and Hallam (1974), Hallam and Gaff (1978) Cactaceae Blossfeldia liliputana South America Barthlott and Porembski (1996) Gesneriaceae Boea hygroscopica F. Muell. Australia Gaff and Latz (1978), Kaiser et al. (1985) Dessication 07 18/3/02 1:57 pm Page 223

Desiccation Tolerance of Vegetative Tissues 223

Table 7.1. Continued

Species Country Reference Haberlea rhodopensis Friv. Southeastern Bewley and Krochko (1982), Müller et Europe al. (1997) Ramonda myconi Reichb. Southwestern Gaff and McGregor (1979), Schwab et (= R. pyrenaica Rich.) Europe al. (1989) Ramonda nathaliae Panc. and Petrov. Southeastern Bewley and Krochko (1982), Müller et Europe al. (1997) Ramonda serbica Panc. Southeastern Markovska et al. (1994) Europe Streptocarpus Lindley spp. Africa Porembski and Barthlott (2000) Labiatae Satureja gilliesii (Benth.) Briq. South America Montenegro et al. (1979) Myrothamnaceae Myrothamnus ﬂabellifolia Welw. South Africa Thoday (1921), Child (1960), Gaff (1971, 1977) Myrothamnus moschata (Baillon) Madagascar Gaff (1977) Niedenzu Scrophulariaceae Chamaegigas intrepidus Dinter ex Heil South Africa Gaff (1971, 1977) Craterostigma monroi S. Moore, South Africa Gaff (1977) C. nanum Engl. Craterostigma plantagineum Hochst. South Africa Gaff (1971, 1977), Schwab et al. (1989) Craterostigma wilmsii Engl. South Africa Gaff (1971, 1977) Ilysanthes purpurascens Hutch., South Africa Gaff (1977) I. wilmsii Engl. and Diels Limosella L. South Africa Porembski and Barthlott (2000) Lindernia All. spp. Tropical Africa Porembski et al. (1997)

large number of desiccation-tolerant plants, and early studies centred on mea- species have been documented from cen- suring the rates of drying and the loss of tral and southern Africa, Australia and moisture in leaves of plants observed in the South America (Gaff, 1971, 1977, 1986, field or laboratory (Child, 1960; Hambler, 1987; Gaff and Ellis, 1974; Gaff and 1961; Hornby and Hornby, 1964). Hallam, 1974; Gaff and Churchill, 1976; Regreening of species that lose chlorophyll Gaff and Latz, 1978; Gaff and Giess, 1986). during desiccation, as well as the use of Although fewer have been described from vital stains, was soon added to confirm tis- Europe, Asia and North America, new des- sue viability after rehydrating (Gaff and iccation-tolerant species continue to be Okon’O-Ogola, 1971; Gaff and Ellis, 1974; found as dry habitats are further explored Gaff, 1977; Hallam and Gaff, 1978), and (Gaff and Bole, 1986; Iturriaga et al., 2000). early measurements of respiration and pho- Because these plants exhibit such a dra- tosynthesis progressed from simple gas matic change in morphology during desic- exchange to IRGA (Stuart, 1968; Vieweg cation and rehydration, revival of normal and Ziegler, 1969; Eickmeier, 1979). Solute appearance after apparent death was, at leakage from desiccated tissue was also first, sufficient to indicate survival of used as a measure of damage (Leopold et Dessication 07 18/3/02 1:57 pm Page 224

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al., 1982). By the 1980s, research exploring promoting soil development and the estab- the relationship of metabolism to the desic- lishment of other species (Child, 1960). cation phenomenon was well under way, In addition to physiological desiccation with a few species being notable models tolerance, some species share water- around which much of this research cen- absorbing or retaining characteristics with tred (e.g. S. lepidophylla, C. plantagineum, desiccation-intolerant species. Xeromorphic M. flabellifolia, Chamaegigas intrepidus, characteristics in the liliaceous desiccation- Polypodium virginianum, Sporobolus stap- tolerant plant Borya nitida and the grass S. fianus, Boea hygroscopica). This has natu- stapfianus help reduce water loss, with the rally led to molecular studies, particularly latter retaining almost 80% RWC after a with Craterostigma plantagineum (see week of desiccation (Gaff and Churchill, reviews by Hartung et al., 1998; Scott, 1976; Vecchia et al., 1998). In other 2000), and these lines of research are exam- species, specialized structures aid in ined in detail in Chapter 12. absorbing or dispersing water. Scales on the underside of the leaf of P. polypodioides appear to function in distributing 7.3.1. Ecological and morphological water over the surface, thereby aiding in adaptations absorption (Pessin, 1924; Stuart, 1968), while dead leaf bases around the stem of A. Resurrection species are often small, low- pilosa help retain water and slow the rate growing plants with short internodes and of drying (Hambler, 1961). Velamen on the compact growth that are found as pioneers roots of some species (Afrotrilepis pilosa, in shallow soils or on rocky outcroppings, Coleochloa setifera, Xerophyta pinnifolia) areas that can experience extreme variations allows for rapid absorption when water is in moisture availability. When the rains do available (Porembski and Barthlott, 1995). come, these plants must be able to rehy- Other resurrection plants, such as drate, photosynthesize and grow before dry- Craterostigma plantagineum, C. wilmsii ing occurs again. At some point, they must and C. nanum, possess few morphological also remain active for a period long enough characteristics which help to avoid or slow to reproduce. Some species are adapted for desiccation, and it is not surprising that seasonal changes in water availability. these show a greater physiological toler- Others dry and are rewetted at more irregu- ance for rapid desiccation than do more lar intervals. Chamaegigas intrepidus grows xeromorphic species (Sherwin, 1995; in southern Africa in situations where water Farrant et al., 1999). It should also be noted collects into ephemeral pools. This aquatic that some desiccation-tolerant species are plant may be subjected to as many as 20 able to grow under conditions of extreme desiccation/rehydration cycles annually in water deficits as well as in areas where addition to surviving through at least 8 soils are richer and water more plentiful, months of dry dormancy (Schiller et al., and that morphologies, such as leaf shape, 1998, 1999). The ability to survive rapid and may differ in the different environments frequent desiccation and to quickly re-estab- (Hornby and Hornby, 1964). lish normal function is a necessity under Although they are adapted to extreme such conditions. desiccation, often in hot, dry environ- As pioneers, resurrection species may ments, not all resurrection plants are toler- be restricted to areas that are uninhabitable ant of either high light levels or high by other vascular species. The sedge temperatures. Many resurrection species Afrotrilepis pilosa forms monospecific do tolerate full sun, but others, especially stands, which persist without encroach- pteridophytes, typically require a shaded ment by less well-adapted species habitat and may be damaged by excessive (Porembski et al., 1996). Plants such as sunlight (Gaff, 1977; Lebkuecher and Myrothamnus flabellifolia, however, pro- Eickmeier, 1991). Similarly, some resurrec- vide shelter and trap organic materials, tion species, again often pteridophytes, fre- Dessication 07 18/3/02 1:57 pm Page 225

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quently occur in thermally buffered micro- Child, 1960; Oppenheimer and Halevy, habitats, such as in rocky crevices or out- 1962; Kappen, 1964; Stuart, 1968; Gaff and croppings. This environment provides Churchill, 1976; Reynolds and Bewley, some shelter from direct sunlight, reducing 1993). While some research on the limits of daytime temperatures while holding heat desiccation tolerance has been done during the cooler nights and winter days. directly on field-dried tissues, in the labo- The optimal temperature for photosynthe- ratory tissues have been air-dried, equili- sis in the desert fern, Notholaena parryi, brated over solutions producing known has been measured as several degrees RHs or dried over desiccating agents, such cooler than the mean air temperature of its as sulphuric acid or silica gel, for more general environment, suggesting a modify- rapid drying. The humidity over silica gel ing effect of its microhabitat (Nobel, 1978). and other strong drying agents is often Moderating the extremes of temperature reported as ‘0% RH’, but this limit (corre- and light can also be important for surviv- sponding to a water potential of ∞) is ing desiccation, since desiccation damage unattainable in practice. can be enhanced by photodamage and high Levels of desiccation tolerance differ temperatures (Eickmeier, 1986; Muslin and among species. Gaff (1977) examined 37 Homann, 1992; Chapter 9). desiccation-tolerant ferns and angiosperms Although research on desiccation toler- for their RH tolerance levels and found ance in vascular species has focused pri- that only 30% of the pteridophytes could marily on the sporophyte, gametophytes of survive equilibration with an RH close to some pteridophytes also display desicca- 0%, while 76% of the angiosperm taxa did tion tolerance. The extent of this phenome- so, reflecting the tendency of pterido- non has not been well documented, but phytes to inhabit somewhat protected several reports indicate that it may be a areas. The least tolerant resurrection fern, survival mechanism in at least some pteri- however, was still able to survive equili- dophyte species (Mottier, 1914; Kappen, bration with 30% RH, significantly lower 1965; Page, 1979; Quirk and Chambers, than the RH tolerated by non-resurrection 1981). Some success has been achieved in plants. The RWCs of most dry resurrection recovering in vitro-grown fern gameto- plants are in the range of 5–10% or less phytes of Adiantum tenerum, Adiantum (Scott, 2000). trapeziforme, Cibotum glaucum, Davallia As with dry seeds, vegetative tissues fejeensis and Drymaria quercifolia after that are sufficiently dry may remain viable exposure to liquid nitrogen when the for relatively long periods of time, gametophytes were first air-dried, indicat- although moisture levels, temperature and ing that the tissues were dry enough to species differences will affect longevity. avoid freezing injury (Pence, 2000). Dried Afrotrilepis pilosa remained viable for a year at room temperature, and also survived storage overnight at 10°C, 7.3.2. The effect of intensity of desiccation whereas undried tissues did not survive freezing (Hambler, 1961). Dry tissues of The vegetative tissues of most vascular seven vascular species survived for at least plants can survive equilibration with RHs 3.5 years as field-dried leaves when they only in the range of 85–98%; the Namibian were sealed in plastic or air-tight glass Welwitschia mirabilis will stand drying (Gaff, 1977), and two of these species, only to a RWC of 56% (Gaff, 1972). Truly Xerophyta squarrosa and C. setifera, desiccation-tolerant species, however, can showed 100% survival even after 5 years. equilibrate with much lower RHs, often As with seeds, lower temperatures might < 10%, and still recover on remoistening. be expected to prolong viability of dry tis- Drying tissues may lose from 70 to 95% of sues, and such tissues should be good can- their original water content, generally over didates for low-temperature storage or a period of several days (Pessin, 1924; cryopreservation. Dessication 07 18/3/02 1:57 pm Page 226

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7.3.3. Effect of rate of desiccation 7.3.4. Morphological and cytological changes that occur with drying In contrast to bryophytes, vascular plants are buffered to varying degrees from ambi- Drying of resurrection plants brings about ent moisture levels by their larger size, the various morphological changes. These presence of a cuticle and stomata, and result from the loss of water at the cellular their ability to access underground mois- level, but provide protection at the whole ture. In addition, desiccation-tolerant plant level. The curling of leaves to form species with xeromorphic characteristics long, threadlike structures and the curling can slow water loss even further. As a of older leaves and stems over younger result, the desiccation-tolerance mecha- leaves and buds slow the rate of drying in nisms in desiccation-tolerant vascular taxa younger, growing tissues, as well as pro- appear to be, in large part, inducible rather tecting inner dried tissues from photodam- than constitutive, as in many bryophytes, age (Pessin, 1924; Child, 1960; Gaff and with tolerance developing over the course Churchill, 1976; Gaff, 1977). Younger leaf of 12–24 h. tissue, in general, appears more tolerant of Natural drying of resurrection species in desiccation (Gaff and Ellis, 1974; Norwood the field generally occurs over a period of et al., 1999). days or even weeks. In Craterostigma plan- Changes in shape at the whole plant tagineum signs of desiccation were visible level are accompanied by a dramatic reduc- within 1 week of the last rain (Hornby and tion in size, loss of turgor and a decrease in Hornby, 1964). Gaff (1977) observed 11 cell volume. Some resurrection species may species in the field in southern Africa for shrink to less than 20% of their original leaf the first signs of water stress in the leaves area upon drying. These species are able to and noted that, for ten of the species, dry- maintain connections between the plasma ing times ranged from 40 to 96 h. The membrane and the cell wall and to undergo exception was Chamaegigas intrepidus, a controlled and extensive folding of the cell small, aquatic plant of ephemeral rock wall during drying, allowing the collapse of pools, which was air-dry within an hour. the tissue without the fatal results seen in Critical evaluation of the maximum tol- non-tolerant species (Hartung et al. 1998; erated rates of drying support the observa- Vicre et al., 1999). tion that natural drying of resurrection Damage from light has been observed in species is generally slow. A number of S. lepidophylla and P. polypodioides when studies have shown that these species do curling of the leaves was manually inhib- not survive rapid desiccation, although ited during illuminated drying (Muslin and slower rates of drying will allow survival Homann, 1992; Lebkuecher and Eickmeier, to very low levels of moisture. For exam- 1993), and it is thought that a number of ple, leaves of Borya nitida which were morphological and physiological processes air-dried could not survive below equili- associated with desiccation are adaptations bration with 85% RH or less. If, however, to minimize damage from light in the dry the leaves were first exposed to 96–98% tissues. In several species, hairs or other RH for 2 days, they were then capable of substances help reflect light from the abax- surviving close to 0% RH (Gaff and ial leaf surfaces, which are exposed during Churchill, 1976). Studies with Selaginella desiccation-induced curling (Nobel, 1978; lepidophylla, Boea hygroscopica and other Sherwin and Farrant, 1998), as well as species have demonstrated similar from adaxial surfaces in species with responses (Eickmeier, 1983; Sgherri et al., leaves that do not curl (Vecchia et al., 1994). Slow drying can be replaced by 1998). Pigments in many desiccation- ABA in several systems, suggesting that it tolerant plants also help to reduce photo- is closely involved with the induction of damage, and colour changes in dried leaves protective mechanisms in resurrection have been reported for a number of species. species (Reynolds and Bewley, 1993). In C. wilmsii, Xerophyta viscosa and Dessication 07 18/3/02 1:57 pm Page 227

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Myrothamnus flabellifolia, anthocyanin remain intact, although the vacuole may levels increased significantly in dried tis- fragment into many small vacuoles. These sues, helping to mask chlorophyll and species are generally adapted to areas that reduce damage by free radicals (Sherwin experience fewer fluctuations in water and Farrant, 1998; Koonjul et al., 2000). availability and thus do not require a rapid Photoprotection by mechanisms associated recovery response. with the production of zeaxanthin has also An inherent problem in cytological stud- been demonstrated (Casper et al., 1993; ies of these desiccated tissues has been that Eickmeier et al., 1993). aqueous fixatives can initiate changes asso- Changes in colour are accentuated in ciated with rehydration in desiccated tis- some species by the loss of chlorophyll sues. Fixatives with high osmolality have during desiccation. Resurrection plants can been effective in some studies (Platt et al., be classified as either poikilochlorophyl- 1998), but the use of freeze-substitution lous desiccation tolerants (PDTs), which techniques has proved most efficient in lose chlorophyll during drying, or maintaining the structure of desiccated homoiochlorophyllous desiccation toler- cells for electron microscopy (Thomson and ants (HDTs), which retain chlorophyll Platt, 1997; Platt et al., 1998). throughout desiccation. Gaff and Hallam (1974) reported that most resurrection pteridophytes and dicots were HDTs, while 7.3.5. Rehydration and recovery about half of the monocots surveyed were PDTs. PDTs avoid photodamage to their The ‘resurrection’ of desiccation-tolerant photosynthetic apparatus by dismantling vascular species centres on the events of it, a trade-off against the greater time rehydration and recovery from apparent needed for the re-establishment of photo- lifelessness. Very little rehydration occurs synthetic activity. Both HDT and PDT from dew, but rains of 10 mm or greater species can withstand severe desiccation generally stimulate rehydration (Gaff, (Gaff, 1989), but HDTs are fully functional 1977). Rapid water uptake may occur within about 24 h of exposure to water, through the leaves, and in experimental while PDTs may take 48–72 h or longer to systems this is often the method used for regreen and re-establish photosynthetic rehydrating desiccated tissues. C. plan- apparatus and function (Gaff and Ellis, tagineum can recover 85% RWC within 1974; Tuba et al., 1993; and see below). 5–6 h when submerged or substantially Although the cells shrink during dehydra- indundated, and desiccation-tolerant tion and the cell walls and membranes grasses regain most of their shape within become convoluted, most of the organiza- 4–5 h of rehydration (Gaff and Ellis, 1974), tion of the chloroplasts and other In nature, however, water uptake will also organelles is maintained in HDT species occur through the roots, and larger species, (Platt et al., 1994; Thomson and Platt, such as M. flabellifolia, which rely primarily 1997). on rehydration from the roots, will take In contrast, desiccation in PDT species longer to rehydrate and recover than species leads to the degeneration of chloroplast taking up water primarily through the membranes and the loss of the grana, leaves. During desiccation, the flow of stroma and thylakoid structure, and the water through the vascular system is dis- unprotected chlorophyll suffers photode- rupted, leaving gaps of air. Upon rehydra- struction (Owoseye and Sanford, 1972; Gaff tion, the xylem must be refilled by et al., 1976; Hallam and Gaff, 1978; Bartley capillary action and/or root pressure in and Hallam, 1980; Bergstrom et al., 1982; order to resume functioning (Sherwin et Tuba et al., 1993; Vecchia et al., 1998). In al., 1998). these species, there is also loss of inner Several factors can influence the rate mitochondrial membranes and cristae. and type of recovery. Species of Selaginella Nuclear, plastid and tonoplast membranes from drier habitats resume photosynthesis Dessication 07 18/3/02 1:57 pm Page 228

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more quickly than those from moister habi- flowers (as in Ranunculus ficaria, tats (Eickmeier, 1980), suggesting a natural Cardamine spp., Saxifraga spp. and Allium adaptation to the less frequent availability spp. (Richens, 1947)). There are also vari- of water in the drier areas. There may also ous kinds of stem and root tubers, and be developmental differences in desicca- viable shoot fragments, which establish as tion tolerance. Recovery is limited to independent plants. Desiccation tolerance young growing tissues in a number of has been noted in tubers of Anemone coro- species, suggesting a greater desiccation naria and Ranunculus asiaticus (Antipov tolerance than in older tissues (Gaff and and Romanyak, 1983). Some other tubers Churchill, 1976; Sherwin and Farrant, and at least some bulbs, bulbils and similar 1996). When C. plantagineum was rehy- structures are likely to be desiccation-toler- drated through the roots, rather than ant too, but there seems to have been little leaves, water uptake was slower and many systematic work to determine which of the of the older leaves did not recover com- more persistent of these are truly desicca- pletely (Bernacchia et al., 1996). tion-tolerant and which are simply highly The time needed for the recovery of resistant to water loss. function will depend on how much of the photosynthetic apparatus was dismantled during the drying process. Photosynthesis 7.4. Concluding Comments is re-established in HDT plants within a few hours to a day after rewatering Various facets of the desiccation responses (Eickmeier, 1979; Bernacchia et al., 1996). of vegetative tissues have parallels in seed HDT species generally retain a portion of biology, though these should be seen as the thylakoid system, as well as chloro- illustrative and a prompt to thought rather phyll, during desiccation, and, within a than as necessarily implying close physio- day of rehydration, functioning grana and logical correspondence. The shutting-down lamellae are re-formed. In contrast, PDT of metabolism on drying and recovery on species lose membrane structure as well as remoistening after moderate periods of des- chlorophyll when dried, and these must all iccation suggest parallels in the maturation be re-formed when water is available and then in the imbibition and germination (Hallam and Gaff, 1978; Markovska et al., of seeds. The gradual loss of viability on 1995; Sherwin and Farrant, 1996). prolonged desiccation and the relation of Chlorophyll synthesis begins within a few this to intensity of desiccation and temper- hours of water availability, but it can take ature have obvious (even if not complete) up to several days to re-establish function parallels in the well-researched field of (Tuba et al., 1993; Drazic et al., 1999). PDT seed storage. Fundamental prerequisites for species are generally adapted to seasonal desiccation tolerance are maintenance (or changes in water availability, while HDT rapid recovery) of membrane integrity, species may experience more frequent fluc- preservation of macromolecules in a func- tuations in moisture. tional state and maintenance of spatial relationships between functional components of the cell. Protective substances 7.3.6. Vegetative propagules: bulbils, corms, such as sucrose and dehydration proteins tubers and plant fragments probably combine to allow vitrification of the cell contents on drying (Crowe et al., Many normal homoiohydric vascular 1998; Buitink, 2000; Chapter 10) to provide plants produce vegetative propagules, a living (and reversible) equivalent of good which often serve to carry the plant fine-structural fixation for electron through unfavourable dry periods. These microscopy. Some other factors, such as include bulbils formed either in the soil (as enhanced activity of anti-oxidant systems in many Oxalis species (Robb, 1963)) or in (Dhindsa and Matowe, 1981; Seel et al., leaf axils above ground, often replacing 1991, 1992a; Smirnoff, 1993), may be seen Dessication 07 18/3/02 1:57 pm Page 229

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as extensions of processes common to all potentials (Gaff, 1980), and there are indica- living cells (Foyer et al., 1994; Alscher et tions that molecular mobility and rates of al., 1997). ageing generally decrease with falling water This underlines the distinction between content, but increase again at water contents effects related to ‘pure’ desiccation toler- in equilibrium with air at less than c. 10% ance, characteristic of (and inseparable RH (Buitink et al., 2000). The results in Fig. from) a drying and rewetting event, and the 7.3 are broadly consistent with the conclu- cumulative damaging effects of longer-term sion that ‘desiccation-tolerant tissues are desiccation. The effects of cumulative desic- often least damaged at values of of about cation damage are certainly diverse and 20 to 40% RH or 1300 to 2200 bars’ complex (see, for example, Chapter 9). Gaff (130 to 220 MPa) (Gaff, 1980). (1980) distinguished a number of possible Bewley (1979) introduced the concept of sources of injury operating at different rates, ‘repair’ in the recovery of bryophytes from of which the slower types may be either desiccation, primarily in relation to the stress-parallel or stress-inverse. Injury at restoration of membrane integrity, and he high water potentials may arise from contin- and Oliver (Oliver and Bewley, 1984, 1997; ued activity of metabolic processes. In Bewley and Oliver, 1992; Oliver et al., osmotic stress experiments, measurable res- 2000) have extended the concept and asso- piration and photosynthesis were found ciated it particularly with the recovery down to c. 10 MPa in Anomodan viticulo- processes in bryophytes. However, as they sus, and between 10 and 20 MPa in point out, ‘repair’ must be an element in Homalothecium lutescens (Dilks and the recovery of all desiccation-tolerant Proctor, 1979); and photosynthetic activity plants, most of all in the poikilochloro- has been detected in the lichen phyllous vascular species. The broad con- Dendrographa minor down to almost 40 cept of ‘repair’ needs to be considered MPa (Lange, 1988). In various lichens, it analytically and quantitatively in terms of was found that incorporation of tritium into the wide range of processes that it must sugar alcohols, amino acids and some TCA- embrace. Some systems, such as protein- cycle intermediates was taking place rather synthesis mechanisms and the photosys- freely at 40 MPa, and still detectable at tems, survive a drying–rewetting event 100 MPa (Cowan et al., 1979). Many essentially intact and are functional within bryophytes, especially those of woodland seconds or minutes of remoistening, but habitats, are remarkably tolerant of being experimental evidence shows that recovery kept at or near full turgor in the dark for of some other systems must be slower, and periods of weeks, but under these condi- full return of cell function to a steady state tions Tortula ruralis dies within a few days. may typically take hours or days. As Fig. 7.2 shows, given a low level of light, Underlying processes of recovery span a it survives well at 3 MPa, but much less similar range of time scales. Reinstatement well at water potentials between 9 and of water into macromolecules and re-estab- 37 MPa. As has been noted for resurrec- lishment of normal membrane integrity are tion vascular plants, slow growth of likely to be primarily physical, and fast, pathogens, especially fungi, may be a com- taking place within seconds or minutes. mon cause of deterioration in this range Other processes are slower, such as re- (Gaff, 1997). At lower water potentials, other establishment of normal water relations in factors are likely to be important. High-light vascular ‘resurrection plants’ and synthesis damage to dry thalli of the forest lichen of the rehydrins (which themselves evi- Lobaria pulmonaria has been demonstrated dently represent a diversity of processes (Gauslaa and Solhaug, 1999), and photon proceeding at varied rates (see Chapter damage is likely to be signiﬁcant under ﬁeld 12)). There are important aspects of recov- conditions for other desiccation-tolerant ery of cell function after desiccation, such organisms too. Very slow stress-parallel as reinitiation of the cell cycle, of which effects may be envisaged at very low water we know very little (Paolillo, 1984). Dessication 07 18/3/02 1:57 pm Page 230

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What determines the success of desicca- carbon ﬁxation, though still fast, is measur- tion-tolerant plants in some habitats and ably slower. For species of shady habitats, their exclusion from others? In broad such as R. loreus, Mnium hornum or terms, desiccation-tolerant plants are char- Polytrichum formosum, recovery is slower acteristic of situations where availability of again. Homoiochlorophyllous desiccation- water is strongly intermittent, and either tolerant vascular plants are especially char- the physical nature of the site or climate acteristic of situations where a thin soil makes a continuous closed cover of vascu- cover allows normal vascular-plant water lar plants impossible. However, there is a relations to function for much of the year, wide range of possibilities within these but the soil is desiccated for more or less constraints. Lichens on exposed rock faces extended periods. The ‘high-inertia’ such as Rhizocarpon geographicum (Ried, extreme is represented by poikilochloro- 1960a,b) or the crustose and endolithic phyllous desiccation-tolerant plants such lichens in the Negev Desert studied by as the Xerophyta species of central and Lange et al. (1970) represent a ‘low-inertia’ southern Africa, which, essentially, are extreme (Tuba et al., 1998). Species with adapted to the switch between a wet and a this pattern of adaptation extend from the dry season. Tropical inselbergs provide the wettest to quite arid climates. Their water habitats for a large proportion of all desic- content tracks closely the incidence of pre- cation-tolerant vascular plants (Porembski cipitation – rain, dew or impacted mist and Barthlott, 2000). droplets – from the atmosphere, and they By contrast with all desiccation-tolerant dry quickly when precipitation ceases. plants, drought-tolerant plants require con- Survival in such situations demands rapid tinued access to soil water at a physiologi- return to a positive carbon balance on cally tolerable water potential. Vascular remoistening after desiccation. Some therophytes, such as desert ephemerals and mosses such as Tortula ruralis respond to temperate winter annuals, are drought changing hydration almost equally quickly. evaders, and succulents are drought For larger lichens and many bryophytes the avoiders. Both groups commonly grow in time scale is longer. Cushions of the com- the same habitats as desiccation-tolerant mon wall-top moss Grimmia pulvinata species, which arguably (Proctor, 2000) store substantial amounts of water follow- may be seen as drought evaders no less ing rain and may take a number of hours to than the therophytes, substituting desicca- dry out (Proctor, 1990; Proctor and Smith, tion-tolerant vegetative tissues for desicca- 1995; Zotz et al., 2000), and recovery of tion-tolerant seeds.

7.5 References

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Proctor, M.C.F. and Smith, A.J.E. (1995) Ecological and systematic implications of branching patterns in bryophytes. In: Hoch, P.C. and Stephenson, A.G. (eds) Experimental and Molecular Approaches to Plant Biosystematics. Missouri Botanical Garden, St Louis, pp. 87–110. Quirk, H. and Chambers, T.C. (1981) Drought tolerance in Cheilanthes with special reference to the gametophyte. Fern Gazette 12, 121–129. Raven, J.A. (1977) The evolution of vascular land plants in relation to supracellular transport processes. Advances in Botanical Research 5, 153–219. Reynolds, T.L. and Bewley, J.D. (1993) Abscisic acid enhances the ability of the desiccation-tolerant fern, Polypodium virginianum to withstand drying. Journal of Experimental Botany 44, 1771–1779. Richards, P.W. (1984) The ecology of tropical forest bryophytes. In: Schuster R.M. (ed.) New Manual of Bryology, Vol. 2. Hattori Botanical Laboratory, Nichinan, pp. 1233–1270. Richens, R.H. (1947) Biological Flora of the British Isles. Allium vineale L. Journal of Ecology 34, 209–226. Ried, A. (1960a) Stoffwechsel und Verbreitungsgrenzen von Flechten. II. Wasser- und Assimilationshaushalt, Entquellungs- und Submersionsresistenz von Krustenflechten benach- barter Standorte. Flora 149, 345–385. Ried, A. (1960b) Nachwirkungen der Entquellung auf den Gaswechsel von Krustenflecthen. Biologisches Zentralblatt 79, 657–678. Robb, S.M. (1963) Oxalis latifolia Kunth. New Phytologist 62, 75–79. Roberts, E.H. (1975) Problems of long-term storage of seeds and pollen for genetic resource conservation. In: Frankel, O.H. and Hawkes, J.G. (eds) Crop Genetic Resources for Today and Tomorrow. Cambridge University Press, Cambridge, UK, pp. 269–296. Rosetto, E.S. and Dolder, H. (1996) Comparison between hydrated and desiccated leaves of a Brazilian resurrection plant. A light and scanning electron microscopic approach. Revista Brasileira de Biologia 56, 553–560. Schiller, P., Heilmeier, H. and Hartung, W. (1998) Uptake of amino acids by the aquatic resurrection plant Chamaegigas intrepidus and its implication for N nutrition. Oecologia 117, 63–69. Schiller, P., Wolf, R. and Hartung, W. (1999) Intracellular pH stability in the aquatic resurrection plant Chamaegigas intrepidus in the extreme environmental conditions that characterize its natural habitat. New Phytologist 140, 1–7. Schnepf, E. and Reinhard, C. (1997) Brachycytes in Funaria protonemata, induction by abscisic acid and fine structure. Journal of Plant Physiology 151, 166–175. Schonbeck, M.W. and Bewley, J.D. (1981a) Responses of the moss Tortula ruralis to desiccation treatments. I. Effects of minimum water content and rates of dehydration and rehydration. Canadian Journal of Botany 59, 2698–2706. Schonbeck, M.W. and Bewley, J.D. (1981b) Responses of the moss Tortula ruralis to desiccation treatments. II. Variations in desiccation tolerance. Canadian Journal of Botany 59, 2707–2712. Schwab, K.B., Schreiber, U. and Heber, U. (1989) Response of photosynthesis and respiration of resurrection plants to desiccation and rehydration. Planta 177, 217–227. Schwabe, W. and Nachmony-Bascomb, S. (1963) Growth and dormancy in Lunularia cruciata (L.) Dum. II. The response to daylength and temperature. Journal of Experimental Botany 14, 353–378. Scott, P. (2000) Resurrection plants and the secrets of eternal leaf. Annals of Botany 85, 159–166. Seel, W., Hendry, G., Atherton, N. and Lee, J. (1991) Radical formation and accumulation in vivo, in desiccation tolerant and intolerant mosses. Free Radical Research Communications 15, 133–141. Seel, W., Hendry, G.A.F. and Lee, J.A. (1992a) Effects of desiccation on some activated oxygen processing enzymes and anti-oxidants in mosses. Journal of Experimental Botany 43, 1031–1037. Seel, W.E., Baker, N.R. and Lee, J.A. (1992b) Analysis of the decrease in photosynthesis on desiccation of mosses from xeric and hydric environments. Physiologia Plantarum 86, 451–458. Sˇesták, Z., Cˇatsky´, J. and Jarvis, P.G. (1971) Plant Photosynthetic Production, Manual of Methods. Dr W. Junk, The Hague, The Netherlands. Sgherri, C.L.M., Loggini, B., Bochicchio, A. and Navari-Izzo, F. (1994) Antioxidant system in Boea hygroscopica, changes in response to desiccation and rehydration. Phytochemistry 37, 377–381. Sharma, B.D. and Purohit, S.N. (1986) Ferns and fern allies of Rajasthan, India. Leaf water deficit in relation to resurrection habit. Indian Fern Journal 2, 57–61. Sherwin, H.W. (1995) Desiccation tolerance and sensitivity of vegetative plant tissue. PhD thesis. University of Natal, Durban, South Africa. Dessication 07 18/3/02 1:57 pm Page 237

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Sherwin, H.W. and Farrant, J.M. (1996) Differences in rehydration of three desiccation-tolerant angiosperm species. Annals of Botany 78, 703–710. Sherwin, H.W. and Farrant, J.M. (1998) Protection mechanisms against excess light in the resurrection plants Craterostigma wilmsii and Xerophyta viscosa. Plant Growth Regulation 24, 203–210. Sherwin, H.W., Pammenter, N.W., February, E., Willigen, C.V. and Farrant, J.M. (1998) Xylem hydraulic characteristics, water relations and wood anatomy of the resurrection plant Myrothamnus flabellifolius Welw. Annals of Botany 81, 567–575. Smirnoff, N. (1993) The role of active oxygen in the response of plants to water deficit and desiccation. New Phytologist 125, 27–58. Stewart, G.R. (1989) Desiccation injury, anhydrobiosis and survival. In: Jones, H.G., Flowers, T.J. and Jones, M.B. (eds) Plants Under Stress. SEB Seminar Series 39, Cambridge University Press, Cambridge, pp. 115–130. Stuart, T.S. (1968) Revival of respiration and photosynthesis in dried leaves of Polypodium polypodioides. Planta 83, 185–206. Thoday, D. (1921) On the behaviour during drought of the leaves of two Cape Species of Passerina with some notes on their anatomy. Annals of Botany 35, 587–601. Thomson, W.W. and Platt, K.A. (1997) Conservation of cell order in desiccated mesophyll of Selaginella lepidophylla ([Hook and Grev.] Spring). Annals of Botany 79, 439–447. Tuba, Z., Lichtenthaler, H.K., Csintalan, Z. and Pócs, T. (1993) Regreening of desiccated leaves of the poikilochlorophyllous Xerophyta scabrida upon rehydration. Journal of Plant Physiology 142, 103–108. Tuba, Z., Csintalan, Z. and Proctor, M.C.F. (1996) Photosynthetic responses of a moss, Tortula ruralis ssp. ruralis, and the lichens Cladonia convoluta and C. furcata to water deficit and short periods

of desiccation, a baseline study at present-day CO2 concentration. New Phytologist 133, 353–361. Tuba, Z., Proctor, M.C.F. and Csintalan, Z. (1998) Ecophysiological responses of homoiochlorophyllous and poikilochlorophyllous desiccation-tolerant plants, a comparison and an ecological perspective. Plant Growth Regulation 24, 211–217. Vassiljev, J.M. (1931) Über den Wasserhaushalt von Pflanzen der Sandwüste im Sudöstlichen Kara- Kum. Planta 14, 225–309. Vecchia, F.D., El Asmar, T., Calamassi, R., Rascio, N. and Vazzana, C. (1998) Morphological and ultrastructural aspects of dehydration and rehydration in leaves of Sporobolus stapfianus. Plant Growth Regulation 24, 219–228. Vicre, M., Sherwin, H.W., Driouich, A., Jaffer, M.A. and Farrant, J.M. (1999) Cell wall characteristics and structure of hydrated and dry leaves of the resurrection plant Craterostigma wilmsii, a microscopical study. Journal of Plant Physiology 155, 719–726. Vieweg, G.H. and Ziegler, H. (1969) Zur Physiologie von Myrothamnus flabellifolia. Berichte der Deutschen Botanischen Gesellschaft 82, 29–36. Werner, O., Espin, R.M.R., Bopp, M. and Atzorn, R. (1991) Abscisic acid-induced drought tolerance in Funaria hygrometrica Hedw. Planta 186, 99–103. Witham, H.V. (1972) Ferns of the Colorado desert. California Native Plant Society Newsletter 8, 10–13. Wittrock, V.B. (1891) Biologiska ormbunkstudier. Acta Horti Bergiani 1, 2–63. Wood, A.J., Duff, R.J. and Oliver, M.J. (1999) Expressed sequence tags (ESTs) from desiccated Tortula ruralis identify a large number of novel plant genes. Plant Cell Physiology 40, 361–368. Zotz, G., Schweikert, A., Jetz, W. and Westerman, H. (2000) Water relations and carbon gain in relation to cushion size in the moss Grimmia pulvinata (Hedw.) Sm. New Phytologist 148, 59–67. Dessication 07 18/3/02 1:57 pm Page 238 Dessication 08 18/3/02 1:57 pm Page 239

8 Systematic and Evolutionary Aspects of Desiccation Tolerance in Seeds

John B. Dickie and Hugh W. Pritchard Seed Conservation Department, Royal Botanic Gardens Kew, Wakehurst Place, Ardingly, West Sussex RH17 6TN, UK

8.1. Introduction 239 8.1.1. Reviews and compilations of seed desiccation tolerance 240 8.1.2. Classiﬁcation of seed storage responses 241 8.1.3. The phylogenetic classiﬁcation of plants 242 8.1.4. Evolution of desiccation tolerance in land plants 243 8.2. Systematics and Evolution of Seed Desiccation Tolerance 244 8.2.1. The complete dataset 244 8.2.2. Gymnosperms 244 8.2.2.1. Araucariaceae 246 8.2.3. Angiosperms 247 8.2.3.1. Fagaceae 249 8.3. Seed Desiccation Tolerance and Ecology sensu lato 250 8.3.1. Are recalcitrant seeds bigger than orthodox seeds? 250 8.3.2. Are desiccation-sensitive seeds morphologically or anatomically distinct from tolerant ones? 251 8.3.3. Are desiccation-sensitive seeds associated with particular habitats? 252 8.4. Future Directions 253 8.5. Conclusion 254 8.6. References 254

8.1. Introduction comparative biology of seed desiccation tolerance, looking at broad patterns in the The ability of seeds to tolerate desiccation occurrence of this functional trait through- is a trait of major adaptive importance to out the spermatophytes and examining sys- their survival and dispersal role. That tematic, ecological and other potentially seeds of some species do not withstand informative correlations. We intend to drying presents a challenge for ex situ con- complement the in-depth mechanistic servation. In this review, we explore the studies on a number of species described © CAB International 2002. Desiccation and Survival in Plants: Drying Without Dying (eds M. Black and H.W. Pritchard) 239 Dessication 08 4/4/02 2:22 pm Page 240

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elsewhere in this volume by discussing (Hong et al., 1998b), hereafter referred to as seed desiccation tolerance in an evolution- the Compendium. ary context. In so doing, we aim to high- light gaps in knowledge and hint at possible new research directions. At a 8.1.1. Reviews and compilations of seed more practical level, a better understanding desiccation tolerance of the evolution of seed desiccation tolerance is likely to suggest approaches to ex Significant compilations of recalcitrant seed situ conservation for those species with data are listed in Table 8.1. The earliest list desiccation-sensitive seeds. of recalcitrant (desiccation-sensitive) seeded Much of the consideration of desicca- species included 73 species from 37 genera tion tolerance in seeds focuses on the (Salix and Swietenia at genus level only) and ‘other side of the coin’, sensitivity to desic- 29 families (King and Roberts, 1979). Within cation, as this appears to be the exception a year, the list had been shortened, firstly to in seeds and leads to a number of practical 68 species/42 genera/29 families (King and problems. Our account relies on a survey of Roberts, 1980) and then to 49 species/36 the literature, as well as some new analysis genera (Juglans and Swietenia at genus level of existing data. In relation to the former, only) and 27 families (Roberts and King, two other recent reviews (Farnsworth, 1980). Major divergences between the lists 2000; Pammenter and Berjak, 2000) have related to, for example, the inclusion/exclu- also dealt with the subject from a compara- sion of Citrus spp., of which at least some tive and evolutionary aspect. For the latter, were now considered to be orthodox, and heavy reliance is placed here on the data Coffea spp., about which there was some accumulated in the Compendium of doubt. A later review identified 186 recalci- Information on Seed Storage Behaviour trant-seeded species across 124 genera, after

Table 8.1. Compilations of recalcitrant-seeded species.

Number of species Number of genera Number of families Reference

73 37 29 King and Roberts (1979) (Salix and Swietenia at genus level only)

68 42 29 King and Roberts (1980) (Malus and Swietenia at genus level only)

49 36 27 Roberts and King (1980) (Juglans and Swietenia at genus level only)

186 124 64 Hofmann and Steiner (1989) (Anthurium, Malus, Mauritia, Oxalis, Roystonea, Sabal, Swietenia and Thrinax at genus level only)

NGa NG 45 von Teichman and van Wyk (1994)

195 143 75 Farnsworth (2000) 514b 192c 65d Hong et al. (1998b)

a NG, full details not given. b Out of a total of 6919 species considered, i.e. 7.4%. c Out of 2146 listed in electronic version, i.e. 9%. d Out of 251 families for which there are seed storage data, i.e. 25.9%. NB: there are c. 462 Angiosperm Phylogeny Group (APG) families. Dessication 08 18/3/02 1:57 pm Page 241

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the exclusion of 19 reclassified species intermediate and recalcitrant. Orthodox (Hofmann and Steiner, 1989). Research since seeds can be dried without damage, to low then indicates that the seeds of numerous moisture contents, usually much lower other species can now be considered not to than those they would normally achieve in be recalcitrant as long as they are treated nature. They can be conserved ex situ for carefully, e.g. Corylus avellana (hazelnut), relatively long periods (at least decades) in Zizania aquatica (Indian wild rice), Elaeis seed banks, and many of them, but not all, guineensis (oilpalm) and Azadirachta indica form persistent seed banks in the soil. Over (neem). The difficulties associated with allo- a wide range of storage environments, their cating seeds to the recalcitrant grouping can longevity increases with reductions in both be gauged by the fact that 15–28% of the moisture content and temperature, in a species in the early lists were reported as quantifiable and predictable way (Ellis and having recalcitrant behaviour that had not Roberts, 1980; Dickie et al., 1990). yet been fully confirmed. Recalcitrant, or desiccation-sensitive, seeds Similarly, there is discrepancy between do not survive drying to any large degree, the more recent lists of species. For exam- although the critical moisture level for sur- ple, 17 species listed by Farnsworth (2000) vival varies among species, from about as recalcitrant or viviparous are shown in 25% to 40% seed/embryo moisture content the Compendium (Hong et al., 1998b) to (fresh mass basis) for cacao and red oak, have seeds which are probably orthodox. respectively (e.g. Leprince et al., 1998). The species are: Amomyrtus luma, Agathis Thus, they are not amenable to long-term robusta, Caltha palustris, Chenopodium storage for conservation, nor are they likely quinoa, Cordia alliodora, Cupressus macro- to form persistent soil seed banks. For this carpa, Dovyalis hebecarpa, Fagopyrum review, this category includes those seeds, esculentum, Fagraea fragrans, Flacourtia of some aquatic species in particular, indica, Hedera helix, Michelia champaca, described as viviparous (Farnsworth, Muntingia calabura, Nyssa aquatica, Piper 2000). Intermediate seeds are more tolerant hispidum, Santalum album and Vochysia of desiccation than recalcitrants, though honurensis. that tolerance is apparently more limited The latest hard-copy version of the than is the case with orthodox seeds, and Compendium (Hong et al., 1998b) draws on when dry they generally lose viability more data for 6919 species, of which 514 (7.4%) rapidly at 0°C and 20°C than at warmer from 65 families are recorded as being temperatures around 15°C (Ellis et al., recalcitrant or likely to be recalcitrant. That 1990, 1991). version is an update of an earlier version Assignment of species to these classes of with very limited circulation, sponsored by response is not always clear-cut and sev- the International Plant Genetic Resources eral ‘likely’ and ‘probable’ epithets are Institute (IPGRI). Readers should note that used in the Compendium. One of the main the analysis described below was actually difficulties relates to a lack of a unified carried out on an electronic version of the approach to measuring the level of desicca- dataset (see IPGRI website for download – tion (in)tolerance. Whilst most studies use www.ipgri.cgiar.org/), consisting of records moisture content on a fresh mass and for 7146 species, and which forms the basis sometimes dry mass basis (e.g. Tompsett, of part of the Royal Botanic Gardens Kew’s 1984a,b; Pritchard and Prendergast, 1986; Seed Information Database see Tweddle et Farrant et al., 1988), estimates of critical al., 2002 for information on Release 2.0. water potentials for desiccation stress have been used more recently (e.g. Roberts and Ellis, 1989; Pritchard, 1991; Poulsen and 8.1.2. Classification of seed storage responses Eriksen, 1992; Pritchard and Manger, 1998; Sun, 1999; Walters, 1999). Although such The Compendium recognizes three main an approach removes the potentially con- categories of storage response: orthodox, founding effects of differing chemical com- Dessication 08 18/3/02 1:57 pm Page 242

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position on the assessment of critical mois- the Seed Information Database at ture contents for desiccation stress, espe- www.rbgkew.org.uk/data/sid). cially the effect of oil content on the sorption properties of seeds (Cromarty et al., 1984), critical water potentials for 8.1.3. The phylogenetic classification of stress have been suggested for the onset, plants mid-point and end of the viability loss response in seed populations (Pritchard, Since Darwin’s Origin of Species, the aim of 1991; Tompsett and Pritchard, 1998; successive generations of plant systematists Dussert et al., 1999; Sun, 1999; Walters, has been to produce a classification that is 1999). In addition, the physiological state ever more ‘natural’, reflecting as closely as of the seeds prior to dehydration is influ- possible the phylogeny or evolutionary enced by developmental age and post-har- descent of species and higher groupings vest storage conditions and this can have a (see, for example, Woodland (2000) or Judd profound effect on the level of desiccation et al. (1999) for an account of the history of tolerance (e.g. Probert and Longley, 1989; plant classification systems and especially Finch-Savage, 1992; Tompsett and the more modern ones, such as those of Pritchard, 1993, 1998; Pammenter et al., Cronquist (1981) or Dahlgren (1983)). The 1998; reviewed by Pammenter and Berjak, development of cladistic methods based on 1999). Finally, the methods of desiccation, parsimony, together with the recent rapid rehydration and germination testing also increase in the availability of DNA sequence impact on our perception of desiccation data, has led to the emergence of robust stress (in)tolerance (Grout et al., 1983; phylogenetic classifications of seed plants. Kovach and Bradford, 1992; Leprince et al., A radically new ordinal classification of the 1998; Sacandé, 2000). flowering plants has arisen from the cooper- The consequent variability in data, and ative work of the Angiosperm Phylogeny interpretation, has led to divided opinion Group (APG, 1998). This classification on whether there is a continuum of seed recognizes only monophyletic families and desiccation tolerances between species orders, most, but not all, of which fit into (Berjak and Pammenter, 1994; Dussert et informal higher-level groups or clades. al., 1999; Sun, 1999) or approximately five Concordance with other, more formal, clas- discrete levels of desiccation tolerance sification systems may be rather forced at (Walters, 1999). In addition, variability in ordinal and higher levels; and family cir- response has tended to a proliferation of cumscriptions are not exactly the same in ‘classes’, for example ‘sub-orthodox’ all cases, e.g. the sinking of Aceraceae into (Bonner, 1990; Dickie and Smith, 1995) Sapindaceae, and Chenopodiaceae into and ‘minimally recalcitrant’ (Berjak et al., Amaranthaceae. Like all classification sys- 1989; Dickie et al., 1991). However, and tems, the APG system is a working hypothe- despite the caveats about assigning species sis, not fact. Nevertheless, we believe that to three classes of storage, the practical sys- its powerful representation of likely evolu- tem of classification used in the tionary pathways and relationships pro- Compendium is retained here. Also, for vides the best background for comparative reasons of simplicity, our analyses of the investigations of physiological or ecological Compendium data rely totally on the com- traits such as seed desiccation tolerance, pilers’ assignment of species to a particular and we use it in the analysis of the flower- seed storage category, and we include those ing plants in the Compendium dataset cases where a classification is likely or below. The classification we use for the probable, as well as those where the gymnosperms is basically the arrangement observed responses are more definite. used by Judd et al. (1999), referring also to Readers are referred to the extensive bibli- two more recent molecular treatments of the ography in the Compendium (and in group (Bowe et al., 2000; Chaw et al., 2000). Dessication 08 18/3/02 1:57 pm Page 243

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8.1.4. Evolution of desiccation tolerance in greatest opportunity for spatial and tempo- land plants ral movement, and having desiccation-tolerant propagules would offer a distinct Vegetative desiccation tolerance is a wide- ecological advantage in environments not spread but uncommon occurrence in plants constantly moist, e.g. seasonally dry tem- (Oliver et al., 2000; Chapters 1, 7). Although perate and tropical habitats. Oliver et al. the algae, lichens and bryophytes contain (2000) speculated that such propagules the most desiccation-tolerant plants, about may have evolved as a modification of veg- 120 spp. of ferns, fern-allies and etative desiccation tolerance, i.e. using angiosperms exhibit vegetative desiccation existing genes. tolerance. The facts of its early evolution As noted by Pammenter and Berjak are not certain, but it probably coincided (2000), little is actually known about the with the colonization of the land by primi- evolution of seed desiccation tolerance tive plants during the Ordovician period itself, though rather more has been (from about 510 million years ago (mya)), inferred. Inference has relied on the associ- although it is frequent in the spores and ation of seed desiccation tolerance with cysts of extant representatives of more various aspects of systematics, ecology primitive organisms. For instance, the (particularly habitat and plant habit) and conidia of Metarhizium flavoviride with- seed and fruit structure, among others (e.g. stand drying, and once dry they respond Tompsett, 1994; Hong and Ellis, 1998; quantitatively to moisture content and tem- Hong et al., 1998b). perature in the same way as orthodox seeds Systematic analyses, mainly based on (Hong et al., 1998a). Oliver et al. (2000) seed structural features that are regarded as argued that desiccation tolerance was the primitive (see later), have led to the pro- ancestral state for early land plants (liver- posal that seed desiccation sensitivity is worts, hornworts and mosses), but that this the ancestral state, with tolerance evolving trait was lost early in the evolution of tra- early, and several times independently (e.g. chaeophytes, possibly beginning in the von Teichman and van Wyk, 1991, 1994; Silurian (from 439 mya). It has been postu- Pammenter and Berjak, 2000). Meanwhile, lated that desiccation tolerance was inde- based on a review of 195 sensitive species, pendently evolved (or possibly re-evolved) Farnsworth (2000) has suggested that the in both Selaginella and ferns, and at least most parsimonious explanation of the cur- eight times in the angiosperms. In the last rent distribution of species’ seed desicca- group, vegetative desiccation tolerance is tion sensitivity is by convergent loss of found in the order Hamamelidales and the tolerance from tolerant ancestors. Oliver et families Poaceae (grasses), Cyperaceae al. (2000) inferred that this may have been (sedges), Velloziaceae, Liliaceae, Labiatae, the case in seeds. We are aware of the Gesneriaceae and Scrophulariaceae (Oliver inherent risks of inferring evolutionary pat- et al., 2000). (Note that Hamamelidales is terns by extrapolating from extant species not recognized as monophyletic by the on purely phylogenetic grounds, and thus APG, and the species concerned, our interpretation of the evolution of seed Myrothamnus flabellifolius, is not assigned desiccation tolerance is placed in a wider to an order, being placed in the Core ecological and functional biology context. Eudicots clade.) As plants adapted further The factors or features concerned are to an existence on land, structural and closely interrelated and it is often difficult morphological modifications permitted to disentangle the relative importance of greater control of plant water status one from another in explaining seed (homoiohydry), together with an increase responses to drying. However, from a prac- in size and growth rate, and vegetative des- tical standpoint the ability to diagnose or iccation tolerance was lost. A stationary predict the response from such diverse existence was countered by the evolution information would be a valuable tool for of dispersal structures that facilitated the seed conservation planning. Dessication 08 18/3/02 1:57 pm Page 244

244 J.B. Dickie and H.W. Pritchard

For convenience we have chosen to con- The evidence so far available indicates sider systematic aspects separately ﬁrst, and that desiccation tolerance is the rule for then to cover ecology in the broad sense, mature seeds of the overwhelming majority including both habitat and parent plant fea- of species. Overall, of the 7146 species tures, as well as seed and fruit structure, listed in the Compendium (electronic ver- including size. We believe that analyses of sion) the great majority (c. 90%) are ortho- such associations will ultimately illuminate dox, with c. 7% recalcitrant and 2% the evolution of the extremely valuable trait intermediate. This is in stark contrast to of seed desiccation tolerance. the tolerance of vegetative desiccation by adult plants: the overwhelming majority of spermatophytes are sensitive (Oliver et al., 8.2. Systematics and Evolution of Seed 2000), and resurrection plants are very Desiccation Tolerance rare.

8.2.1. The complete dataset 8.2.2. Gymnosperms Species covered in the Compendium probably represent less than 2.5% of all seed Among the gymnosperms, seed desiccation plant species, and the issues of coverage sensitivity is just as uncommon as it is and potential bias (e.g. under-representa- overall and as it is in angiosperms (see tion of tropical moist forest species) in the Section 8.2.3), with the same relative pro- dataset are real. The low level of sampling portions found among the species repre- of terminal taxa is of particular concern in sented (87% tolerant, 6% intolerant and light of the incidence of variation of behav- 4% limited tolerance). Of the gym- iour within genera; for example, the fol- nosperms represented in the Compendium lowing genera contain species with both the majority are conifers (200 species), and orthodox and recalcitrant seeds: Acer of these 174 are recorded as orthodox. (see also Hong and Ellis, 1990, 1992a; Though their extant members are few and Dickie et al., 1991), Agathis, Araucaria, sampling is limited, all the cycads, ginkgos Calophyllum, Castanopsis, Citrus, and gnetophytes so far examined have des- Coprosma, Diospyros, Garcinia, Magnolia, iccation-tolerant (or at least not desicca- Pittosporum, Spondias, Vitex. However, tion-sensitive) seeds. With the demise of the Compendium is substantially larger the ‘anthophyte hypothesis’ (Bowe et al., than any of the earlier compilations used 2000; Chaw et al., 2000; Qiu et al., 2000), it by other authors as the basis for review is not now so easy to suggest that the puta- (e.g. Hofmann and Steiner, 1989; von tive sister group to the angiosperms (gneto- Teichman and van Wyk, 1994), and is more phytes), and hence a common ancestor, rigorous in its assignment of species to possessed only orthodox seeds. However, storage category. For example, apparently the ancestral status of seed desiccation toler- mainly on account of its preference for ance within the gymnosperms still has sig- moist habitats, Caltha palustris was classi- niﬁcant support, from the fact that ﬁed as having recalcitrant seeds by recalcitrant behaviour has not been observed Hofmann and Steiner (1989); and this has in the cycads or ginkgos. These are regarded been perpetuated in subsequent reviews by as basal in the group (e.g. Bowe et al., 2000; von Teichman and van Wyk (1994) and by Chaw et al., 2000). Indeed, so far, desiccation Farnsworth (2000). Yet seeds of this sensitivity appears to be restricted to two species have been successfully stored air- derived conifer families, Podocarpaceae and dry and frozen for a number of years in the Araucariaceae. In the Podocarpaceae only Royal Botanic Gardens Kew (now Podocarpus usambarensis is recorded as Millennium) Seed Bank at Wakehurst Place possibly orthodox, of ten species covered, – its seeds are clearly orthodox and the remainder having no or limited toler- recorded as such in the Compendium. ance of desiccation (seven recalcitrant, two Dessication 08 18/3/02 1:57 pm Page 245

Systematic and Evolutionary Aspects of Desiccation Tolerance 245

intermediate). Within the Araucariaceae, bution (South America, Australia, New seed storage behaviour is polymorphic, Zealand, New Caledonia, New Guinea and with orthodox, intermediate and recalci- other South Pacific Islands) (Setoguchi et trant behaviour shown by congeners in al., 1998). In a comprehensive survey of both Agathis and Araucaria (Tompsett and seed storage responses in this family, Kemp, 1996). Tompsett and Kemp (1996) showed that In the gymnosperm fossil record, while four of 14 species investigated had recalci- well-preserved gametophytic tissue is trant seeds. All the recalcitrant species sometimes contained in Late Palaeozoic belong to the genus Araucaria, which con- sediments, embryos are rarely found. Post- stitutes about two-thirds of the species in zygotic development in the earliest gym- the family. The genus can be split into nosperms is thought to have been rapid Sections, with Eutacta being the largest. All and continuous, making it less likely that investigated species of this Section (eight in embryos would be fossilized (Mapes et al., total) produce seeds that are tolerant of des- 1989). Thus, the appearance in the Permo- iccation to low levels, from 15 to 2%. In Carboniferous (c. 370–240 mya) strata of contrast, species investigated in the Sections well-developed cotyledonary embryos in Araucaria (Araucaria araucana, Araucaria relatively small seeds (6–7 mm long) has angustifolia), Intermedia (Araucaria hun- been used to suggest the development of a steinii) and Bunya (Araucaria bidwillii) pro- quiescent phase in embryo growth, i.e. the duce recalcitrant seeds. Based on the embryos may have been dormant (Mapes et consensus tree of the 20 equally parsimo- al., 1989). Thus, by Permo-Carboniferous nious ones for Araucariaceae, the cpDNA times, conifer seeds may have already rbcL sequence information shows a major evolved two functionally important traits – branching of Agathis from Araucaria after a dormancy and, possibly, desiccation toler- split from Wollemia (Setoguchi et al., 1998). ance. Readers should note that Mapes et al. However, there is no agreement as to (1989) used the word ‘dormancy’, as does whether Wollemia is closer to either Farnsworth (2000), in its broad sense, to Araucaria or Agathis (Gilmore and Hill, denote the capability for embryonic devel- 1997; Setoguchi et al., 1998). Interestingly, opmental arrest, no matter how long sus- Wollemia, Agathis and the non-Eutacta tained or controlled, in contrast to Araucaria have two cotyledons, whilst the ‘vivipary’. Most seed biologists use the Eutacta Araucaria have four. All araucar- word ‘quiescence’ to describe this capabil- ian fossil species from the Mesozoic (c. ity, reserving ‘dormancy’ for specific physi- 245–65 mya) have dicotyledonous embryos, cal or physiological mechanisms that delay suggesting that the common ancester of the seed germination, even when hydration Section Eutacta had two cotyledons. As and the typical temperature requirements Agathis, the Eutacta Araucaria and are satisfied (e.g. Baskin and Baskin, 1998). Wollemia have desiccation-tolerant seeds, As we discuss below, there is a general we can postulate that the increase from two inverse relationship between dormancy to four cotyledons was not associated with and desiccation sensitivity (an exception is the loss of desiccation tolerance. Aesculus hippocastanum). Desiccation tolerant seeds ( 15% moisture content) of the Section Eutacta vary in size or mass from about 300 mg for 8.2.2.1. Araucariaceae Araucaria cunninghamii to 1870 mg for This family includes 41 species across Araucaria heterophylla (Tompsett and three genera, Agathis, Araucaria and Kemp, 1996). Similarly, desiccation-tolerant Wollemia (Farjon, 1998). Wollemia is repre- seeds of Agathis sp. are around 200 mg and sented by one species, the Wollemi pine those of W. nobilis measure about 1.1 (Wollemia nobilis) discovered in the last 10 0.9 cm (Offord et al., 1999). In contrast, years (see Offord et al., 1999). The family the desiccation-sensitive seeds of the primarily has a southern hemisphere distri- Sections Araucaria, Intermedia and Bunya Dessication 08 18/3/02 1:57 pm Page 246

246 J.B. Dickie and H.W. Pritchard

are generally bigger, varying from 580 mg for seeds (Collinson, 1999; Eriksson et al., A. hunsteinii to c. 10,000 mg in A. bidwillii 2000). At least for angiosperms, the behav- (Dooley, 1990). There are abundant palaeo- iour of modern species would predict that botanical data for this family, which have such ancient seeds were orthodox, i.e. tol- allowed an assessment to be made between erant of desiccation. the evolution of seed size and current trends In the angiosperms, all but two of the in the level of seed desiccation tolerance. monophyletic orders recognized by the APG The fossil record for Section Bunya are covered in the Compendium dataset (see seeds indicates a relatively small seed size Fig. 8.1), and all of those contain some for Araucaria mirabilis (c. 1 0.4 cm), species with orthodox seeds. Seed desicca- Araucaria brownii (0.8 0.3 cm) and tion sensitivity, or recalcitrance, is spread Araucaria sphaerocarpa (1.6 0.7 cm) across all major clades, but, with the excep- (see Setoguchi et al., 1998, and references tion of Ericales, is quite rare in the asterids. therein). This is in stark contrast to the Examples of ‘hot spots’ for this state are seeds of the extant species A. bidwillii at c. Malvales, Arecales and Laurales in the 5 3 cm, and it has been suggested, on the eudicots, monocots and basal angiosperms, basis of other morphological features, that respectively, and others are indicated in this species should be treated separately Fig. 8.1. Of the most basal ‘ANITA’ group from Mesozoic (c. 245–65 mya) araucarians (Amborellaceae, Nymphaeales, Illicales, assigned to the Section Bunya. Trimeniaceae and Austrobalyaceae; see The current molecular data suggesting a Barkman et al., 2000; Qiu et al., 2000), rep- monophyletic origin for the Sections resentation is restricted to Nymphaea spp. Araucaria, Intermedia and Bunya agree (see below), and the other families and with the fossil record, which suggests their orders should be targeted for study. Moving evolution into Sections was possibly com- to the other basal orders, less than half the plete before South America separated from species listed from the Magnoliales have Antarctica during the Eocene at the latest. recalcitrant seeds and, of the few Piperales Thus, it is possible that desiccation-sensi- (see also Vázquez-Yanes and Orozco- tive seeds in these Sections of the family Segovia, 1982) examined, none has them. may have been present at least 40–60 mya. Seeds of Ceratophyllum demersum Molecular data also suggest that Section (Ceratophyllales), while difﬁcult to germi- Eutacta (containing extant species with nate, are almost certainly desiccation- desiccation-tolerant seeds) is older than the tolerant (Hong et al., 1998b; Hay et al., other Sections. Although there is the need 2000; F. Hay, Ardingly, 2001, personal com- to review Mesozoic fossils of Eutacta, our munication). Thus, the extant members of working hypothesis is that desiccation the basal groups do not show anything like tolerance may have been associated with exclusively, or even predominantly, recalci- an ancestral small-seeded state in trant behaviour. Araucariaceae. At the family level, Table 8.2 shows the percentages of the three types of seed storage behaviour occurring in a selection (44) 8.2.3. Angiosperms of the largest angiosperm families. These are ordered by % species coverage in the From comparative studies of DNA amounts Compendium, to give some idea of the cur- (Leitch et al., 1998), it seems likely that rent sampling levels. In all, about half of all angiosperms at least originated as rapid- angiosperm families are represented, and cycling species of ephemeral habitats of these only c. 25% have members with (Midgley and Bond, 1991), where seed des- desiccation-sensitive seeds. Mean coverage iccation tolerance would have been a dis- of genera in families is around 18%, rang- tinct advantage. It is also likely that the ing from 88% in the Fagaceae to less than earliest angiosperms were early succes- 2% in the Melastomataceae. Species cover- sional herbs or shrubs with relatively small age within family goes from a low of Dessication 08 18/3/02 1:57 pm Page 247

Systematic and Evolutionary Aspects of Desiccation Tolerance 247

0/1 Ceratophyllales 28/42 Laurales 12/42 Magnoliales 0/7 Piperales (Acorales) 4/31 Alismatales 3/199 Asparagales 0/18 Dioscoreales (Pandanales) 0/22 Liliales Monocots 25/97 Arecales 4/584 Poales 0/10 Commelinales Commelinoids 1/15

Angiosperms Zingiberales 0/158 Ranunculales 2/81 Proteales 2/404 Caryophyllales 2/7 Santalales 0/80 Saxifragales 0/17 Geraniales 31/200 Malpighiales Eudicots 4/10 Oxalidales 15/1133 Fabales 25/338 Rosales Eurosid I 4/69 Cucurbitales 69/161 Fagales 22/314

Rosids Myrtales 2/569 Brassicales 97/296 Eurosid II Malvales 75/285 Sapindales Core eudicots 0/30 Cornales 32/202 Ericales 0/1 Garryales 6/137 Gentianales 5/430 Euasterid I Lamiales 0/181 Solanales 0/8 Asterids Aquifoliales 4/107 Apiales 0/489 Euasterid II Asterales 0/62 Dipsacales

Fig. 8.1. Seed storage behaviour at ordinal level. No data for the two orders in parentheses. All other orders contain some species with desiccation-tolerant seeds. Numbers of desiccation-sensitive species are shown as fractions of total no. examined – orders having at least one sensitive sp. are shown in bold. Tree derived from several cladistic analyses by the Angiosperm Phylogeny Group – see www.rrz.uni-hamburg.de/biologie/ b_online/apg/APG.html

0.04%, again in the Melastomataceae, to a family. Families with a high incidence high of 17% in the Brassicaceae, and a (> 10% of species examined) of recalcitrant mean value between 2 and 3%. The overall species are comparatively rare (ten, or less percentage occurrence of species with des- than a quarter of those listed) and widely iccation-sensitive seeds is around 10% per scattered taxonomically (examples in Table Dessication 08 18/3/02 1:57 pm Page 248

248 J.B. Dickie and H.W. Pritchard

Table 8.2. Representation of seed storage ‘groups’ in 44 plant families in relation to the number of genera and species for which there are data. Bold signiﬁes families with the highest percentage (10.9–80.2%) of recalcitrant species out of the total species investigated.

Representation Species’ seed storage behaviour Genera Species Orthodox Intermediate Recalcitrant Family (%) (%) (%) (%) (%)

Brassicaceae (incl. Capparaceae) 37.5 16.9 99.6 0.2 0.2 Fagaceae 87.5 8.2 17.4 1.2 80.2 Amaranthaceae (incl. Chenopodiaceae) 20.7 7.1 100.0 0.0 0.0 Caryophyllaceae 27.6 7.1 100.0 0.0 0.0 Rosaceae 46.3 7.0 96.5 0.0 1.5 Fabaceae 34.1 6.3 98.6 0.2 1.2 Sapindaceae (incl. Aceraceae, Hippocastanaceae) 16.8 5.8 63.1 1.2 31.0 Cucurbitaceae 18.2 5.4 95.1 0.0 4.9 Poaceae 22.6 5.3 98.6 0.6 0.8 Solanaceae 23.2 4.9 100.0 0.0 0.0 Proteaceae 23.4 4.7 90.7 0.0 2.7 Malvaceae (incl. Sterculiaceae, Bombaceae, Tiliaceae) 24.5 4.2 91.1 1.2 5.9 Polygonaceae 26.5 4.0 93.2 0.0 2.3 Arecaceae 30.5 3.6 27.8 12.4 25.8 Ranunculaceae 29.0 3.6 100.0 0.0 0.0 Moraceae 28.9 3.6 51.2 0.0 48.8 Rutaceae 16.7 3.6 50.0 28.1 10.9 Myrtaceae 25.6 3.1 83.9 0.0 14.7 Clusiaceae 20.0 2.8 57.9 0.0 42.1 Gentianaceae 12.0 2.5 100.0 0.0 0.0 Sapotaceae 18.9 2.4 3.8 23.1 65.4 Scrophulariaceae 12.8 2.3 100.0 0.0 0.0 Apiaceae 11.2 2.2 98.7 0.0 0.0 Ericaceae 15.9 2.1 100.0 0.0 0.0 Lamiaceae 17.5 2.0 100.0 0.0 0.0 Convolvulaceae 10.9 1.8 100.0 0.0 0.0 Boraginaceae 15.4 1.8 100.0 0.0 0.0 Asteraceae 12.6 1.8 99.6 0.0 0.0 Iridaceae 11.0 1.4 100.0 0.0 0.0 Lauraceae 28.8 1.2 5.7 0.0 77.1 Celastraceae 9.1 1.2 80.0 13.3 6.7 Bromeliaceae 22.0 1.0 100.0 0.0 0.0 Cyperaceae 10.6 0.9 100.0 0.0 0.0 Apocynaceae (incl. Asclepiadaceae) 6.3 0.9 88.9 0.0 4.4 Urticaceae 8.3 0.7 100.0 0.0 0.0 Annonaceae 7.8 0.6 62.5 0.0 12.5 Euphorbiaceae 8.2 0.5 85.4 0.0 6.3 Cactaceae 3.1 0.4 100.0 0.0 0.0 Rubiaceae 4.6 0.4 83.9 5.4 7.1 Orchidaceae 5.5 0.3 63.9 25.3 0.0 Araceae (incl. Lemnaceae) 10.2 0.3 83.3 0.0 8.3 Zingiberaceae 6.3 0.3 66.7 33.3 0.0 Piperaceae 12.5 0.2 83.3 16.7 0.0 Melastomataceae 1.1 0.0 100.0 0.0 0.0 Dessication 08 18/3/02 1:57 pm Page 249

Systematic and Evolutionary Aspects of Desiccation Tolerance 249

8.2 indicated by bold type). This ‘top ten’ The Fagaceae consist of eight or nine list is quite familiar to students of seed des- genera (depending on authority) and close iccation sensitivity – Fagaceae, Lauraceae, to 1000 species. What is particularly inter- Sapotaceae, Moraceae, Clusiaceae, Sapin- esting about this family is that desiccation daceae (including Aceraceae), Arecaceae tolerance is mainly delimited by genera (= Palmae), Myrtaceae, Annonaceae and (note that this is the case in a number of Rutaceae; and all of these also have at least other families). Species in the genera Fagus some, and usually many, orthodox species. and Nothofagus are desiccation-tolerant, Other ‘hotspots’ for recalcitrant seeds in whilst those in Quercus and Castanea are smaller families, not shown in the table, desiccation-sensitive – although one species include Anacardiaceae, Dipterocarpaceae, of Quercus (Quercus emoryi) appears to be Meliaceae and Rhizophoraceae. All the relatively desiccation-tolerant (Hong et al., extant Nymphaeaceae (a basal family – see 1998b). above) are aquatic herbs, and intuitively The last complete descriptive treatment might be expected to have desiccation-sen- of the family dates from about 150 years sitive seeds (see remarks on ecology ago and various infrafamilial schemes have below), yet Nymphaea gigantea is reported been proposed, with notable character par- to have orthodox seeds (Ewart, 1908, cited allelisms and disagreement on the origin in the Compendium). and evolution of the cupule. Thus, it is not Below the family level, there are also possible to be categorical about the evolu- some recognizable patterns in the distribution of the family. None the less, there is tion of seed storage types. Intergeneric fossil evidence from North America variation in seed desiccation tolerance is (Buchannan locality) to suggest that the present in c. 25% of families for which two subfamilies – Castaneoideae and there are data (i.e. c. 12% of all families). Fagoideae – may have split no later than By overlaying the data from the the Palaeocene (c. 58 mya), and that the Compendium on the APG cladogram, we Fagaceae probably originated in the late have been able to identify a number of fam- Cretaceous (c. 65 mya) (Nixon and Crepet, ilies of particular interest with respect to 1989). Fagus (desiccation-tolerant) appears the level or proportion of species with des- to be basal for the subfamily Fagoideae, iccation-sensitive seeds. These include the with Quercus arising later (Crepet and monocotyledon family Arecaceae (c. 4% of Nixon, 1989). Thus, there is the possibility species investigated and c. 26% recalci- that the ancestral seed state in this subfam- trant) and the dicotyledon family Fagaceae ily may well have been desiccation- (of the 8% of species investigated, 80% are tolerant. Moreover, the palaeoclimate at recalcitrant). this locality is generally considered to have been warm temperate to tropical with seasonal drought. This type of environment is 8.2.3.1. Fagaceae also consistent with the modern distribu- The family Fagaceae belongs to the order tion of many species of Castaneoideae, Fagales, which has 43% recalcitrant species including Castanopsis (variable desicca- (i.e. 69 out of 161 species investigated) (Fig. tion tolerance!) in Asia. The mean seed 8.1). The vast majority of these recalcitrant weight for the > 50 species with recalci- species (53) belong to this family. The trant seeds (principally in the genera Fagaceae are distributed across temperate Quercus and Castanea) is close to 4 g. In parts of the northern hemisphere and South contrast, seed weight for the handful of America, and as far east as Malesia, orthodox species (in Fagus, Castanopsis Australia and New Zealand, and adjacent and Nothofagus) is around 0.3 g. Thus, areas. In most parts of the range, the family orthodox Fagaceae seeds tend to be smaller is a very prominent part of broadleaved than the recalcitrants, as was found to be forests (Govaerts and Frodin, 1998). the case in Araucariaceae. Dessication 08 4/4/02 2:23 pm Page 250

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8.3. Seed Desiccation Tolerance and 8.3.1. Are recalcitrant seeds bigger than Ecology sensu lato orthodox seeds?

The ability of seeds of the vast majority of Seed size varies over ten orders of magni- extant spermatophyte species (so far exam- tude (Harper et al., 1970), with a number of ined) to tolerate desiccation is presumably potential ecological and evolutionary a trait of major adaptive importance to their causes, as well as phylogenetic constraints. survival and dispersal role. Assuming that The case has been made above, for two it is an ancient trait (see above), what eco- families, that seeds of the recalcitrant logical trade-offs have induced certain species are generally larger than those of species to relinquish it from time to time the related orthodox species. But is this the during evolution? Seed desiccation sensi- case across all species so far investigated? tivity is not a problem for those species that The answer is ‘yes’, with intermediate have it, only for people wishing to store the seeds somewhere in between. Figure 8.2 seeds. From a practical point of view, the shows the seed mass distributions for 1080 association between seed storage behaviour species for which ‘seed’ weights are given and plant ecology and structural character- in the Compendium (1000-seed weights istics has long been of interest (e.g. Roberts cited). The mean seed weight for recalci- and King, 1980) in relation to the search for trant seeds (3958 mg) is greater than that predictors of seed storage behaviour. This for intermediates (900 mg), which is greater issue is bound up in the complex of inter- than for orthodox seeds (329 mg). However, acting factors defining the ‘regeneration as in all large-scale comparisons of seed niche’ (Grubb, 1977) of particular species. mass, the degree of overlap is considerable As such, simple associations with single (see Leishmann et al., 2000), and except at factors will not have broad applicability, the extremes it would be a relatively poor and this has been recognized, for example predictor of seed storage behaviour. ‘Seed’ by Hong and Ellis (1997, 1998), in propos- mass is easy to measure. However, it is a ing the use of multiple criteria. With partic- broad term, including true seeds as well as ular reference to Meliaceae they suggested a variety of indehiscent fruits, confounded four – seed size, shape and moisture con- by variation in the proportion of covering tent at maturity, together with ‘plant ecol- structures removed in postharvest clean- ogy’. The latter is not a single criterion, but ing, leading to the use by some authors of presumably means ‘habitat’ in this context. terms such as ‘dispersule’ and ‘germinule’ Desiccation-sensitive seeds, so-called recal- (e.g. Grime et al., 1988). It is strongly asso- citrants, are widely held to be generally ciated with a number of seedling character- large and ‘fleshy’, more likely to occur in istics, habitat type and dispersal mode, and forest-tree species and more frequently in the strength of the relation between growth the moist tropics, or in aquatic species, and form depends on location, but woody possibly in certain taxonomic groups. The plants and climbers generally have heavier first part of this review has confirmed that seeds than graminoids and forbs (see seed desiccation sensitivity is more fre- Leishmann et al., 2000). Seed size is also quent in certain plant families, but that larger in tropical floras, independent of those families are not exclusively recalci- growth form and dispersal mode (Lord et trant, nor is there any systematic pattern to al., 1997). Thus, everything that might be that distribution. The occurrence of recalci- positively associated with seed desiccation trance is much more likely to result from sensitivity is associated with seed weight, convergence in relation to ecological condi- which in turn is broadly associated with tions. This section of the chapter examines seed desiccation sensitivity. the validity of such broad statements by The very earliest seeds appeared in the looking at the available evidence in the fol- late Devonian–Mississippian era (c. 400 lowing, sometimes obviously overlapping, mya) on seed ferns and similar plants and, categories. while there may be doubts about the types Dessication 08 18/3/02 1:58 pm Page 251

Systematic and Evolutionary Aspects of Desiccation Tolerance 251

30 Recalcitrant n = 205 20

30 Intermediate n = 46 20

% Frequency 10

30 Orthodox n = 839 20

0 0.01 0.1 1 10 100 1000 10,000 100,000

Seed mass (mg) (log scale)

Fig. 8.2. Seed size distributions for storage types in the Compendium. The number of species per storage category is shown. NB: seed mass values shown are central between the ticks.

of environment in which they evolved, 8.3.2. Are desiccation-sensitive seeds they were relatively quite small (volume morphologically or anatomically distinct mostly less than 10 mm3) (Tiffney, 1986), from tolerant ones? and thus, by reference to extant species, more likely rather than less likely to be Are recalcitrant seeds generally ‘ﬂeshy’? desiccation-tolerant. This argument centres to some extent on Dessication 08 18/3/02 1:58 pm Page 252

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size and degree of hydration at dispersal, and have recalcitrant embryos (see Pritchard as well as the proliferation of certain tis- et al., 1995). sues. Many orthodox seeds are borne in The morphology of the dispersal unit fleshy fruits, presumably animal-dispersed, coat (testa and/or pericarp, depending on and are at high moisture content at matu- species) may offer some resistance to dehy- rity/fruit shedding (e.g. tomato), though the dration in some recalcitrant seeds, e.g. seeds themselves are not especially large Dipterocarpus tuberculatus (Tompsett, and many of them are known to be ortho- 1992) and Acer pseudoplatanus (Dickie et dox, e.g. apples (Dickie and Bowyer, 1985). al., 1991), compared with their more ortho- Among gymnosperms there is also at least dox relatives, such as Dipterocarpus alatus one example, Taxus brevifolia (Walters- and Acer platanoides, respectively. Such a Vertucci et al., 1996), where seeds sur- property may have provoked the sugges- rounded by a fleshy false fruit (aril) are tion that recalcitrant seeds are homoiohy- nevertheless orthodox. Hong and Ellis drous (Berjak et al., 1989), but recalcitrant (1998) regarded moisture content at the seeds do equilibrate to ambient relative time of dispersal as a good marker of seed humidities, even if equilibration times are storage type, along with other factors, but sometimes longer than expected. Short term of itself it is unlikely to be particularly avoidance of desiccation is likely to be a informative, and Dussert et al. (2000) viable alternative to tolerance, as a survival found that it was not correlated with the mechanism under certain conditions. level of seed desiccation tolerance in nine Coffea spp. Corner (1951, 1976) introduced the term 8.3.3. Are desiccation-sensitive seeds ‘overgrown seeds’ initially to describe seeds associated with particular habitats? of a number of leguminous species that are relatively large, non-endospermic and have There are general associations between habi- a poorly developed testa. The list includes tat or macroclimate and seed storage genera such as Castanospermum and responses, at least for some species. Pentaclethra, known to have recalcitrant Tompsett (1994) noted that the more ortho- seeds, along with others such as Bauhinia, dox-seeded members of the Aracauriaceae Millettia and Pithecellobium, from which and Dipterocarpaceae were found in season- desiccation-sensitive seeds have so far not ally dry tropical woodland. Dickie et al. been recorded (Hong et al., 1998b). Von (1992) drew a similar conclusion from a lim- Teichman and van Wyk (1991, 1994) ited survey of seed storage behaviour in the attempted to relate recalcitrant seed behav- Arecaceae, and Dussert et al. (2000) have iour to large size resulting from over- examined the relation between seed desicca- developed chalazal tissue, along with several tion sensitivity and climate in the native other seed structural characters presumed to environments of nine Coffea species. The be primitive (e.g. bitegmic, crassinucellate desiccation-tolerant species of Meliaceae are ovules, presence of substantial endosperm). often located in savannah regions of Africa However, Corner (1992) doubted that pachy- (Tompsett, 1994; Hong and Ellis, 1998). chalazy itself is primitive. However, recalcitrant-seeded species are There is no evidence that the gross mor- known in the tropical drylands, e.g. phological disposition of reserve materials, Vitellaria paradoxa (Pritchard and Daws, i.e. either endospermic or cotyledonous, is 1997). It could be that such species are his- associated with seed storage behaviour. In torical relics of an earlier more hydric envi- Arecaceae (Palmae), Cocos nucifera is ronment and/or that the reproductive endospermic and recalcitrant, Washingtonia strategy of the species is finely tuned to the sp. are endospermic and orthodox (Dickie et dynamics of the local microenvironment, al., 1992). In Fabaceae (Leguminosae) including the availability of seed dispersers. Pisum spp. are non-endospermic and ortho- Vázquez-Yanes and Orozco-Segovia (1984) dox, whilst Inga spp. are non-endospermic and Vázquez-Yanes et al. (2000) have com- Dessication 08 18/3/02 1:58 pm Page 253

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piled much of the available evidence show- tolerance, e.g. in Fagaceae, Arecaceae and ing that perhaps a majority of species, espe- Rubicaceae (e.g. Coffea sp.), opens up the cially the dominant trees, of humid moist prospect of creating a molecular cladogram forests have seeds that germinate rapidly to for desiccation (in)tolerance in closely produce a carpet of dormant or very slowly related species using existing methodolo- growing seedlings. Many of these may be gies. Some of the families identified here recalcitrant, but a number do persist as soil appear suitable for further investigation in seed banks (e.g. Cao et al., 2000), especially this context, and in particular the basal pioneer and gap species (Vázquez-Yanes et groups or species in those families. al., 2000). The current Compendium dataset The existence of variation in seed desic- almost certainly under-represents tropical cation response among species within a moist forest species and more data are genus has been remarked upon above (see needed. That such vegetation will have qual- also Hong and Ellis, 1995). Of particular itatively more recalcitrant species than most interest recently has been information on other vegetation types is perhaps beyond the inheritance of desiccation tolerance in dispute, but by how much more is another interspecific crosses of Coffea (Dussert et al., question. There are a number of different 1999). Furthermore, there is growing evi- types of tropical moist forests and many gra- dence of the potential for variation within dations into drier vegetation types, where species. This follows the isolation of a grow- the proportions of different functional types ing number of viviparous and seed desicca- of trees (e.g. pioneer and gap versus canopy tion-sensitive mutants in Arabidopsis (e.g. and emergent) may vary considerably. Ooms et al., 1993) and several crop species Aquatic vegetation is the other main type (maize, rice, peppers), of which the wild- assumed to contain a high proportion of types bear orthodox, desiccation-tolerant species with desiccation-sensitive seeds. seeds. However, there are no reports of seed This is largely on the grounds that there is desiccation-tolerant mutants in species with plenty of water around, seeds would not recalcitrant seeds. The work on desiccation- need to survive drying out and there may be sensitive mutants (e.g. Ooms et al., 1993) selection pressure for very rapid germination suggests that relatively few genes seem to be and establishment (Farnsworth, 2000). In associated with sensitivity. Moreover, it can fact, this is frequently not the case, and the be assumed that desiccation tolerance in all example of Caltha palustris has been cited orthodox seeds is limited to a ‘developmen- above. Hay et al. (2000) have shown that, of tal window’ from maturation to germination all the aquatic and marsh species investi- (see Hong and Ellis, 1992b), more or less gated, over 90% are orthodox, with the per- coincident with embryo ‘dormancy’ (used in centage recalcitrant at 7%, with the the broad sense and including quiescence remainder having limited desiccation toler- (e.g. Mapes et al., 1989; Farnsworth, 2000)). ance, e.g. Najas flexilis (Hay and Muir, 2000). In addition, it is known that many species These proportions are no different from those have variable desiccation tolerance between in seed plants as a whole (see earlier). tissues of the sporophyte (seed and vegeta- Perhaps this is less surprising when it is tive tissues) and gametophyte (pollen), indi- remembered that the seeds of many aquatic cating that transcriptional, translational or species are dispersed over long distances on post-translational control of existing genes the feet of birds, for instance, and others in is most probably involved in the expression marshes may have to survive fluctuating of desiccation tolerance. It is probable that moisture levels and seasonal desiccation. seeds of the few angiosperm resurrection plants capable of surviving vegetative desiccation in the adult phase are desiccation- 8.4. Future Directions tolerant. Published data showing whether or not the desiccation tolerance of the sporo- The existence of congeneric species with phyte continues unbroken from mature distinctly different levels of desiccation embryo to adult plant have been lacking. Dessication 08 18/3/02 1:58 pm Page 254

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However, J.M. Farrant (Cape Town, 2001, help with understanding as well as diagnos- personal communication) has found that ing and predicting the condition. Apart from seedlings of Craterostigma wilmsii, the highlighting by Hong and Ellis (1997) of Xerophyta viscosa and Eragrostis nindensis the possible role of moisture content at go through a desiccation-sensitive phase maturity, little has yet been done to compile post-germination. Interestingly, very prelim- the large but widely scattered amount of inary results suggest that in E. nindensis at information on seed and fruit dispersal (see, least one gene expressed in the desiccation- for example, Jordano, 2000; Stiles, 2000; tolerant seed and adult phases is not Willson and Traveset, 2000) in the context of expressed during the desiccation-sensitive seed desiccation tolerance. This will be a seedling stage (J.M. Farrant, Cape Town, significant component of the Royal Botanic 2001, personal communication). Moreover, Gardens Kew’s (Millennium Seed Bank) it appears that species vary in the level of Seed Information Database (Tweddle et al., desiccation sensitivity displayed during the 2002). Another profitable area to pursue seedling window, with relative tolerance in would be the ecological trade-offs involved C. wilmsii possibly related to high levels of in seed size, provisioning and protection and up-regulation of protection systems in that seedling establishment in relation to desicca- species compared with others (Farrant et al., tion sensitivity (see, for example, Garwood, 1999). 1996; Mazer, 1998; Leishmann et al., 2000). Clearly, the identification of a set of spe- Likewise, there is evidence of increasing cific (homologous) genes or products asso- interest in the importance of ripening phe- ciated with the loss or acquisition of nology in relation to environment at seed desiccation tolerance, e.g. through gene dispersal (e.g. Dussert et al., 2000; Rodríguez micro-array technology, will permit com- et al., 2000). parative physiology and molecular screening of diverse species to give us greater insight into the evolution of regulatory 8.5. Conclusion pathways for stress tolerance. Some molecules of potential value, e.g. late embryoge- Seed desiccation tolerance is a complex, nesis abundant proteins, are already under labile trait, which is at least as likely to be extensive investigation in plant and micro- ancestral as is sensitivity in the evolution bial systems (see Chapters 1, 5 and 10). of seed plants. In view of its frequent and Such comparative physiological studies wide occurrence in both gymnosperms and need to be to a common standard to permit angiosperms, especially among extant incorporation into a taxon-based database. members of basal groups, parsimony dic- Such information will come from many tates that it is the likely ancestral state in sources, including the Millennium Seed seed plants. The analysis here gives con- Bank project (Royal Botanic Gardens Kew, siderable support to a similar suggestion by UK), which may contribute to an increase Farnsworth (2000). Applying Dollo’s law in seed storage information for species (see Judd et al., 1999, p. 21), parallel origin from an existing level of around 70 families seems unlikely, and it is quite conceivable to around > 200 families by the year 2010. that this trait has been lost repeatedly, pos- Greater understanding of the ecology of sibly through single gene loss, in response species with desiccation-sensitive seeds will to one or more ecological trade-offs.

8.6 References

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Judd, W.S., Campbell, C.S., Kellogg, E.A. and Stevens, P.F. (1999) Plant Systematics: a Phylogenetic Approach. Sinauer Associates, Sunderland, Massachusetts, 464 pp. King, M.W. and Roberts, E.H. (1979) The Storage of Recalcitrant Seeds – Achievements and Possible Approaches. International Board for Plant Genetic Resources, Rome, Italy, 96 pp. King, M.W. and Roberts, E.H. (1980) Maintenance of recalcitrant seeds in storage. In: Chin, H.F. and Roberts, E.H. (eds) Recalcitrant Crop Seeds. Tropical Press, Kuala Lumpur, Malaysia, pp. 53–89. Kovach, D.A. and Bradford, K.J. (1992) Imbibitional damage and desiccation tolerance of wild rice (Zizania palustris) seeds. Journal of Experimental Botany 43, 747–757. Leishmann, M.R., Wright, I.J., Moles, A.T. and Westoby, M. (2000) The evolutionary ecology of seed size. In: Fenner, M. (ed.) Seeds: the Ecology of Regeneration in Plant Communities. CAB International, Wallingford, UK, pp. 31–57. Leitch, I.L., Chase, M.W. and Bennett, M.D. (1998) Phylogenetic analysis of DNA C-values provides evidence for a small ancestral genome size in ﬂowering plants. Annals of Botany 82 (Suppl. A), 85–94. Leprince, O., van Aelst, A.C., Pritchard, H.W. and Murphy, D.J. (1998) Oleosins prevent oil-body coa- lescence during seed imbibition as suggested by a low-temperature scanning electron microscope study of desiccation-tolerant and -sensitive oilseeds. Planta 204, 109–119. Lord, J., Egan, J., Clifford, H.T., Jurado, E., Leishmann, M., Williams, D. and Westoby, M. (1997) Larger seeds in tropical ﬂoras: consistent patterns independent of growth form and dispersal mode. Journal of Biogeography 24(2), 205–211. Mapes, G., Rothwell, G.W. and Howarth, M.T. (1989) Evolution of seed dormancy. Nature 337, 645–646. Mazer, S.J. (1998) Rainforest plants protect their investments. Trends in Ecology and Evolution 13(12), 471–473. Midgley, J.J. and Bond, W.J. (1991) Ecological aspects of the rise of angiosperms – a challenge to the reproductive superiority hypothesis. Biological Journal of the Linnaean Society 44, 81–92. Nixon, K.C. and Crepet, W.L. (1989) Trigobalanus (Fagaceae): taxonomic status and phylogenetic relationships. American Journal of Botany 76, 828–841. Offord, C.A., Porter, C.L., Meagher, P.F. and Errington, G. (1999) Sexual reproduction and early plant growth of the Wollemi pine (Wollemia nobilis), a rare and threatened Australian conifer. Annals of Botany 84, 1–9. Oliver, M.J., Tuba, Z. and Mishler, B.D. (2000) The evolution of vegetative desiccation tolerance in land plants. Plant Ecology 151, 85–100. Ooms, J.J.J., Léon-Kloosterziel, K.M., Bartels, D., Koornneef, M. and Karssen, C.M. (1993) Acquisition of desiccation tolerance and longevity in seeds of Arabidopsis thaliana: a comparative study using abscisic acid-insensitive abi3 mutants. Plant Physiology 102, 1185–1191. Pammenter, N.W. and Berjak, P. (1999) A review of recalcitrant seed physiology in relation to desiccation-tolerance mechanisms. Seed Science Research 9, 13–37. Pammenter, N.W. and Berjak, P. (2000) Evolutionary and ecological aspects of recalcitrant seed biology. Seed Science Research 10, 301–306. Pammenter, N.W., Greggains, V., Kioko, J.I, Wesley-Smith, J., Berjak, P. and Finch-Savage, W.E. (1998) Effects of differential drying rates on viability retention of recalcitrant seeds of Ekebergia capensis. Seed Science Research 8, 463–471. Poulsen, K.N. and Eriksen, E.N. (1992) Physiological aspects of recalcitrance in embryonic axes of Quercus robur L. Seed Science Research 2, 215–221. Pritchard, H.W. (1991) Water potential and embryonic axis viability in recalcitrant seeds of Quercus rubra. Annals of Botany 67, 43–49. Pritchard, H.W. and Daws, M. (1997) Preliminary screening results: Butyrospermum paradoxum (Vitellaria paradoxa). The Project on Handling and Storage of Recalcitrant and Intermediate Tropical Forest Tree Seeds, Newsletter No. 2, 6. IPGRI/DFSC. Pritchard, H.W. and Manger, K.R. (1998). A calorimetric perspective on desiccation stress during preservation procedures with recalcitrant seeds of Quercus robur L. CryoLetters 19 (Suppl. 1), 23–30. Pritchard, H.W. and Prendergast, F.G. (1986) Effects of desiccation and cryopreservation on the in vitro viability of embryos of the recalcitrant seed species Araucaria hunsteinii K. Schum. Journal of Experimental Botany 37, 1388–1397. Dessication 08 18/3/02 1:58 pm Page 258

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Pritchard, H.W., Haye, A.J., Wright, W.J. and Steadman, K.J. (1995) A comparative study of seed viability in Inga species: desiccation tolerance in relation to the physical characteristics and chemical composition of the embryo. Seed Science and Technology 23, 85–100. Probert, R.J. and Longley, P. (1989) Recalcitrant seed storage physiology in three aquatic grasses (Zizania palustris, Spartina anglica and Porteresia coarctata). Annals of Botany 63, 53–63. Qiu, Y.L., Lee, J., Bernasconi Quadroni, F., Soltis, D.E., Soltis, P.S., Zanis, M., Zimmer, E.A., Chen, Z., Savolainen, V. and Chase, M.W. (2000) Phylogeny of basal angiosperms: analyses of ﬁve genes from three genomes. International Journal of Plant Sciences 161, S3–S27. Roberts, E.H. and Ellis, R.H. (1989) Water and seed survival. Annals of Botany 63, 39–52. Roberts, E.H. and King, M.W. (1980) Storage of recalcitrant seeds. In: Withers, L.A. and Williams, J.T. (eds) Crop Genetic Resources – the Conservation of Difﬁcult Material. Proceedings of an international workshop held at the University of Reading, UK, 8–11 September, 1980. International Union of Biological Sciences, Serié B42, pp. 39–48. Rodríguez, M.D., Orozco-Segovia, A., Sánchez Coronado, M.E. and Vázquez-Yanes, C. (2000) Seed germination of six mature neotropical rain forest species in response to dehydration. Tree Physiology 20, 693–699. Sacandé, M. (2000) Stress, storage and survival of neem seed. PhD thesis, University of Wageningen, The Netherlands, 124 pp. Setoguchi, H., Osawa, T.A., Pintaud, J.-C., Jaffré, T. and Veillon, J.-M. (1998) Phylogenetic relationships within Araucariaceae based on rbcL gene sequences. American Journal of Botany 85, 1507–1516. Stiles, E.W. (2000) Animals as seed dispersers. In: Fenner, M. (ed.) Seeds: the Ecology of Regeneration in Plant Communities. CAB International, Wallingford, UK, pp. 111–124. Sun, W.Q. (1999) Desiccation sensitivity of recalcitrant seeds and germinated orthodox seeds: can germinated orthodox seeds serve as a model system for studies of recalcitrance? In: Marzalina, M., Khoo, K.C., Jayanthi, N., Tsan, F.Y. and Krishnapillay, B. (eds) IUFRO Seed Symposium 1998. Recalcitrant Seeds. Proceedings of the Conference, 12–15 October 1998. Forest Research Institute Malaysia, Kuala Lumpur, pp. 29–42. Tiffney, B.H. (1986) Evolution of seed dispersal syndromes according to the fossil record. In: Murray, D.R. (ed.) Seed Dispersal. Academic Press, Sydney, Australia, pp. 273–305. Tompsett, P.B. (1984a) Desiccation studies in relation to the storage of Araucaria seed. Annals of Applied Biology 105, 581–586. Tompsett, P.B. (1984b) The effect of moisture content and temperature on the seed storage life of Araucaria columnaris. Seed Science and Technology 12, 801–816. Tompsett, P.B. (1992) A review of the literature on storage of dipterocarp seeds. Seed Science and Technology 20, 251–267. Tompsett, P.B. (1994) Capture of genetic resources by collection and storage of seed: a physiological approach. In: Leakey, R.R.B. and Newton, A.C. (eds) Tropical Trees: the Potential for Domestication and the Rebuilding of Forest Resources. HMSO, London, pp. 61–71. Tompsett, P.B. and Kemp, R. (1996) Database of Tropical Tree Seed Research (DABATTS). Database Contents. Royal Botanic Gardens, Kew, UK, 263 pp. Tompsett, P.B. and Pritchard, H.W. (1993) Water status changes during development in relation to the germination and desiccation tolerance of Aesculus hippocastanum L. seeds. Annals of Botany 71, 107–116. Tompsett, P.B. and Pritchard, H.W. (1998) The effect of chilling and moisture status on the germination, desiccation tolerance and longevity of Aesculus hippocastanum L. seeds. Annals of Botany 82, 249–261. Tweddle, J.C., Turner, R. and Dickie, J.B. (2002) Seed Information Database (Release 2.0, Jan 2002). http://www.rbgkew.org.uk/data/sid Vázquez-Yanes, C. and Orozco-Segovia, A. (1982) Germination of the seeds of a tropical rain forest shrub, Piper hispidum Sw. (Piperaceae) under different light qualities. Phyton 42, 143–149. Vázquez-Yanes, C. and Orozco-Segovia, A. (1984) Ecophysiology of seed germination in the tropical humid forests of the world: a review. In: Medina, E., Mooney, H.A. and Vázquez-Yanes, C. (eds) Physiological Ecology of Plants of the Wet Tropics. Dr. W. Junk, The Hague, The Netherlands, pp. 37–50. Vázquez-Yanes, C., Orozco-Segovia, A., Sánchez-Coronado, M.E., Rojas-Aréchiga, M. and Batis, A.I. (2000) Seed ecology at the northern limit of the tropical rain forest in America. In: Black, M., Dessication 08 18/3/02 1:58 pm Page 259

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Bradford, K.J. and Vazquez-Ramos, J. (2000) Seed Biology: Advances and Applications. CAB International, Wallingford, UK, pp. 375–388. von Teichman, I. and van Wyk, A.E. (1991) Trends in the evolution of dicotyledonous seeds based on character associations, with special reference to pachychalazy and recalcitrance. Botanical Journal of the Linnean Society 105, 211–237. von Teichman, I. and van Wyk, A.E. (1994) Structural aspects and trends in the evolution of recalcitrant seeds in dicotyledons. Seed Science Research 4, 225–239. Walters, C. (1999) Levels of recalcitrance in seeds. In: Marzalina, M., Khoo, K.C., Jayanthi, N., Tsan, F.Y. and Krishnapillay, B. (eds) IUFRO Seed Symposium 1998. Recalcitrant Seeds. Proceedings of the Conference, 12–15 October 1998. Forest Research Institute Malaysia, Kuala Lumpur, pp. 1–13. Walters-Vertucci, C., Crane, J. and Vance, N.C. (1996) Physiological aspects of Taxus brevifolia seeds in relation to seed storage characteristics. Physiologia Plantarum 98, 1–12. Willson, M.F. and Traveset, A. (2000) Ecology of seed dispersal. In: Fenner, M. (ed.) Seeds: the Ecology of Regeneration in Plant Communities. CAB International, Wallingford, UK, pp. 85–110. Woodland, D.W. (2000) Contemporary Plant Systematics, 3rd edn. Andrews University Press, Berrien Springs, Michigan. Dessication 08 18/3/02 1:58 pm Page 260 Dessication 09 18/3/02 1:58 pm Page 261

Part IV

Mechanisms of Damage and Tolerance Dessication 09 18/3/02 1:58 pm Page 262 Dessication 09 18/3/02 1:58 pm Page 263

9 Desiccation Stress and Damage

Christina Walters,1 Jill M. Farrant,2 Norman W. Pammenter3 and Patricia Berjak3 1USDA-ARS National Seed Storage Laboratory, 1111 South Mason Street, Fort Collins, CO 80521, USA; 2Department of Molecular and Cellular Biology, University of Cape Town, 7700, South Africa; 3School of Life and Environmental Sciences, University of Natal, Durban 4041, South Africa

9.1. Introduction 263 9.2. Water Stress 264 9.2.1 Drought vs. desiccation 264 9.2.2. Exacerbating stresses 265 9.2.3. Degrees of stress 265 9.3. Desiccation Damage 269 9.3.1. Mechanical strains and structural damage 269 9.3.1.1. Cellular and subcellular scales 269 9.3.1.2. Molecular scale 273 9.3.2. Metabolically derived damage 278 9.4. Perspectives on the Kinetics of Desiccation Damage 280 9.5. Conclusion 281 9.6. Acknowledgements 282 9.7. References 282

9.1. Introduction organism structures when water was not available to provide physical support and Terrestrial plants became established in the acquiring nutrients when the lack of water Silurian Period (459–409 million years limited the movement of both organisms ago), a few hundred million years after the and the necessary resources. The loss of ﬁrst appearance of multicellular organisms mobility also required plants to develop on earth (Late Precambrian Period: mechanisms to tolerate a spectrum of other 900–545 million years ago) (Strickberger, stresses associated with life on land, partic- 2000). The time required for the necessary ularly temperature extremes and high lev- adaptions to arise attests to the harshness els of radiation. The requirements for water of a water-limited environment. The two are so fundamental (and obvious) that most major challenges were maintaining cell and research has focused on the strategies used © CAB International 2002. Desiccation and Survival in Plants: Drying Without Dying (eds M. Black and H.W. Pritchard) 263 Dessication 09 18/3/02 1:58 pm Page 264

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to address the challenges of life in non- what are generally considered to be aquatic environments (i.e. protective mech- ‘drought-tolerant’ organisms, which usu- anisms) rather than the physical evidence ally resist water loss by having imperme- of failure – collapse and starvation. able outer coverings and reducing surface Studies of cellular responses to water area-to-volume ratios. Drought-stressed stress mostly focus on what cells need to organisms may grow relatively slowly, per- tolerate or resist water loss. Direct evidence haps because of the reduced turgor pres- concerning the damaging process is sparse, sure for cell expansion, but also because of with the mechanisms of damage often the tremendous metabolic costs of main- made by inference from the presence of taining structures that block water loss to putative protectants. Often it is unclear the environment (e.g. Pimienta-Barrios and whether a change in morphology, ultra- Nobel, 1998), supporting root structures structure or metabolism is a simple conse- that seek water, and accumulating compati- quence of drying, a protective strategy or a ble solutes that keep osmotic potentials sign of damage. For example, cessation of low (Jones and Gorham, 1983). The degree metabolism is considered a component of of drought tolerance can be based on how all three possibilities (Vertucci and much the organism resists water loss (i.e. Leopold, 1984; Leprince et al., 1999, 2000; the minimum water potential sustained), Salmen Espindola et al., 1994, respec- the duration that the organism sustains low tively). Damage by desiccation is often water potentials, or the productivity measured by an irreversible change or a (growth) of the organism during the stress. failure of the organism to revive once water When drought-tolerance mechanisms fail, is plentiful again. These rather crude the organism either loses water essential assays do not detect damage that is repara- for structure or compromises metabolism ble, though the suite of repair enzymes pro- to an unsupportable level. Either of these duced de novo upon rehydration attests to consequences is considered a subset of the the turnover of cellular constituents (Oliver strains associated with desiccation damage. et al., 1998). A better understanding of the The distinction between drought and des- nature of desiccation stress and the result- iccation tolerance lies in the protection ing strains is required if we are to under- mechanisms – mechanisms conferring tol- stand fully the nature of protection and erance of drought avoid water removal repair and, ultimately, exploit millions of while mechanisms conferring tolerance of years of evolutionary adaptation to pro- desiccation enable the organism to survive duce plants more capable of withstanding in spite of the water loss. the basic challenges of life in a water- In the above context, drought tolerance limited environment. is really desiccation avoidance. Because the mechanisms required to scavenge and sequester water may differ from those that 9.2. Water Stress enable the organism to exist without it, tolerance of drought does not necessarily 9.2.1. Drought vs. desiccation imply tolerance of desiccation. None the less, both desiccation- and drought-tolerant Most terrestrial organisms can grow (by organisms accommodate life at low water mitotic divisions and cell expansion) at potentials (1 MPa). Mild drops in water potentials greater than about 1 MPa water potential (from 1 to about 3 MPa) (Levitt, 1980; Vertucci and Farrant, 1995, coincide with a series of metabolic changes and references therein). Organisms that that make cells more tolerant of the water successfully deal with lower water poten- stress (Ingram and Bartels, 1996; Bray, tials can either cope with limited water 1997; Oliver et al., 1998; see Chapter 11). availability while maintaining high inter- The products of these metabolic changes nal water concentration, or cope with (antioxidants, low-molecular-weight carbo- water loss. The former class constitutes hydrates, late embryogenesis abundant Dessication 09 18/3/02 1:58 pm Page 265

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(LEA)-like proteins, heat-shock proteins) tective pigments such as anthocyanins are putative protectants for both drought (Smirnoff, 1993; Foyer et al., 1994; and desiccation, even though the mecha- Sherwin and Farrant, 1998; Farrant, 2000; nism of protection is quite different for the Vander Willigen et al., 2001), increases in two types of stress. Future research should xanthophyll pools and conversion to the be directed towards resolving this apparent de-epoxide forms (Smirnoff, 1993; Foyer et contradiction. al., 1994; Kranner and Grill, 1997) and production of free-radical-scavenging enzymes (Foyer et al., 1994; Wise, 1995; Pammenter 9.2.2. Exacerbating stresses and Berjak, 1999). Low temperatures tend to intensify Water-stressed plants ( w 1 MPa) are water stress. The classic case describes predisposed to damage by other stresses. temperatures at which water freezes extra- Free-radical production appears to be a cellularly, thereby creating a water poten- common effect of numerous stresses tial gradient and forcing intracellular water including drought and desiccation, ageing, to migrate out of the cell (Meryman, 1974; freezing, pollution, temperature extremes Steponkus, 1979). Lowering the tempera- and radiation (Elstner et al., 1988; ture requires an increase in the water con- McKersie et al., 1988; Puntarulo et al., tent of cells to maintain a constant w 1991; Hendry, 1993; Leprince et al., 1993; ( w 10 MPa). This requirement for more Foyer et al., 1994; Wise, 1995; Bowler and water at lower temperatures is related to Fluhr, 2000), and so it is likely that these the exothermic nature of water condensa- stresses may be cooperative or synergistic. tion on macromolecular surfaces at low Stressed plants are particularly susceptible water potentials (Walters, 1998). to photo-oxidative damage (Elstner et al., Consistently, critical water contents that 1988; Foyer et al., 1994; Wise, 1995). Light lead to desiccation damage are greater at energy, which was efﬁciently harvested, lower temperatures (Kovach and Bradford, transduced and assimilated in non-water- 1992; Vertucci et al., 1995; Eira et al., stressed cells, may be absorbed by the pho- 1999a). Indeed, the moisture content giving tosynthetic apparatus and dissipated as rise to changes in membrane phase behav- reactive oxygen molecules that damage cel- iour, the most often cited consequence of lular constituents (Bewley and Krochko, desiccation stress in model systems, 1982; Vertucci et al., 1985; Kaiser, 1987; increases with decreasing temperature Elstner et al., 1988; McKersie et al., 1988; (Crowe et al., 1989; Crowe and Crowe, Smirnoff, 1993; Foyer et al., 1994; Tuba et 1992; Hoekstra and Golovina, 1999; al., 1996, 1998; Sherwin and Farrant, 1998; Hoekstra et al., 1999; Bryant et al., 2001). Csintalan et al., 1999; Farrant, 2000; Vander Willigen et al., 2001). Desiccation- tolerant plants initiate many processes that 9.2.3. Degrees of stress are considered to be protective against photochemical damage. These processes There are many ways to measure water loss include dismantling of the photosynthetic in cells. Water content (absolute or relative) apparatus (Gaff and Hallam, 1974; (e.g. Berjak et al., 1992; Sun et al., 1994; Hetherington et al., 1982a,b; Öquist and Farrant, 2000), water potential and related Strand, 1986; Gaff, 1989; Demmig-Adams functions (Roberts and Ellis, 1989; Vertucci and Adams, 1992; Vertucci and Farrant, and Roos, 1990; Tompsett and Pritchard, 1995; Tuba et al., 1996, 1998; Sherwin and 1993; Vertucci and Farrant, 1995; Vertucci Farrant, 1998; Farrant et al., 1999; Farrant, et al., 1995; Farrant and Walters, 1998), cell 2000), chlorophyll shading by leaf folding volume (Meryman, 1974; Steponkus, 1979; or rolling (Dalla Vecchia et al., 1998; Murai and Yoshida, 1998a,b), intracellular Sherwin and Farrant, 1998; Farrant et al., viscosity (Vertucci and Roos, 1990; Koster, 1999; Farrant, 2000), accumulation of pro- 1991; Williams et al., 1993; Leopold et al., Dessication 09 18/3/02 1:58 pm Page 266

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1994; Buitink et al., 1998b; Leprince and and Bradford, 1992) while other laborato- Hoekstra, 1998; Bryant et al., 2001) inter- ries show detrimental effects of drying at molecular proximity (Lis et al., 1982; much higher water contents (Probert and Steponkus et al., 1995; Wolfe and Bryant, Longley, 1989; Vertucci et al., 1995). 1999; Bryant et al., 2001) and structural Similar discrepancies are reported for cof- water (Ladbrooke and Chapman, 1969; fee (Coffea arabica), lemon (Citrus limon), Vertucci and Leopold, 1984, 1987; Crowe neem (Azadirachta indica) and tea et al., 1990; Pammenter et al., 1991) all (Camellia sinensis) (e.g. Ezumah, 1986; change with desiccation (Fig. 9.1; see Ellis et al., 1990; Chaudhury et al., 1991; Chapter 2). Each of these parameters has Berjak et al., 1993; Hong and Ellis, 1995; been used to define the level of water Dussert et al., 1999; Eira et al., 1999a; stress, but it is unclear which parameter(s) Sacandé et al., 2000). Even when the recal- causes the stress and which is merely a citrant category is undisputed, there are correlate of the stress. The distinction is differences among species in how much important as it reveals the nature of the water can be removed and how long a strain and the damage. A better under- water-stressed seed can survive (Berjak et standing of the nature of desiccation stress al., 1989, 1990; Farrant et al., 1989, 1997; and strain will also reveal whether damage Vertucci and Farrant, 1995; Pammenter and accrues continuously, whether it occurs Berjak, 1999). To accommodate the vari- when the stress or strain reaches a thresh- ability in desiccation tolerances observed old, and whether numerous different among species, the categories of seed strains result from the removal of water behaviour were further divided to distin- from the cell. In other words, is damage by guish highly recalcitrant, recalcitrant, desiccation a single event, a continuous intermediate, sub-orthodox and orthodox; event or a series of insults to the cell or and Pammenter and Berjak (1999) have organism? suggested that, in reality, a continuum Desiccation tolerance/sensitivity has tra- exists among seed species, based on desic- ditionally been regarded as a qualitative cation response. feature: cells either do or do not survive Not only are there differences among drying. The definition of ‘dry’ varies among species in response to desiccation, but laboratories or experiments (i.e. 90% water there are also differences for seeds of the 1 loss; water contents 10% (0.10 g H2O g same species and among tissues as a func- dry weight or fresh weight); water contents tion of developmental stage. Studies of the in equilibrium with 75% or maybe even acquisition and loss of desiccation toler- 15% relative humidity (RH); water con- ance during embryogenesis and germina- tents achieved after a material has been tion have demonstrated that tolerance to freeze-dried or held in a laminar flow hood desiccation progressively increases with for some period of time), and most studies seed maturation (Berjak et al., 1990, 1992, use just one drying level. The binary 1993; Finch-Savage, 1992b; Farrant et al., approach suggested that damage was a sin- 1993; Sun and Leopold, 1993; Sun et al., gle event that either happened or did not 1994; Tompsett and Pritchard, 1993; happen. Seeds were assigned to one of two Vertucci et al., 1995; Farrant and Walters, categories, orthodox or recalcitrant, to dis- 1998; reviewed by Vertucci and Farrant, tinguish between those that survived or did 1995; Pammenter and Berjak, 1999; see not survive drying (Roberts, 1973; see Chapter 5) and decreases with germination Chapter 5). Classification of seeds of cer- (Sargent et al., 1981; Senaratna and tain species as recalcitrant has been dis- McKersie, 1983; Leprince et al., 1990; puted as laboratories around the world Reisdorph and Koster, 1999). Correlative have demonstrated variable success in dry- evidence suggests that there are similar ing them. For example, some groups report stages of tolerance in vegetative tissues of survival of Zizania palustris at water con- desiccation-tolerant angiosperms (Farrant, 1 tents as low as 0.07 g H2O g dw (Kovach 2000; Vander Willigen et al., 2001; J.M. Dessication 09 18/3/02 1:58 pm Page 267

Desiccation Stress and Damage 267 ost

(1983), Vertucci

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Walters Walters

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structures fail structures Measures of cytoplasmic accounted for.

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, 1997) at full hydration and an assumption that 8% of the , 1997) at full hydration

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Membranes Osmotic excursions Osmotic et al. I II V III IV level Hydration MPa –0.1 –1 –10 –100 –1000 1 99 90 50 10 Stress 99.9 (1998b, 2000). Conceptual models of hydration are taken from Rupley (1998b, 2000). Conceptual models of hydration 99.99 RH (%) et al. 3.0 2.0 1.0 0.5 0.1 0.05 0.01 ) –1 17 7.0 3.5 1.8 0.15 0.07 1.0 0.93 0.84 0.82 0.76 0.76 0.76 Strain 1.0 Volume (%)Volume WC (g g 0.49 0.33 0.22 0.13 0.12 0.12 0.05 immature mature immature mature ) and the proportion of space occupied by cell organelles (Farrant cell organelles (Farrant ) and the proportion of space occupied by 1 0.1 0.2 0.4 1.5 63 410 <410 ) –2 axes (Farrant and Walters, 1998). Changes in volume are calculated for immature and mature bean axes from the amount of water l are calculated for immature and mature bean axes from the amount of water 1998). Changes in volume Walters, and axes (Farrant 0.06 ? ? ? ? ? ? (N s m Viscosity immature mature Scales of water stress (water potential and relative humidity (RH)), water loss (water content) and strain (volume and viscosit (volume content) and strain loss (water humidity (RH)), water potential and relative stress (water Scales of water (1990) and Vertucci and Farrant (1995). Different mechanisms of damage are as described in the text. (1995). Different mechanisms and Farrant Vertucci (1990) and Fig. 9.1. Fig. volume of the cytoplasmic matrix at full hydration is dry matter; the effect of positive turgor pressure on cell volume is not turgor pressure on cell volume is dry matter; the effect of positive matrix at full hydration of the cytoplasmic volume (1998) and Buitink viscosity are taken from Leprince and Hoekstra (Leopold, 1986). Water contents as a function of water potential are described for mature pea axes (Vertucci and Leopold, 1987; potential are described for mature pea axes (Vertucci contents as a function of water Water (Leopold, 1986). conceptual models of hydration (shaded rectangles) and projected damage to cells (white box). The ﬁgure is modiﬁed from a simil The box). (shaded rectangles) and projected damage to cells (white conceptual models of hydration Aesculus hippocastanum density is 1 g ml (assuming water Dessication 09 18/3/02 1:58 pm Page 268

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Farrant, unpublished data). Vegetative tis- w) in most embryos (Avicennia marina, sues from resurrection and cold-tolerant and perhaps other highly recalcitrant seeds, angiosperms require time to adapt to water- excepted (Farrant et al., 1992, 1993)) was stress situations (Steponkus et al., 1995; found to decline to about 10 to 15 MPa Oliver et al., 1998; Farrant et al., 1999; see (Pritchard, 1991; Finch-Savage, 1992a; Chapter 7), presumably to induce protec- Tompsett and Pritchard, 1993; Vertucci and tive mechanisms. It is surmised that in vege- Farrant, 1995; Farrant and Walters, 1998). tative tissues of angiosperms there is also a Embryos that are recalcitrant cannot be developmental programme in response to dried below this level. During the final stress that leads to progressively greater des- stages of maturation, species defined as iccation tolerance. The variability in critical having intermediate postharvest physiology water contents among different species, dur- (e.g. coffee, citrus and papaya) acquire the ing maturation or germination of embryos ability to tolerate between about 60 and and during adjustment of vegetative tissues, 80 MPa (Dussert et al., 1999; Eira et al., leads to the general conclusion that toler- 1999a; Sacandé et al., 2000) (neem is con- ance/sensitivity is a quantitative feature. sidered to be in the intermediate category The quantitative nature of desiccation as it has diminishing longevity at lower tolerance/sensitivity suggests two broad water potentials (see Ellis et al., 1990)). possibilities for the mechanism(s) of desic- Truly orthodox species survive the immedi- cation damage in organisms. Desiccation ate effects of complete water loss, but suc- damage may occur by a single mechanism cumb more rapidly if they are dried below with species and tissue types differing in about 190 to 250 MPa (Vertucci and how much damage they can accrue or how Leopold, 1987; Vertucci and Roos, 1990; much stress they can endure. Alternatively, Walters, 1998). Vegetative tissues of resur- damage by different mechanisms may rection plants acquire the same degree of occur at multiple levels of stress and extreme tolerance (Bewley, 1979; Gaff, species and tissue types differ in which 1989; Oliver et al., 1998). Cells that have stresses, or combinations of stresses, they the genetic capacity to induce tolerance can withstand. The former possibility sug- mechanisms progress towards tolerance gests that a desiccation-tolerant organism and, with sufficient time, complete the requires only a simple cadre of protectants, developmental programme leading to full while the latter situation suggests that a tolerance of desiccation (e.g. Finch-Savage, complex suite of protectants, each with a 1992b). different function, is required for an organ- The differing levels of desiccation sensi- ism to be truly tolerant of dehydration. tivity among developmental stages and seed Evidence is accumulating to suggest that categories appear to correspond to levels of desiccation damages cells and organisms by physiological activity documented in desic- many mechanisms and that different types cation-tolerant organisms with studies of of damage occur at different levels of stress. metabolic activity and properties of the When expressed in terms of water poten- aqueous medium (Vertucci and Farrant, tial, developing embryos acquire tolerance 1995). Five hydration levels designate the stepwise (Vertucci and Farrant, 1995; cells’ ability to support growth (Level V, 0 Farrant and Walters, 1998). During histodif- to 1.5 MPa), to photosynthesize and effect ferentiation, embryos are damaged by water stress-related metabolism (Level IV, 1.8 to potentials less than about 1.2 to 2 MPa 4 MPa), to respire (Level III, 5 to about (Vertucci and Farrant, 1995, and references 12 MPa), to carry out catabolic reactions therein). During dry matter accumulation, (Level II, about 15 to 190 MPa), and to embryos tolerate water potentials as low as be almost in stasis (Level I, 220 MPa) about 4 to 5 MPa ( Farrant et al., 1992, (Fig. 9.1) (Clegg, 1986; Roberts and Ellis, 1993; Farrant and Walters, 1998). Following 1989; reviewed by Vertucci and Farrant, vascular separation, the water potential at 1995; Farrant, 2000; Vander Willigen et al., which damage is first measured (i.e. critical 2001). These hydration levels correspond to Dessication 09 18/3/02 1:58 pm Page 269

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other parameters that measure the extent of by a reduced cell size or lack of integrated desiccation (Fig 9.1) (Chapters 2 and 4). In metabolism, do not in themselves imply Hydration Level V, turgor pressure is posi- damage. They may be purely consequences tive and water behaves as it would in a of water removal and may be completely dilute solution. At lower hydration levels, reversible once water is added back to the cells shrink (Meryman, 1974; Steponkus, system. Therefore, damage from desicca- 1979; Steponkus et al., 1995), the properties tion is not indicated by differences of water change (Rupley et al., 1983; between the hydrated and dry state, but Vertucci, 1990) and the aqueous matrix rather by the resumption of normal activity becomes more viscous having properties of upon rehydration. syrups (Level IV), rubbers (Level III) and The number of different stresses that leathers and glasses (Level II) (Vertucci and can be associated with removal of water Roos, 1990; Slade and Levine, 1991; from cells can be attributed to the multiple Williams et al., 1993; Leopold et al., 1994; roles that water plays in supporting life. Buitink et al., 1998b; Leprince and Water plays a structural role: at the cellular Hoekstra, 1998). Viscosity is minimized at scale, water, fills spaces and provides tur- the transition from Level II to I (Vertucci gor, while, at the molecular scale, water and Roos, 1990; Buitink et al., 1998b), a provides hydrophilic and hydrophobic moisture level that also corresponds to a associations and controls intermolecular discrete change in the heat capacity of distances that determine the conformation water (Rupley et al., 1983; Vertucci, 1990; of proteins, polar lipids and the partition- Buitink et al., 1996; M.T.S. Eira, unpub- ing of molecules within organelles. With lished data) and poorly understood charac- water present, reactive surfaces of metals teristics of water sorption (Vertucci and or molecules are not as exposed, and this Leopold, 1987; Vertucci and Roos, 1990; limits reactivity among molecules. Water Vertucci et al., 1994; Buitink et al., 1998a,b; also plays a role in controlling metabolism, Walters, 1998; Eira et al., 1999b). With the as it is a reactant and product of many exception of Hydration Level I (Vertucci reactions. As a dilutant, water affects the and Leopold, 1987; Eira et al., 1999b; chemical potential of other molecules, M.T.S. Eira, L.S. Caldas and C. Walters potentially shifting the likelihood of reac- unpublished data), the relationships tions. Water also provides the fluid matrix between physical properties of water and that allows diffusion of substances to reac- water potential appear similar among tive sites. Changes in water concentration diverse cells (Fig. 9.1), perhaps with only affect viscosity of the matrix and the over- subtle differences to distinguish desiccation- all mobility of dissolved or suspended mol- tolerant from less tolerant materials (Koster, ecules. The drier the medium becomes, the 1991; Berjak et al., 1993; Farrant and more viscous it becomes, until it is essen- Walters, 1998; Leprince et al., 1999). If des- tially a solid matrix, trapping molecules iccation stress occurs when cells traverse (Slade and Levine, 1991; Williams et al., critical water potentials or hydration levels, 1993; Leopold et al., 1994; Buitink et al., then the stresses experienced by desiccat- 1998b; Wolfe and Bryant, 1999). As one ing cells are similar among organisms, but would expect from all the roles of water, the responses to those stresses (i.e. damage there will be a number of strains that the versus protection) differ with desiccation tissues undergo when water is removed. tolerance (Pammenter et al., 1991). 9.3.1. Mechanical strains and structural damage 9.3. Desiccation Damage 9.3.1.1. Cellular and subcellular scales As water is removed from cells, the physical and physiological properties of the cells The first sign of desiccation/drought stress change. These changes, often characterized is the loss of turgor pressure. This occurs at Dessication 09 18/3/02 1:58 pm Page 270

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water potentials of about 1 to 2 MPa, mature embryos (Fig. 9.1), this strain of cell coinciding with the water potential range contraction can be avoided by accumulat- designated as ‘permanent wilting point’ for ing dry matter. non-transpiring vegetative tissue (Levitt, Differences in the degree to which cell 1980). At lower water potentials, cells lose walls contract compared with protoplasm water and shrink (Meryman, 1974, may cause mechanical stress and damage Steponkus, 1979; Levitt, 1980; Steponkus to the plasmalemma or plant cells during and Lynch, 1989; Steponkus et al., 1995). dehydration. The tight attachment of the Osmotic adjustments, which lessen the plasmalemma to the cell wall is believed to water potential difference between cells create tension to the membrane in shrink- and the environment and augment the ing cells (e.g. Murai and Yoshida, 1998b), amount of dry matter in cells, can prevent which is most profound at the cell water loss and cell contraction at water wall–plasmalemmma attachments near the potentials between 1 and 2.5 MPa plasmodesmata (Iljin, 1957; Bewley and (Levitt, 1980; Jones and Gorham, 1983). Krochko, 1982). Plasmolysis, where the Osmotic adjustments are fairly ineffective plasma membrane separates from the cell at reducing strains when cells are exposed wall, appears to mitigate damage to whole to lower water potentials (Wolfe and cells during severe water stress (Murai and Bryant, 1999). In slow-freezing experi- Yoshida, 1998b), and there is some evi- ments, believed to mimic dehydration dence to suggest that cells in desiccation- stress, protoplasts can undergo reversible tolerant seeds are slightly plasmolysed contraction–expansion cycles, or ‘osmotic (Perner, 1965). Observations of plasmolysis excursions’, when slowly cooled and may be an artefact of the aqueous fixatives ≈ warmed from > 0°C ( w 0.5 MPa) to used to study dry organisms (Öpik, 1985; temperatures of 2 to 5°C ( 2.5 w Platt et al., 1997; Wesley-Smith, 2001). In 6 MPa) (Meryman, 1974; Steponkus, studies using anhydrous chemical fixation 1979; Steponkus and Lynch, 1989). A (Öpik, 1985) or freeze substitution (Wesley- 60–80% reduction in cell volume occurs Smith, 2001, Wesley-Smith et al., 2001), when the water potential of cells decreases the plasma membrane remained closely from about 0.5 MPa to about 4.5 to appressed to the cell walls, and both the 6 MPa (4 to 5°C) (Meryman, 1974; cell wall and the plasmalemma became Steponkus, 1979; Steponkus and Lynch, highly convoluted during desiccation of 1989). Similar contraction was calculated tolerant cells. Öpik (1985) demonstrated for immature embryo cells in which 88% that the plasmalemma separated from the of the cell volume was occupied by water cell wall during rehydration as a result of (Fig. 9.1). However, cells filled with dry differential swelling or weakening of the matter reserves (mature embryos in Fig. cell wall–plasmalemma association caused 9.1) do not contract as much as highly vac- by detergents such as dimethylsulphoxide. uolated cells (immature embryos in Fig. The mechanical properties of the cell wall, 9.1). For a similar reduction in water including its elasticity, ability to fold and potential to 5 MPa, the cells of fully associations with plasmodesmata, influ- mature bean axes contract only by about ence the degree of plasma membrane dis- 18%, and complete desiccation only causes ruption consequent upon contraction or a 24% reduction in volume in these cells expansion (Webb and Arnott, 1982; Öpik, (Fig. 9.1). When cells that have not been 1985; Murai and Yoshida, 1998b; Vicré et acclimatized to the water stress shrink by al., 1999). 50–80%, they burst when returned to the Cell membranes must fold or vesiculate original water potential. This observation to accommodate the volume changes dur- led to the concept of ‘minimum critical ing cell contractions. Conservation of mem- volume’ (Meryman, 1974), which describes brane surface area during contraction is the limits to which a cell can contract in a critical for successful rehydration. If the reversible osmotic excursion. As seen for surface area of the plasmalemma is Dessication 09 18/3/02 1:58 pm Page 271

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reduced too much, the cell bursts upon water stress, surviving to water potentials rehydration, suggesting that there is a criti- of 12 MPa or less, compared with the cal minimum surface area, rather than a benchmark of 5 MPa described above for critical minimum volume, to which cells protoplasts from non-acclimatized cells. can survive (Steponkus, 1979; Steponkus None the less, drying results in some and Lynch, 1989; Steponkus et al., 1995). degree of cell contraction, which is mostly Protoplasts from cells that are not acclima- completed when the water potential of the tized to the cold contract through invagina- cell is reduced to 12 MPa (Fig. 9.1). In tions of the plasma membrane, which cells that survive water potentials of about eventually form endocytotic vesicles that 12 MPa but not lower, both endo- and cannot be reincorporated into the plas- exocytotic vesicles have been observed (P. malemma upon warming (Steponkus and Berjak and N.W. Pammenter, unpublished Lynch, 1989; Steponkus et al., 1995). The date). These observations are not reported plasma membrane of protoplasts from cells in extremely dried embryos, perhaps more tolerant of water stress (i.e. acclima- because of technical problems of fixation. tized by low temperatures) contracts In severely dried cells of fully desiccation- through exocytotic extrusions which tolerant seeds, the plasmalemma stays remain continuous with the plasma mem- intact and closely attached to the cell wall brane and help to conserve the membrane as this folds, suggesting that the membrane surface area (Steponkus and Lynch, 1989; surface area remains relatively constant Steponkus et al., 1995). High phospho- during drying even though the cell volume lipid:sterol ratios and high amounts of is diminished (Öpik, 1985). Some mem- diunsaturated fatty acids in the plas- brane constituents may be removed during malemma appear to facilitate exocytotic cell contraction as evidenced by whorls of folding in shrinking protoplasts and greater membrane close to the plasmalemma in elasticity of the expanding membranes seed cells (Webster and Leopold, 1977; (Steponkus and Lynch, 1989; Steponkus et Öpik, 1985; Wesley-Smith et al., 2001) and al., 1995). Protoplasts with these properties circular membrane structures and plas- tend to survive to lower water potentials toglobuli within chloroplasts in sections of (Steponkus et al., 1995). leaf tissue from desiccation-tolerant The mechanism by which membrane angiosperms (Farrant et al., 1999; Farrant surface area is conserved in intact cells is 2000; Mundree et al., 2000). These mem- largely unknown. There are some studies brane bodies have been proposed to pro- of the effect of dehydration on cell volume vide additional membrane reserves upon and membrane configuration in cells from rehydration (Webster and Leopold, 1977; plant embryos, but these are often con- Farrant et al., 1999; Mundree et al., 2000), founded by problems associated with using although mechanisms by which they aqueous fixatives (Platt et al., 1997; would be reinserted are not clear and their Wesley-Smith et al., 2001). In addition, the very presence may be artefacts of aqueous studies often use mature embryos (recalci- fixation. Alternatively, these membrane trant or orthodox) where > 50% of the cell abnormalities may arise from other volume is occupied by dry matter (e.g. organelles, such as endoplasmic reticula, Farrant et al., 1997). These cells will not and may participate in autophagy or vac- experience the same degree of shrinkage as uole formation (Wesley-Smith et al., 2001). highly vacuolated cells (Fig. 9.1), and so The shapes of nuclei, mitochondria and the need for conserving membrane surface plastids in dried cells of desiccation-toler- area is not as critical. Circumventing the ant seeds are irregular and convoluted, sug- problem of cell shrinkage may explain why gesting that the surface area of the most orthodox and recalcitrant embryos membranes of these organelles are also (except for A. marina and other recalcitrant conserved simply by folding (Öpik, 1985). seeds with highly vacuolated cells (Farrant The membranes of cell vacuoles are et al., 1992, 1993)) are fairly tolerant of likely to experience tensions similar to Dessication 09 18/3/02 1:58 pm Page 272

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those described for protoplast membranes mature orthodox seeds lack defined cristae during osmotic excursions, and so are (Bergtrom et al., 1982; Thomson and Platt- prone to rupture, with lethal consequences, Aloia, 1984; Farrant et al., 1997) and mito- following exposure to water potentials of chondrial proteins are easily extractable 2.5 to 5 MPa (Murai and Yoshida, from dried pollen (Hoekstra and van 1998a). Highly vacuolated cells of imma- Roekel, 1983). Conversely, mitochondria ture seeds (Berjak et al., 1984, 1994; from immature embryos and recalcitrant Farrant et al., 1989, 1997) and desiccation- seeds are more defined, and the greater dif- sensitive vegetative tissue (Farrant and ferentiation has been linked to greater sen- Sherwin, 1997; Farrant, 2000) are particu- sitivity to desiccation (Farrant et al., 1997). larly sensitive to tonoplast dissolution. Chloroplast structure also degrades during Replacing the water in vacuoles with solid water stress. Dried leaves of the desicca- material reduces the degree to which vac- tion-tolerant grasses Borya nitida and uoles must contract, thereby lessening the Xerophyta humilis become yellow, concur- tension on tonoplast membranes during rent with the loss of grana stacks in the drying. Dry matter reserves naturally accu- chloroplasts (Gaff and Hallam, 1974; mulate during embryogenesis in orthodox Farrant, 2000). Using fluorescence-induc- and some recalcitrant seeds, and may tion kinetics to study partial processes of explain the progressive tolerance to low photosynthesis, researchers found a water contents in developing embryos decrease in the efficiency of photosystem II (Vertucci and Farrant, 1995; Farrant et al., at water potentials between 3 and 4 1997; Farrant and Walters, 1998). There is MPa (Wiltens et al., 1978; Hetherington, et also accumulation of dry matter in vac- al., 1982b; Vertucci et al., 1985; Sherwin uoles of vegetative tissues in many of the and Farrant, 1998; Tuba et al., 1998; desiccation-tolerant angiosperm species Csintalan et al., 1999) or during acclimati- during acclimatization to water stress zation to winter (Öquist and Strand, 1986). (Farrant, 2000). This decline could be a consequence of Water loss results in a general contrac- photochemical damage, but is more likely tion of cell volume. The plasmalemmae of to be a reflection of protective dismantling plant cells can be damaged if they are of photosystem II (Demmig-Adams and sheared from cell walls, which contract less Adams, 1992; Farrant, 2000). Indeed, the than protoplasm, or if contraction results dismantling of the photosynthetic appara- in an irreversible loss of membrane surface tus during drying of B. nitida and X. area. In addition to protection by filling humilis is required for survival: plants cells with dry matter (described above), the dried too rapidly stay green and do not consequences of volume changes can also recover (Gaff and Hallam, 1974; Farrant et be lessened by initial high surface area-to- al., 1999). volume ratios of cells and vacuoles (Iljin, Slight water stress ( 1 w 3 MPa) 1957; Bewley, 1979) and may explain why enhances the protein synthesis that is cells from non-vascular plants, which usu- believed to be important for conferring tol- ally have small vacuoles and lack plasmo- erance (Ried and Walker-Simmons, 1993; desmata, do not appear to suffer physical Vertucci and Farrant, 1995; Ingram and damage upon contraction (reviewed by Bartels, 1996; Oliver et al., 1998; Mundree Bewley and Krochko, 1982). Damaging et al., 2000; Whittaker et al., 2001). Further effects of cell contraction are usually mani- drying reduces the rate of protein synthesis fested during rehydration, suggesting that in both tolerant and sensitive cells (Bewley the stress and damage are not direct effects and Krochko, 1982; Salmen Espindola et of desiccation, but rather indications of al., 1994; Ingram and Bartels, 1996; Oliver rehydration stress and mechanical failure. et al., 1998; Mundree et al., 2000; A dismantling of mitochondria and Whittaker et al., 2001), perhaps because of chloroplasts is associated with severe a dismantling of endoplasmic reticulum, water stress. Mitochondria observed in dictyosomes and polysomes (Webster and Dessication 09 18/3/02 1:58 pm Page 273

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Leopold, 1977; Thomson and Platt-Aloia, occur. A consequence of these molecular 1984; Farrant et al., 1997; Wesley-Smith et aggregations is an increased ordering of al., 2001). molecular structures, and it may seem Indirect evidence from recalcitrant ironic that primary lesions during drying seeds suggests that, during dehydration, are directly attributed to order rather than the cytoskeleton is disrupted at fairly high to loss of it. Drying-induced compaction of water potentials (3.8 MPa for Trichilia molecules requires greater packing effi- dregeana and 3.5 MPa for Quercus robur) ciency, resulting in localized enrichments leading to an abnormal distribution of of similar-type molecules in a process organelles within cells (Berjak et al., 1999; known as demixing (Lis et al., 1982; Bryant Mycock et al., 2000). Although it is tacitly and Wolfe, 1989; Rand and Parsegian, assumed that cytoskeletal disassembly 1989; Bryant et al., 1992). Molecules remix must occur during dehydration in desicca- upon rehydration, but the reactions that tion-tolerant seeds (and vegetative tissues), occurred in the desiccated state may have it is its failure to reconstitute that charac- irreversible consequences. terizes this aspect of dehydration-related Intermolecular associations of polar injury in recalcitrant material (Mycock et lipids are intrinsically linked to the water al., 2000). content of the medium. Under aqueous There is clearly a general trend towards conditions, polar lipids spontaneously contraction or disassembly of cellular align to form micelles or bilayer structures machinery during water stress to about 5 depending on the polar head group of the MPa. In most desiccation experiments, lipid. Acyl chains within bilayers are plant materials are stressed further and cell more-or-less mobile, giving considerable survival is assayed by whether or not fluidity to the structure and allowing pro- organelles reassemble upon rehydration. In teins and other constituents to be inserted. desiccation-sensitive cells that do not rup- Drying brings membrane bilayers into close ture, the protein-synthesizing machinery proximity and causes membrane con- does not recover, nor do mitochondria and stituents to segregate laterally into different chloroplasts resume normal function; domains enriched with particular lipid organelles become irregularly shaped and classes or proteins (Lis et al., 1982; Bryant disorganized (reviewed by Bewley and and Wolfe, 1989; Rand and Parsegian, Krochko, 1982; Farrant et al., 1989; Berjak 1989; Bryant et al., 1992; Crowe and et al., 1990; Mycock et al., 2000). The con- Crowe, 1992; Steponkus et al., 1995; traction and dismantling of organelles Hoekstra and Golovina, 1999) (Fig. 9.2). described above are clearly signs of water The closer packing between membranes stress, but it is unclear whether these and among membrane constituents results changes are symptoms of damage occurring in greater rigidity of the fatty acid domain at 5 MPa, or means of protection when within the bilayer. There are two mecha- water stress intensifies. It is also unclear nisms, based on either intra- or interlamel- whether the failure to reconstitute lar events, used to explain why fatty acid organelles indicates a primary site of dam- domains become more rigid. If water mole- age or a general debilitation when cells die. cules are removed from between adjacent These cause and effect arguments have led polar head groups, the associated fatty acids researchers to study the primary effects of compress because of the increased strength dehydration on the structure of macromol- of van der Waals attractions (Crowe et al., ecules. 1990; Crowe and Crowe, 1992; Hoekstra and Golovina, 1999). Alternatively, as different bilayers come into close apposition, 9.3.1.2. Molecular scale strong repulsive hydration forces keep Removing water from cells pushes cellular them separate, but create isotropic tensions constituents together, causing them to that lead to lateral compression within the interact in ways that might not otherwise acyl domain (Lis et al., 1982; Wolfe, 1987; Dessication 09 18/3/02 1:58 pm Page 274

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Rand and Parsegian, 1989; Bryant and branes, but has been demonstrated in cells Wolfe, 1992; Wolfe and Bryant, 1999). from non-acclimatized leaves that were ≈ Increased rigidity eventually leads to phase lethally cooled to 5°C (oat, w 6 MPa) ≈ transitions within the membrane from a or 10°C (rye, w 12 MPa) (Steponkus et fluid to a gel state (Ladbrooke and al., 1995) and more frequently in animal Chapman, 1969; Cullis and de Kruijff, cells (Cullis and de Kruijff, 1979; Crowe and 1979). While these phase transitions are Crowe, 1992). Evidence of cell fusion, but completely reversible, they are believed to not via hexagonal-phase changes, is common interfere with the semi-permeable proper- in desiccation-damaged cells, protoplasts ties of membranes. Permanent damage and liposomes (e.g. Crowe et al., 1986; comes from the exclusion of proteins from Steponkus et al., 1995). In oat and rye leaves parts of the bilayer (Rand and Parsegian, acclimatized to cold (but clearly not fully 1989; Bryant and Wolfe, 1992; Crowe and desiccation-tolerant), fusion of plasmalemma Crowe, 1992; Hoekstra and Golovina, 1999) and endomembrane systems is suggested at (Fig. 9.2). Transient damage occurs upon temperatures between 10 and 40°C (12 rehydration: the rush of water on to an w 48 MPa), depending on the level of inelastic membrane may cause it to rupture cold tolerance achieved (Steponkus et al., (Murphy and Noland, 1982; Steponkus et 1995). Upon rehydration, improperly fused al., 1995; Hoekstra et al., 1999) or imper- membranes produce vesicles that exclude fect packing among different domains may cell constituents or are combinations of dif- cause leakage of cellular constituents ferent membrane systems (e.g. the plas- (Crowe and Crowe, 1992; Hoekstra et al., malemma fuses with chloroplast outer 1999). membrane or with endoplasmic reticulum) The close approach of membrane systems (Fig. 9.2). Because the osmotic balance and the lateral demixing of membrane com- inside and outside the cells has been components can lead to an even greater threat to pletely disrupted, vesicles produced from membranes than lamellar fluid-to-gel transi- membrane fusions are identified by their tions. Membranes can fuse together, causing inability to expand during rehydration the complete loss of compartmentation (Steponkus et al., 1995). within the cell (Crowe et al., 1986; Crowe Most of our understanding of how polar and Crowe, 1992; Steponkus et al., 1995) lipids behave in water-stressed situations (Fig. 9.2). Fusion is known to occur among comes from model studies of liposomes liposomes and native membrane fractions, with known composition. In these systems, although the mechanism that causes polar phase transitions are usually studied, even lipids to cross over to a different bilayer is though they may only be harbingers of real unclear. In principle, the hydration charac- damage. Phase transitions of prepared teristics of individual lipids and lipids in a membrane systems occur at a range of mixture, the intrinsic curvature of different water contents and temperatures depend- head groups, the water content and the tem- ing on the saturation of the acyl chains and perature allow the formation of inverted the presence of non-phospholipids (e.g. micelles within closely appressed bilayers Ladbrooke and Chapman, 1969; Cullis and (Cullis and de Kruijff, 1979; Crowe et al., de Kruijff, 1979; Crowe et al., 1989; 1986; Steponkus et al., 1995) (Fig. 9.2). In Steponkus et al., 1995). A water potential domains enriched with non-bilayer-forming of about 12 MPa is often cited as critical. lipids such as phosphatidylethanolamine- It has been suggested that structural water diglycerides or monogalactosyl-diglycerides, needed for the proper spacing of polar the polar head groups coalesce into rings and head groups is removed at w 12 MPa the acyl chains extend radially outwards in (Ladbrooke and Chapman, 1969; Crowe et ≈ what is known as a hexagonal phase (Cullis al., 1990). Also, at w 12 MPa, large, and de Kruijff, 1979; Siegel et al., 1994; potentially deforming hydration forces Steponkus et al., 1995). Fusion via hexago- result from the close approach of mole- nal-phase changes is rare in native mem- cules (Wolfe, 1987). Dessication 09 18/3/02 1:58 pm Page 275

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Plasma membrane

Hydrated membrane systems Cytoplasm Endomembrane

Exclusion of intrinsic proteins –H2O Demixing

Plasma membrane

Dehydrated Membrane membranes appression

Endomembrane

Plasma membrane

Non-bilayer phase formation Dehydrated and membrane fusion membranes

Endomembrane

+H2O

Plasma membrane

Cytoplasm Rehydrated membranes

Leakage of cell Endomembrane contents

Fig. 9.2. Schematic drawing of the effect of dehydration on cellular membranes. Different membrane systems may become closely appressed, leading to demixing of lipids and proteins and the loss of proteins from parts of the bilayer. Closely appressed membranes may then form non-bilayer structures that lead to fusion between different membrane systems. Upon rehydration, cellular contents leak out and fused membrane particles do not swell (i.e. they are ‘osmotically unresponsive’). (Adapted from Steponkus et al. (1993), with permission.) Dessication 09 18/3/02 1:58 pm Page 276

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There is little information for comparing loss between molecular surfaces, relieving membrane phase behaviour among ortho- the size of hydration forces (Wolfe and dox and recalcitrant embryos, maturing Bryant, 1999; Koster et al., 2000; Bryant et embryos as they become more tolerant of al., 2001). As dehydration proceeds, the desiccation, or leaves from desiccation-tol- concentration within the interfaces erant angiosperms as they adjust to low increases, with a concomitant increase in water potentials. Changes in bilayer spac- viscosity (Fig. 9.2). The high viscosity of ings or lamellar ﬂuid-to-gel transitions these interfacial solutions provides have been detected in both desiccation- mechanical resistance to the further com- tolerant and sensitive plant cells during pression of macromolecules (Wolfe and dehydration, with little difference in Bryant, 1999; Koster et al., 2000; Bryant et behaviour detected with degree of toler- al., 2001). In both protective models, the ance (McKersie and Stinson, 1980; goal is to keep molecules separated so that Seewaldt et al., 1981; Priestley and de harmful interactions are prevented. Sugars Kruijff, 1982; Singh et al., 1984; Kerhoas et accomplish this capably in model mem- al., 1987; Crowe et al., 1989; Hoekstra et brane systems (Crowe et al., 1986, 1989, al., 1991, 1992; Sun et al., 1994; Hoekstra 1990; Crowe and Crowe, 1992; Wolfe and and Golovina, 1999). In tolerant soybean Bryant, 1999; Koster et al., 2000; Bryant et cotyledons, a gel-like transition occurred al., 2001). However, the presence of ade- when seeds were dried to less than 0.2 g quate quantities of sugars in cells and the 1 H2O g dry mass (Seewaldt et al., 1981), a vitriﬁcation of cellular constituents do not water content that corresponds to a water appear to prevent polar lipid phase potential of about 12 MPa (e.g. Vertucci changes in desiccation-tolerant cells and Roos, 1990) (Fig. 9.1). Water potentials (Seewaldt et al., 1981; Priestley and de between 10 and 15 MPa also mark the Kruijff, 1982; Crowe et al., 1989; Hoekstra survival limit for recalcitrant seeds et al., 1989, 1992, 1999; Leopold et al., (described above). A membrane-mediated 1994) or damage in desiccation-sensitive mechanism is often invoked to explain cells (Berjak et al., 1992, 1993; Sun and damage in desiccation-sensitive embryos Leopold, 1993; Still et al., 1994; Sun et al., and pollen because the membrane integrity 1994; Vertucci and Farrant, 1995; Vertucci of these cells appears to be compromised et al., 1995; Farrant and Walters, 1998; upon rehydration (McKersie and Stinson, Wolkers et al., 1998a; Hoekstra and 1980; Senaratna and McKersie, 1983; Golovina, 1999; see Chapter 10). Changing Vertucci and Leopold, 1987; Berjak et al., the composition of membranes (reviewed 1992, 1993; Poulsen and Eriksen, 1992; by Steponkus et al., 1995) and reducing Sun and Leopold, 1993; Sun et al., 1994; their surface area by dismantling Wolkers et al., 1998a). endomembrane systems (described above) The different views of dehydration may be the important tools for maintaining stress (i.e. removal of structural water ver- compartmentation in drying cells. sus enhancement of hydration forces) have Structural changes of proteins with promoted different ideas for the mecha- hydration have received wide attention in nisms of protection. According to the the literature. Early work using a variety of ‘Water Replacement Hypothesis’, if struc- proteins showed that protein structure was tural water is removed, small hydrophilic conserved during drying to extremely low molecules such as sugars must be inserted levels (Schneider and Schneider, 1972; between polar lipid head groups to main- Kuntz and Kauzmann, 1974; Ruegg and tain proper intermolecular spacings and Hani, 1975; Ruegg et al., 1975; Fujita and membrane integrity (Clegg, 1986; Crowe et Noda, 1978; Careri et al., 1980; Takahashi al., 1990; Crowe and Crowe, 1992). An et al., 1980; Jaenicke, 1981; Rupley et al., alternative, but not mutually exclusive, 1983). In parallel studies, it was demon- model suggests that high concentrations of strated that some proteins even maintained compatible solutes can help resist water functional activity (albeit at low levels) Dessication 09 18/3/02 1:58 pm Page 277

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when dry (Acker, 1969; Potthast, 1978; al., 1987, 1990; Franks et al., 1991; Labuza, 1980; Rupley et al., 1983). Prestrelski et al., 1993). Enzymes such as Secondary structure of cytoplasmic pro- lactate dehydrogenase and polypeptides teins (extracted from desiccation-tolerant such as poly-L-lysine are particularly labile pollen) was conserved upon drying in the (Prestrelski et al., 1993), and damage is absence of protectant sugars, demonstrat- exacerbated if molecules are freeze-dried ing innate stability perhaps because of the rather than air-dried (Franks et al., 1991). high degree of -helical structures (Wolkers Rate of drying also has a large effect on the and Hoekstra, 1995). The reversibility of conservation of protein structure, with sorption–desorption isotherms of numer- greater preservation achieved by rapid dry- ous proteins supported the idea that con- ing conditions (Wolkers et al., 1998a,b). formational changes of proteins during Often, desiccation-labile proteins are used hydration were slight and reversible, mak- to study the effects of protectants ing proteins an ideal model for studying (Carpenter et al., 1987, 1990; Prestrelski et hydration properties of biological materials al., 1993). Clearly, these studies are essen- (Bull, 1944; D’Arcy and Watt, 1970). Slight, tial to the pharmaceutical industry, but reversible changes in protein structure, similar mechanisms of protection must not particularly secondary structure, have been be presumed to apply in vivo in dehydrat- attributed to volumetric changes from the ing plants. A tremendous amount of work loss of water rather than to changes in the has demonstrated that proteins are rather native structure of proteins. These changes robust; thus, a need for protection must be occur at fairly low moisture levels demonstrated before a protective mecha- 1 (between 0.2 and 0.1 g H2O g dry mass or nism is implied. Studies must show that 70 to 200 MPa) (Ruegg and Hani, 1975; desiccation-labile enzymes exist in vivo, Griebenow and Klibanov, 1995). Drying, in that they are not produced de novo during fact, stabilizes protein structures, making rehydration and that they are irreversibly them particularly resistant to ageing damaged in desiccation-sensitive cells. (Franks et al., 1991; Costantino et al., 1998) The structure and activity of proteins are and heat denaturation (e.g. Echigo et al., compromised if they are stored under 1966; Ruegg et al., 1975; Fujita and Noda, extremely dry conditions of approximately 1 1978; Takahashi et al., 1980; Jaenicke, 0.1 g H2O g dry matter or about 200 1981; Leopold and Vertucci, 1986; Wolkers MPa or less (Kuntz and Kauzmann, 1974; and Hoekstra, 1997). The extreme stability Luscher-Mattli and Ruegg, 1982; Sanches et of protein structure with low hydration al., 1986; Labrude et al., 1987). Substantial may be attributed to stronger intramolecu- deterioration of the lattice of protein crystals lar associations compared with the situa- was attributed to the refolding of polypep- tion of polar lipids. Such interactions tide chains to increase packing efﬁciency would reduce the need for hydrogen bond- (Kuntz and Kauzmann, 1974; Luscher-Mattli ing with water to maintain structural and Ruegg, 1982). Other studies have shown integrity (obviating the need for water that severe drying exposes haem groups on replacement by sugars as suggested by proteins, promoting free radical production Crowe and co-workers (e.g. Crowe and (Sanches et al., 1986; Labrude et al., 1987). Crowe, 1992)) and/or provide mechanical At such low water contents, proton strength that resists deformation when exchanges among charged amino acids molecules are compressed (obviating the could be measured, suggesting that these need for mechanical barriers to compres- sites were exposed (Careri et al., 1980; sion as suggested by Wolfe and co-workers Rupley et al., 1983). Deterioration at similar (e.g. Wolfe and Bryant, 1999)). water potentials and in similar time frames The conformations of some proteins and is observed in stored seeds and pollen (e.g. polypeptides are irreversibly damaged by Vertucci and Leopold, 1987; Vertucci and drying or freeze-drying in the absence of Roos, 1990; Buitink et al., 1996). Although protectants (Hanafusa, 1969; Carpenter et these organisms survive the initial stress of Dessication 09 4/4/02 2:24 pm Page 278

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complete water removal, they age progres- material (Pammenter and Berjak, 1999) (see sively more rapidly when stored at also Chapter 12). w 220 MPa (< 20% RH). Perhaps mech- When unprotected cells are dried, anisms suggested to cause damage in pro- organelles and macromolecules experience teins at low water contents (e.g. exposure of mechanical or structural damage. This type reactive sites on the proteins, increased of desiccation damage is termed sensu relaxation of molecular structures as they stricto because the primary stress is water ﬁll voids left by water, or relaxation of the removal (Pammenter and Berjak, 1999; glassy matrix that embeds the proteins) are Walters et al., 2001). Membrane structures responsible for the deterioration of stored appear more prone to desiccation damage seeds and pollen. Protein structure is stable sensu stricto than do proteins or DNA, per- in seeds stored at about 30% RH (Golovina haps because of the intense hydrogen et al. 1997), but stability of protein struc- bonding within proteins and nucleic acid tures in seeds stored at lower humidities structures. Protection from damage often has not been documented. Increased ageing lies in the ability of the structure or the rates of seeds and pollen stored below a crit- surrounding medium to offer mechanical ical water content have also been attributed resistance to the stress or accommodate the to reduced viscosity of the aqueous medium stress through enhanced elasticity. in cells that are almost completely dry (Buitink et al., 1998b). Upon dehydration, the same destabiliz- 9.3.2. Metabolically derived damage ing forces that perturb lipid and same protein structures may also affect nucleic acid Loss of turgor precipitates a number of structure (Rau et al., 1984). DNA is a partic- changes in metabolic pathways of plant

ularly stable molecule (Wayne et al., 1999) cells. Assimilation of CO2 (if the tissue is which maintains its structure in the absence photosynthetic) and growth are impaired. of water and reversibly unfolds at high tem- Often protein synthesis is temporarily peratures (Bonner and Klibanov, 2000). The stimulated during mild water stress intermolecular distances of dehydrating (reviewed by Farrant et al., 1989; Ingram DNA strands are comparable to those of and Bartels, 1996; Oliver et al., 1998), with condensed DNA in hydrated nuclei (Rau et a switch in metabolism believed to lead to al., 1984), suggesting that DNA structures the production of putative protection are resistant to perturbations resulting from mechanisms (reviewed by Vertucci and dense packing. When DNA is replicated and Farrant, 1995; Ingram and Bartels, 1996; so is decondensed during germination, the Oliver et al., 1998; Chapters 1, 5 and 11). cells concomitantly become susceptible to Observations of increased polysomes and desiccation injury (Deltour and Jacqmard, rough endoplasmic reticulum in slightly 1974; Crèvecoeur et al., 1988) and rapidly water-stressed recalcitrant embryos suggest dividing cells during embryogenesis also that certain (possibly similar) metabolic appear to be sensitive to desiccation (Myers pathways may also be induced in seeds et al., 1992). Desiccation did not affect the that do not acquire full tolerance of desic- structure of condensed or decondensed cation (Berjak et al., 1984; Farrant et al., chromatin in desiccation-tolerant or sensi- 1989; Pammenter et al., 1998). These tive maize embryos, respectively (Leprince changes in metabolism do not indicate that et al., 1995a). However, in those studies, the cells have already experienced damage; chelation of Ca2+ (and other divalent when brieﬂy stressed, most organisms cations) by the ethylenediamine tetra-acetic resume normal metabolism once the water acid (EDTA) present in the medium used for stress is relieved. However, prolonged mild chromatin spreading, may have relaxed pre- stress (which could be considered akin to viously condensed chromatin, possibly drought) is deleterious to both vegetative accounting for the reportedly similar results and embryonic tissues. Many recalcitrant from desiccation-tolerant and sensitive seeds lose viability if maintained for long Dessication 09 18/3/02 1:58 pm Page 279

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periods at constant high water contents A by-product of continued respiration (e.g. Chin and Roberts, 1980; Berjak et al., and light harvesting when other metabolic 1989; Pammenter et al., 1994; Walters et processes are shut off is the accumulation al., 2001), and similar damage is observed of high-energy intermediates that leak out in orthodox seeds (Walters et al., 2001). of mitochondria and plastids and form The loss of viability has been associated reactive oxygen species (ROS) and free rad- with the continuation of metabolism icals (Puntarulo et al., 1991; Dean et al., (including cell division) (Farrant et al., 1993; Hendry, 1993; Leprince et al., 1993, 1989), which will ultimately lead to a 1994, 1995b; Smirnoff, 1993; Foyer et al., greater demand for water to maintain high 1994; Halliwell and Gutteridge, 1999). water potentials (Berjak et al., 1989; Reactive oxygen species and free radicals Pammenter et al., 1994). react with proteins, lipids and nucleic Metabolism slows at water potentials acids, causing permanent damage to less than about 2 MPa, but not all reac- enzymes (Wolff et al., 1986; Dean et al., tions are affected by dehydration in the 1993; Halliwell and Gutteridge, 1999), same way. Protein synthesis slows down at membranes (Senaratna and McKersie, relatively high water potentials (reviewed 1983, 1986; Chan, 1987; McKersie et al., by Bewley and Krochko, 1982; Clegg, 1986; 1988, 1989; Finch-Savage et al., 1996; Salmen Espindola et al., 1994; Ingram and Halliwell and Gutteridge, 1999; Leprince et Bartels, 1996; Mundree et al., 2000; al., 2000) and chromosomes (Dizdaroglu, Whittaker et al., 2001), while respiration 1994). Peroxidation of lipids decreases the continues to much lower levels (Vertucci ﬂuidity within membranes (McKersie et and Leopold, 1984; Vertucci and Roos, al., 1988, 1989), interfering with their 1990; Salmen Espindola et al., 1994; selective permeability upon rehydration (as Leprince and Hoekstra, 1998; Leprince et described above). Upon dehydration, high al., 1999; Farrant, 2000; Walters et al., levels of free radicals have been detected in 2001). Various reactions within photosyn- desiccation-sensitive embryos (Senaratna thetic (Wiltens et al., 1978; Hetherington et and McKersie, 1983, 1986; McKersie et al., al., 1982b; Vertucci et al., 1985; Vertucci 1988; Hendry et al., 1992; Leprince et al., and Leopold, 1986; Farrant, 2000) and res- 1993, 1994, 1995b, 1999, 2000; reviewed piratory (Vertucci and Leopold, 1986; by Vertucci and Farrant, 1995; Pammenter Leprince and Hoekstra, 1998; Leprince et and Berjak, 1999). The origin and sequence al., 2000) pathways respond differently to of events following the appearance of these low water contents. The differing responses toxic compounds are still unclear. They to water stress among and within metabolic may be produced by the water-stressed cell pathways can lead to imbalances in metab- (Leprince et al., 1994, 1995b, 1999, 2000; olism. Metabolic imbalances may be con- Leprince and Hoekstra, 1998) or as a result founded by the respiration of fungi that of the associated fungi (Goodman, 1994; occurs at water potentials as low as 20 Finch-Savage, 1999), and they may precede MPa in orthodox and recalcitrant seed tis- (or precipitate) damage (Finch-Savage et sues (Mycock and Berjak, 1990; Goodman, al., 1996; Leprince et al., 2000) or arise 1994; Calistru et al., 2000). Damage by after the cell has already died (Finch- metabolic stress is most pronounced in Savage, 1999). cells at water potentials between 2 and There are several ways that cells can 5 MPa with a diminishing effect as cells protect themselves from metabolic imbal- are dried to 12 MPa (Leprince et al., 2000; ance and ROS-mediated damage. At higher Walters et al., 2001). Both desiccation-sen- moisture levels, free-radical-scavenging sitive and -tolerant organisms are damaged enzymes efﬁciently detoxify ROS (Bewley, when stored at intermediate water poten- 1979; Dhindsa, 1987; Hendry, 1993; tials, though the time-dependency of the Smirnoff, 1993; Foyer et al., 1994; Kranner damage varies considerably among species and Grill, 1997; Sherwin and Farrant, 1998; and tissues (Walters et al., 2001). Pammenter and Berjak, 1999; Farrant, Dessication 09 18/3/02 1:58 pm Page 280

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2000). These enzymes appear ineffective at changes of cellular constituents, may lead low water contents, and tocopherol and to metabolically derived damage and vice ascorbic acid may be more effective versa. Most model studies suggest that (reviewed by McKersie et al., 1988; changes in molecular conformations with Pammenter and Berjak, 1999). dehydration are reversible. Dehydration Amphipathic molecules such as tocopherol slows chemical reactions and so organisms can partition between aqueous and lipid that are dried sufficiently rapidly should domains according to the water content of experience few, if any, changes in the the cell and polarity of the molecule chemistry of their cells. Thus, it seems (Golovina et al., 1998). A controlled shut- likely that the primary lesions resulting down of metabolism upon drying may also from water stress, whether they are physi- mitigate the consequences of unbalanced cal or chemical, are minor. It also appears metabolism (as reviewed by Leprince et al., that the primary lesions are not exclusive 1993; Vertucci and Farrant, 1995; Hand to desiccation-sensitive cells. Gel-phase and Hardewig, 1996; Pammenter and lipid transitions occur in both tolerant and Berjak, 1999). Cells with more organelles sensitive pollens (e.g. Hoekstra and and greater definition of organelle structure Golovina, 1999; Hoekstra et al., 1999); appear to be more sensitive to desiccation metabolic imbalances occur in both desic- (Bewley, 1979; Hetherington, 1982a; Gaff, cation-tolerant pea and desiccation- 1989; Berjak et al., 1990; Farrant et al., sensitive tea (Walters et al., 2001); changes 1997; Farrant and Walters, 1998; Farrant, in the secondary structures of proteins are 2000), either because there are more mem- comparable in both mature and immature brane structures to protect (described maize embryos (Wolkers et al., 1998a). above) or because the higher metabolism Irreparable desiccation damage must then leads to greater ROS production. result from a cascade of reactions, initiated Conditions that reduce metabolism such as by primary but subtle lesions, which per- low temperature (Leprince et al., 1995b) or turb organization within the cell and ulti- highly complex substrates (Leprince et al., mately lead to cell death. The extent of 1990) also tend to reduce sensitivity to des- desiccation damage can then be viewed as a iccation. Desiccation-sensitive cells respire function of the rapidity at which the cas- at comparatively greater rates than tolerant cade of deleterious reactions occurs. From cells at the same water content (Leprince et this perspective, desiccation damage is a al., 1999; Walters et al., 2001), which may time-dependent process – an ageing phe- reflect properties of the mitochondria nomenon. themselves or of the cellular matrix. It has Dehydration has contrasting effects on been suggested that changes in viscosity the kinetics of both physical and chemical with dehydration are not as marked in des- deteriorative reactions. Removing water iccation-sensitive cells, and so metabolism from cells increases the concentrations of is not as restricted (Leprince and Hoekstra, reactants but slows the molecular motions 1998). It has also been suggested that the necessary for reactions to occur. The degree packing of macromolecules during dehy- to which cells are damaged by desiccation, dration of desiccation-sensitive cells is not and by extension the critical moisture level as dense (Wolkers et al., 1998a,c), and this to which they survive drying, is deter- might facilitate the diffusion of oxygen mined by the treatment duration, the con- through the cell matrix. centration of the reactants and the physical barriers (e.g. viscosity and compartmentation) to thermodynamically favourable 9.4. Perspectives on the Kinetics of reactions. Given the same experimental Desiccation Damage time, cells that tolerate more stress have either fewer reacting substances or greater This chapter has described how desicca- barriers to harmful reactions. For example, tion damage, incurred from structural according to the Water Replacement Dessication 09 18/3/02 1:58 pm Page 281

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Hypothesis, the concentration of reacting ing to fusion occur in milliseconds (Siegel phospholipids is reduced by inserting sug- et al., 1994). Drying within 2–3 min ars between head groups (Clegg, 1986; reduces protein denaturation of purified Hoekstra et al., 1989, 1991; Crowe and protein (Wolkers et al., 1998a,b), and dry- Crowe, 1992; Hoekstra and Golovina, ing within a few hours allows recalcitrant 1999). Alternatively, vitrification between embryos to survive greater amounts of bilayer interfaces provides a mechanical water loss (Pammenter et al., 1991, 1998; resistance to the compression of bilayers Walters et al., 2001). Despite valiant (Wolfe and Bryant, 1999; Koster et al., attempts, we have not been able to dry 2000; Bryant et al., 2001). According to recalcitrant embryos in less than 10 min these two hypotheses, more tolerant organ- and have yet to observe a recalcitrant isms have either proportionally more sug- embryo survive 15 MPa. This failure to ars to insert (Hoekstra et al., 1989) or more extend the lower limits of survivable water strength to repel (Wolfe and Bryant, 1999). contents in recalcitrant seeds suggests that Continuing this line of thinking may help the damaging reaction that occurs between to further distinguish between the two 10 and 15 MPa is extremely fast in des- hypotheses. According to the Water iccation-sensitive cells and relatively slow Replacement Hypothesis, once a finite (and perhaps non-existent) in tolerant number of sites is filled, the membrane or cells. The low rate of damage at this water cell should be able to tolerate complete potential in tolerant cells cannot be drying and maintain structure through time explained by glass formation per se (e.g. since reactive sites are protected. On the Leopold et al., 1994), as glasses form in other hand, vitrification is likely to provide seeds and pollen at lower water potentials imperfect protection, and macromolecular ( w 70 MPa) (see Chapter 10). However, structures will eventually be compromised the viscosity of leathery and rubbery states given sufficient time or stress. This latter (Fig. 9.1) may effectively impede molecular possibility is consistent with observations of movements leading to membrane damage at naturally occurring desiccation-tolerant higher moisture levels. The greater viscos- systems: desiccation-tolerant seeds (Vertucci ity measured in more desiccation-tolerant and Roos, 1990), pollen (Buitink et al., cells (Leprince et al., 1999) may contribute 1998b) and other phylogenetically diverse to greater structural stability. life forms such as Artemia cysts, leaves from resurrection angiosferms, and yeast (C. Walters, unpublished data) are progressively 9.5. Conclusion damaged when dried to water potentials less than about 200 MPa. Clearly desiccation sensitivity is not an ‘all The effect of time on desiccation-related or nothing’ or qualitative feature as it was damage has been explored at higher mois- once treated. Macromolecules, cells and ture levels ( 12 w 3 MPa) where organisms succumb at a range of stress lev- metabolism is believed to play a role els over a range of times. The primary (Pammenter et al., 1998; Leprince et al., lesion may be slight and reversible by our 1999, 2000; Walters et al., 2001) and at standards of measurement and may occur extremely low moisture levels ( w 220 in both tolerant and sensitive cells. This MPa) where mechanical properties are lesion, which arises from the contraction of compromised and/or reacting substances the aqueous volume and the consolidation are exposed (Vertucci and Roos, 1990; of cellular constituents, may lead to the Buitink et al., 1996, 1998a,b; Walters, irreversible loss of plasmalemma surface 1998). In these systems, the time scale for area, disruption of normal metabolism, measuring damage is days or years, respec- fusion of unrelated membrane systems and tively. However, some damaging reactions protein aggregation. As deleterious reac- may appear to be almost instantaneous. For tions cascade, it becomes difficult to deter- example, membrane-phase transitions lead- mine the earliest sources of damage. Dessication 09 18/3/02 1:58 pm Page 282

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Protectants either mitigate unbalanced 9.6. Acknowledgements metabolism or prevent the contraction and consolidation of cellular constituents. In The authors gratefully acknowledge Drs doing so, a level of quiescence and Peter L. Steponkus (Cornell University), mechanical rigour is imposed. These Folkert Hoekstra (Wageningen University) appear to be the most distinguishing and, especially, Karen L. Koster (The characteristics of desiccation-sensitive and University of South Dakota) for helpful and -tolerant angiosperms. thought-provoking discussions.

9.7. References

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10 Biochemistry and Biophysics of Tolerance Systems

Julia Buitink,1 Folkert A. Hoekstra2 and Olivier Leprince1 1UMR Physiologie Moléculaire des Semences, Institut National d’Horticulture, 16 Bd Lavoisier, F49045 Angers, France; 2Laboratory of Plant Physiology, Department of Plant Sciences, University of Wageningen, Arboretumlaan 4, 6703 BD Wageningen, The Netherlands

10.1. Introduction 293 10.2. Repression of Metabolism 294 10.3. Antioxidant Defence 295 10.4. Partitioning of Amphiphilic Compounds 296 10.5. Macromolecule Stabilization 298 10.5.1. Preferential hydration 298 10.5.2. DNA integrity and chromatin condensation 299 10.5.3. Water replacement hypothesis 299 10.5.4. Vitrification 302 10.5.4.1. Role of vitrification in desiccation tolerance and longevity 303 10.5.4.2. Composition of glasses in desiccation-tolerant organisms 305 10.6. Roles of Specific Compounds in Stability 306 10.6.1. Sucrose/oligosaccharides 306 10.6.2. Late embryogenesis abundant (LEA) proteins 307 10.6.3. Heat-shock proteins (HSPs) 309 10.7. Conclusion and Outlook 310 10.8. References 311

10.1. Introduction arrangements are lost when the water in which they are formed is removed. For Most living cells only survive dehydration instance, when a biological membrane is to a limited extent. Water has a profound dehydrated, irreversible changes occur in its inﬂuence on the association of amphiphilic structural and functional integrity (reviewed phospholipids in bilayers and the folding of by Crowe et al., 1997a). Similarly, many proteins (Tanford, 1978). These molecular labile proteins lose their functional and © CAB International 2002. Desiccation and Survival in Plants: Drying Without Dying (eds M. Black and H.W. Pritchard) 293 Dessication 10 18/3/02 1:58 pm Page 294

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probably structural integrity when they are lowed by partitioning of amphiphilic com- desiccated (reviewed by Carpenter et al., pounds (Section 10.4). The mechanisms 1992). Also, removal of water from desicca- through which the macromolecules are station-sensitive organisms leads to metabolic bilized at different hydration levels are dis- imbalances, resulting in the production of cussed in Section 10.5, together with an free radicals and oxidative damage. Whereas overview of the protective substances that Chapter 9 discussed the damage linked to have been identified so far in relation to desiccation, this chapter deals with the desiccation tolerance (Section 10.6). biochemical and biophysical changes that are part of the mechanisms by which desiccation-tolerant organisms (anhydro- 10.2. Repression of Metabolism biotes) cope with dehydration. The acquisition of desiccation tolerance is correlated The most reported degradative reactions with the accumulation of considerable linked with desiccation sensitivity in seeds quantities of non-reducing di- and are the extensive peroxidation and de-esteri- oligosaccharides, compatible solutes and fication of phospholipids, leading to the loss specific proteins such as the late embryoge- of membrane integrity (Senaratna et al., 1987; nesis abundant (LEA) and heat-shock pro- Hendry et al., 1992; Leprince et al., 1994). teins (HSPs) (see Chapters 1, 5 and 11). The cause of peroxidative damage is thought Two strategies that confer desiccation to originate from an increased formation of

tolerance have been identified. The first reactive O2 species (ROS) as a result of the strategy involves an initial avoidance of the impairment of the electron transport chains accumulation of desiccation-induced dam- during drying (see Chapter 9). age accompanied by the presence of pro- Characteristically, desiccation-tolerant tecting factors such as sugars or LEA tissues suffer less from oxidative damage proteins. The protecting factors can be pre- than do sensitive tissues (Leprince et al., sent before or synthesized during drying 1993, 1994; Vertucci and Farrant, 1995; (Vertucci and Farrant, 1995). Desiccation Pammenter and Berjak, 1999). The produc- tolerance probably depends on these tion rate of ROS is dependent on a number responses acting in synergy during drying. of factors (Skulachev, 1996). It increases For example, it has been shown that pro- with an increased lifetime of the electron tective disaccharides do not exert their pro- carriers in the reduced state, with the tective effects on dried membranes that depletion of ADP and with an increase in contain more than 15% free fatty acids, a respiration rate. It has been surmised that product that accumulates during desicca- desiccation-tolerant organisms reduce or tion-induced oxidative stress (Senaratna adapt their metabolic activities to diminish and McKersie, 1986; Crowe et al., 1989b). the chance of generating ROS (Leprince et The second strategy is based on activa- al., 1994; Vertucci and Farrant, 1995; tion of efficient repair mechanisms upon Pammenter and Berjak, 1999). Evidence rehydration. This second strategy is dealt supporting this hypothesis is increasing, with in Chapter 12. This chapter will give a but has been indirect so far. Rogerson and critical assessment as to how desiccation- Matthews (1977) observed for garden pea tolerant organisms have adapted their seeds that during maturation the ability to strategies to cope with the stresses that withstand desiccation was preceded by a occur during dehydration. The different fall in respiration rate. Furthermore, desic- strategies will be discussed in sequence of cation-tolerant axes of pea and cucumber

hydration level at which they are thought were found to exhibit a much reduced CO2 to be active, from the hydrated to the dry production before dehydration compared state, starting with the avoidance of oxida- with germinated, desiccation-sensitive tive damage by means of regulation of axes, and this difference was maintained metabolism (Section 10.2) and the presence during drying (Leprince et al., 2000; see of antioxidant systems (Section 10.3), fol- Table 10.1). Respiration rates in the desicca- Dessication 10 18/3/02 1:58 pm Page 295

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Table 10.1. CO2 production rates during drying of desiccation-tolerant cucumber axes (24 h imbibed at 20°C; DT), desiccation-sensitive axes (72 h imbibed at 20°C, radicle 3 mm protruded; DI) and germinated cucumber axes that were incubated in polyethylene glycol (PEG) for 7 days to reinduce desiccation tolerance (72 h imbibed at 20°C with a radicle 3 mm protruded, then 7 days in PEG (1.5 MPa) at 10°C; DT PEG). (Adapted from Leprince et al., 2000.)

1 1 CO2 production rate ( l h g dry weight) 1 g H2O g dry weight DT DI DT PEG

3.0 1.7 4.5 1.1 2.0 1.3 3.2 0.5 1.0 0.7 1.5 0.4

tion-sensitive cotyledons of Castanea sativa as anoxia, freezing and dehydration (Hand Mill. increased at the onset of drying and and Hardewig, 1996; Hardie et al., 1998). decreased only with the loss of membrane However, thus far there is only limited evi- integrity. In contrast, dehydration of the dence for metabolic depression in develop- more desiccation-tolerant axes resulted in a ing seeds (Kollöffel and Matthews, 1983)

rapid decline in O2 uptake rates at the onset and somatic embryos (Tetteroo et al., 1995). of drying (Leprince et al., 1999). In general, Some indications that coordinated down- mature recalcitrant seeds have relatively regulation might be essential to confer des- high respiration rates (Salmen Espindola et iccation tolerance in seeds comes from al., 1994; Leprince et al., 1999; Pammenter Leprince et al. (2000). Dehydration of desic- and Berjak, 1999). Thus, the high respira- cation-sensitive axes resulted in an increase tion rates in desiccation-sensitive seeds in the emission rates of acetaldehyde and may promote free-radical-induced injury ethanol, which peaked well before the during drying. The relationship between onset of membrane damage. Tolerant axes the extent of metabolic activity and desicca- did not exhibit acetaldehyde or ethanol tion sensitivity is strengthened by the production. The question remains as to observation that treatments that limit rates whether controlled down-regulation is of metabolism also reduce the incidence of exerted before drying (i.e. during seed mat- desiccation injury (Leprince et al., 1995b). uration) or also during drying. If metabolism must be down-regulated for desiccation tolerance to be acquired, the nature of this regulation is unknown. 10.3. Antioxidant Defence Leprince et al. (1994, 1995b, 2000) suggested that a coordinated down-regulation Since susceptibility to peroxidation may of energy metabolism in seeds early during increase with drying (see Vertucci and drying may play an important role in avoid- Farrant, 1995, for a review; Chapter 9), one ing oxidative stress conditions and/or accu- may reason that free-radical-scavenging sys- mulation of by-products of metabolism to tems are an important component among

toxic levels. During drying, the O2 availabil- the mechanisms of desiccation tolerance. ity decreases with the increase in cellular ROS are natural by-products of the metabo- viscosity. It is likely that to avoid metabolic lism, which are particularly present in imbalances there will be a ﬁne tuning chloroplasts and mitochondria. Thus, between repression of metabolic activity plants are well endowed with antioxidant

and O2 availability (Leprince et al., 2000). molecules and scavenging systems (Larson, Down-regulation of metabolism appears to 1988; Hendry, 1993). Enzymatic free- be an ancient and widespread regulatory radical-processing systems include super- mechanism that allows aerobes to with- oxide dismutase (SOD), which catalyses the stand severe environmental stresses, such dismutation of superoxide (O2 ) into H2O2 Dessication 10 18/3/02 1:58 pm Page 296

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and O2, and those that are involved in the radical-processing systems in seeds rely on detoxification of H2O2 (i.e. catalase, glu- enzymatic activities, it is likely that they are tathione reductase and peroxidases). mainly efficient early during drying and Several studies have demonstrated the link may not function in reduced hydration con- between tolerance of oxidative stress ditions. At the lower water contents, one induced by water deficit and rise in antioxi- may expect molecular antioxidants (e.g. glu- dant concentrations in photosynthetic tathione, ascorbate, tocopherol) to play a plants (Winston, 1990; Price and Hendry, preponderant role in alleviating oxidative 1991). In vegetative tissues, removal of the stress. This is supported by a study on ger- cytotoxic products resulting from oxidative minating seeds of soybean, which showed events is considered to be of prime impor- that their desiccation tolerance was associ- tance for survival of drought stress because ated with the presence of an unknown lipid- genes encoding enzymatic antioxidants soluble antioxidant in extracted membranes become up-regulated during drying (Ingram (Senaratna and McKersie, 1986). However, and Bartels, 1996). In the resurrection plant in germinating maize, no correlation was Craterostigma plantagineum, an inhibitor found between the presence of lipid-soluble of lipoxygenase (the activity of which and hydrophilic antioxidants and desicca- results in lipid hydroperoxide formation) tion tolerance (Leprince et al., 1990b). A accumulates in the leaves during desicca- comparative study on ascorbate and dehy- tion (Bianchi et al., 1992). In Craterostigma dro-ascorbate showed that the concentra- wilmsii and Xerophyta viscosa, the activity tions of these antioxidants were higher in of ascorbate peroxidase increases during recalcitrant seeds than in orthodox seeds dehydration. During rehydration, the activ- (Tommasi et al., 1999). Future studies aimed ity of SOD and glutathione reductase at establishing the importance of free-radi- increases (Sherwin and Farrant, 1996). cal-processing systems in relation to desic- Furthermore, C. wilmsii was also found to cation tolerance should take into account accumulate large amounts of anthocyanins, both the hydration level during drying and which have antioxidant capabilities. the fact that free-radical production will be The protective role of antioxidants in localized within the mitochondria and/or desiccation tolerance of seeds is far from microsomal membranes. Thus, efforts resolved (reviewed by McKersie, 1991; should concentrate on antioxidant systems Hendry, 1993; Leprince et al., 1993). within these organelles. Furthermore, coin- Vertucci and Farrant (1995) have suggested cidental antioxidants such as quinones, that it is particularly in the water content polyols, carbohydrates, amphipathic mole- range corresponding to water potentials of cules (flavonoids, phenolics) and proteins 3 to 11 MPa that unregulated metabolic (e.g. peroxiredoxin) also deserve particular events result in the first wave of free-radi- attention. cal generation, although the upper limit could be higher. Thus, it is assumed that antioxidant systems should be maximally 10.4. Partitioning of Amphiphilic effective during the initial stages of the Compounds maturation drying of developing orthodox seeds (Arrigoni et al., 1992). Cells may contain various cytoplasmic Despite several studies that assessed in metabolites that have amphiphilic proper- vitro (i.e. under hydrated conditions) the ties. Hoekstra et al. (1997) proposed that activity of free-radical-processing systems desiccation may increase the transfer of extracted from drying tissues, it remains to these amphiphiles from the polar cyto- be ascertained whether these systems are plasm into the lipid phase, i.e. the mem- also active at low water contents in vivo. branes and lipid bodies. On the one hand, Considering that the metabolism is virtu- such partitioning into membranes could ally nil in cells containing less than 0.3 g seriously perturb membrane structure, with 1 H2Og dry weight (dw), and that free- effects on permeability properties as the Dessication 10 18/3/02 1:58 pm Page 297

Biochemistry and Biophysics of Tolerance Systems 297

result. On the other hand, partitioning into or liposomes with hydrated ones, the aver- membranes might be extremely effective at age rigidity was similar, even in the pres- automatically inserting amphiphilic ence of a protective sugar. This has been antioxidants into membranes upon dehy- interpreted to mean that dry membranes dration, which could promote desiccation inside anhydrobiotes are fluidized, presum- tolerance and extend storage longevity. The ably by amphiphilic guest molecules presumed transfer of amphiphiles has been (Hoekstra and Golovina, 1999). In isolated experimentally tested in pollen and seeds membranes, such amphiphiles become lost with electron paramagnetic resonance during the isolation procedure. Recent data (EPR) spectroscopy, using amphiphilic based on the number of spin-probe mole- spin probes inserted into the cytoplasm cules in either phase during dehydration (Golovina et al., 1998; Buitink et al., (Golovina and Hoekstra, 2001) indicate that 2000d; Hoekstra and Golovina, 2000). The there is already a considerable transfer of advantage of this method is that the EPR amphiphiles from the cytoplasm to mem- spectra can be used to derive where the branes at the beginning of drying. This spin probe resides – in the cytoplasm or in would mean that fluidization occurs early the lipid phase. It has been demonstrated during dehydration. Indeed, an increase in that amphiphilic probes partition into the molecular mobility at the membrane surface lipid phase with drying, in proportion to was observed early during dehydration, the reduction of the cytoplasmic volume, probably brought about by endogenous and vice versa with rehydration (Golovina amphiphiles. While this mobility decreases et al., 1998; Hoekstra and Golovina, 2000). in desiccation-tolerant organisms on further A similar partitioning behaviour with drying, it remains high in desiccation-sensi- drying is plausible for endogenous cyto- tive organisms until almost all water has plasmic amphiphiles. Extracted endoge- disappeared (Golovina and Hoekstra, 2001). nous amphiphiles reversibly partitioned Endogenous biologically relevant into liposomal membranes with drying, amphiphiles that might undergo partition- which made the liposomes transiently ing similar to the spin probes are phenolic leaky for entrapped polar compounds acids and flavonoids. These molecules are (Golovina et al., 1998). Thus, partitioning abundantly present in dry seeds, pollen and into membranes is possible and has been resurrection plants (Larson, 1988; Oliver et used to explain the transient leakage of al., 1998; Shirley, 1998) and may play sev- cytoplasmic solutes from rehydrating anhy- eral roles in relation to desiccation toler- drobiotes. Plasma membrane permeability ance. A wide array of amphiphiles present of pollen, for example, was indeed elevated in plants are known to be potent antioxi- as long as the amphiphiles resided in the dants (Larson, 1988; Saija et al., 1995; Rice- lipid phase. On sufficient rehydration, Evans et al., 1997). Partitioning of such when the amphiphiles had mainly returned amphiphilic antioxidants from the cyto- to the aqueous cytoplasm, permeability plasm into membranes during drying might returned to the low level observed before prevent desiccation-induced oxidative dehydration, and leakage rates became low. damage. A dehydration test with the antiox- This transiently increased permeability idant flavonoid rutin on liposomes indi- upon imbibition lasted approximately 10 s cated that rutin depresses the average in the case of pollen (Hoekstra et al., 1999). phase-transition temperature of the lipo- There are more indications that in dry somes (Hoekstra and Golovina, 2000). From anhydrobiotes endogenous amphiphiles this it has been inferred that amphiphiles reside in membranes. In situ Fourier trans- might have a role in controlling the mem-

form infrared (FTIR) spectroscopy data has brane-phase transition temperature (Tm), indicated that the average rigidity of mem- along with the non-reducing di- and branes in the gel phase is less when the oligosaccharides (see Section 10.5.3). organism is dry than when it is hydrated. Although partitioning seems beneﬁcial, When comparing dried isolated membranes as indicated above, it appears that the trans- Dessication 10 18/3/02 1:58 pm Page 298

298 J. Buitink et al.

fer must be controlled in order to confer It has been shown in model experi- desiccation tolerance (Buitink et al., 2000d). ments that these substances stabilize pro- This is because amphiphiles can perturb tein structure and activity against thermal membrane functions (Herbette et al., 1983; denaturation (Arakawa and Timasheff, Takahashi et al., 1998), which might be par- 1985). Further, they protect isolated pro- ticularly detrimental at high water contents. teins (Carpenter and Crowe, 1988; For example, if mitochondrial respiration is Carpenter et al., 1990) and (functional) affected, there is the likelihood of an vesicles (Rudolph and Crowe, 1985; enhanced production of free radicals. Anchordoguy et al., 1987, 1988) during freeze–thawing by minimizing denaturation and the vesicles by preventing fusion. 10.5. Macromolecule Stabilization Coming from chemically dissimilar classes, these substances have in common Macromolecule stabilization, such as the that they are preferentially excluded from retention of phospholipid bilayers or contact with the surface of proteins in proper folding of proteins, depends on the aqueous solution, which makes it thermo- presence of water. Upon dehydration, these dynamically unfavourable for proteins to macromolecules need to be protected from unfold (Arakawa and Timasheff, 1985; the deleterious effects that are normally Carpenter and Crowe, 1988; Carpenter et associated with the removal of water. The al., 1992). In other words, these substances stabilization of the molecules in the cells is keep the macromolecules preferentially realized through one or more of the mecha- hydrated. In contrast, compounds such as nisms that are discussed below, in the urea and guanidine–HCl, which preferen- order of the mechanisms that are func- tially bind with proteins, destabilize pro- tional at high hydration levels to those that teins in solution. This means that when are operational at low water contents. the bulk water is removed (below 0.3 1 H2Og dw), this mechanism would fail to work because there is no water left for 10.5.1. Preferential hydration preferential hydration (Crowe et al., 1990). Indeed, most of the compatible solutes, Many plants and microorganisms accumu- except a few sugars, are unable to protect late organic osmolytes in response to envi- proteins and membranes against air-drying ronmental stresses that cause cellular or freeze-drying. In the case of sugars, it is dehydration, such as drought, freezing and envisaged that, after the bulk water is lost, osmotic shock. This accumulation corre- hydrogen bonding and glass formation are lates with improved stress tolerance. the mechanisms by which proteins and Evidence of a causal relationship between membranes are structurally and function- elevated levels of these so-called compati- ally preserved (see Sections 10.5.3 and ble solutes and stress tolerance has come 10.5.4). from the results of enrichment experiments One has to realize that dehydrating (e.g. Saranga et al., 1992) and from the anhydrobiotes pass through stages of behaviours of transgenic organisms engi- drought during which the mechanism of neered to accumulate these compounds preferential exclusion may be effective. It (e.g. Takagi et al., 1997; Strom, 1998). is, therefore, not surprising that anhydro- Among these compatible solutes are pro- biotes are endowed with large amounts of line, serine, glutamate, glycine-betaine, car- compatible solutes, e.g. proline and nitine, mannitol, sorbitol, fructans, polyols, sucrose in pollen (Zhang et al., 1982; trehalose, sucrose and oligosaccharides. Hoekstra et al., 1992), di- and oligosaccha- The absolute osmolyte concentrations, rides in seeds (Amuti and Pollard, 1977), however, are unlikely to mediate osmotic and proline, glycine-betaine and trehalose adjustment (Hare et al., 1998). in microorganisms and yeast. Dessication 10 18/3/02 1:58 pm Page 299

Biochemistry and Biophysics of Tolerance Systems 299

10.5.2. DNA integrity and chromatin undergo reversible changes in condensation condensation states, indicating that certain protective substances are necessary to confer stability The maintenance of genetic information is to the chromatin during drying in desicca- central to survival upon dehydration and tion-tolerant tissues (Leprince et al., 1995a). rehydration (see Chapter 12). As a dynamic While the stability of DNA and chro- and hydrated molecule in vivo, DNA can matin in the dehydrated state must be a assume different conformational structures, prerequisite for desiccation tolerance in depending on the water activity, the base seeds and resurrection plants, much sequence and the presence of speciﬁc bind- remains to be ascertained about factors ing proteins (Osborne and Boubriak, 1994). contributing to the genetic stability and the Determination of the integrity of extracted responses to dehydration in both desicca- DNA in embryos of seeds and in wind-dis- tion-tolerant and sensitive material. persed pollen during the transition from the desiccation-tolerant to the sensitive stage showed that only DNA from desicca- 10.5.3. Water replacement hypothesis tion-tolerant cells retained integrity when cells were subjected to drying regimes. It Studies on model liposome systems com- was further proposed that the attainment of posed of pure phospholipids (PLs) show stable secondary structures that are resistant that drying induces a phase transition in to degradation in vivo at low water poten- the membranes from the ﬂuid liquid-crys- tials is a likely accessory to desiccation tol- talline phase to the solid gel phase (Crowe erance (Osborne and Boubriak, 1994). et al., 1997a; see Chapter 9). The rise of the Another genetic factor that changes as a membrane-phase transition temperature

function of hydration is the structure of (Tm) with drying commences with the dis- chromatin. In the desiccation-tolerant sipation of the last 10–12 water molecules phase of developing and germinating ortho- per PL molecule, i.e. below 0.2–0.3 g 1 dox seeds, the chromatin is in a condensed H2Og dw. The removal of water mole- state. This state appears to be retained dur- cules from the head groups leads to a reducing early rehydration, while seeds are still tion in the lateral spacing between the PL desiccation-tolerant (Leprince et al., 1995a; molecules and, consequently, to the forma-

Pammenter and Berjak, 1999). During ger- tion of a gel phase. Thus, the Tm of model mination, the rehydration stage correspond- membranes composed of phosphatidyl- ing to the resumption of DNA replication is choline increases by as much as 70°C during associated both with chromatin deconden- dehydration. A liquid-crystal-to-gel-phase sation and loss of desiccation tolerance transition of membrane PLs during dehy- (Deltour, 1985). Orderly, reversible chro- dration has been detected in intact pollen, matin compaction and rehydration-induced using FTIR spectroscopy (Crowe et al., redispersion of condensed chromatin thus 1989a). Similar to the model liposome sys-

appear to be associated with desiccation tems, the average Tm values of membranes tolerance. Evidence for this comes from in pollen increased with dehydration.

electron microscope observations on seeds Comparison of the Tm value in dried cattail (Crèvecoeur et al., 1976; Deltour, 1985) and pollen (32°C) with that of dry membranes resurrection plants (Hallam and Luff, 1980), isolated from this pollen (58°C) indicated

and from a biophysical characterization of that the Tm in intact pollen was depressed isolated chromatin from germinating maize by 26°C (Hoekstra et al., 1991). Apparently, embryos (Leprince et al., 1995a). Electron in the dry, intact organism there is a mech- microscopy studies show that irreversible anism that depresses the dehydration-

chromatin compaction accompanies injuri- induced increase of Tm, which is lost when ous dehydration of desiccation-sensitive the membranes are isolated. Upon adding material. Extracted chromatin from dried, sucrose to the isolated membranes before

desiccation-sensitive tissues was unable to drying, the Tm of the dried membranes was Dessication 10 18/3/02 1:58 pm Page 300

300 J. Buitink et al.

again depressed from 58 to 31°C. The In the presence of trehalose (a common mechanism by which this depression is disaccharide often found in anhydrobiotic considered to work is discussed below. microscopic animals and yeasts (Crowe et Experiments on model systems have al., 1984)) polar compounds trapped given some insight into the mechanism by inside the liposomes do not leak during

which sugars depress the Tm of dry mem- drying and rehydration (Crowe et al., branes. It has been established that during 1986). Also, other disaccharides (sucrose) desiccation soluble sugars interact with the and oligosaccharides such as stachyose polar head groups and replace the water and rafﬁnose are effective at retaining molecules. Phospholipid molecules thus compounds trapped inside the liposomes. largely retain the original spacing between It is thought that the leakage does not one another, and the desiccation-induced occur because a phase change is prevented

increase in Tm is circumvented (Fig. 10.1). (Fig. 10.1). In the light of these in vitro Interaction with sugars can even lead to studies, the presence of sucrose and

depression of Tm to values considerably oligosaccharides in desiccation-tolerant below the Tm of the hydrated specimens organisms is thought to be similarly (Crowe et al., 1996). As the size of the solu- involved in the depression of Tm in situ ble carbohydrate molecule increases, the and the prevention of leakage. chance of interaction between the phos- From air-drying experiments with lipo- phate and the carbohydrate decreases, and somes in the presence of disaccharides, it

the depression of Tm is less. Thus, it has has become clear that a mass ratio of 5 : 1, been demonstrated that hydrogen-bonding sugar : PL, satisﬁes the head group hydrogen- between the head group PO and sugar OH bonding capacity (Hoekstra and Golovina,

groups is pivotal for the depression of Tm 1999). Such mass ratios often occur in in dry membranes (Crowe et al., 1996). anhydrobiotic pollen and seeds, but excep-

Water Sugars Rehydration

Hydrated No leakage, liquid crystal no fusion

+ sugars

Desiccation Hydrated, liquid crystal

– sugars

Rehydration Extensive leakage + fusion

Dry gel

Fig. 10.1. Schematic representation of the phase behaviour of phospholipids in a bilayer as inﬂuenced by desiccation in the presence or absence of a disaccharide. The disaccharide maintains the original spacing between the phospholipid molecules and, thus, prevents the transition from the liquid crystalline phase to the gel phase. Dessication 10 18/3/02 1:58 pm Page 301

Biochemistry and Biophysics of Tolerance Systems 301

tions with lower mass ratios have been during drying before the formation of a found, which are nevertheless desiccation- glass, thereby increasing the chance of

tolerant (Hoekstra et al., 1997). The effec- solute loss. Thus, depression of Tm may tive availability of sugars to PLs in situ prevent a loss of membrane permeability might be less than expected, because sug- during drying. Furthermore, depression of

ars also interact with other constituents of the Tm in dry anhydrobiotes is also impor- the cells and with one another. This tant to reduce the risk of leakage during restricted availability of the sugars for rehydration (also called imbibitional leak- interaction with the head groups would be age), apart from evading leakage during in line with the observation that depres- drying (see Chapter 12).

sion in situ of Tm in dry organisms is often Another role for sugars in relation to not to the low value measured in the desiccation tolerance is their stabilizing hydrated specimens (Hoekstra and effect on dried proteins (see Chapter 9). Golovina, 1999). It has been suggested that, Sugars are special in that they allow the besides sugars, other molecules might aid removal of the closely associated water

in the depression of Tm in desiccation-tol- from proteins without this leading to con- erant organisms, for example by the trans- formational changes and loss of enzymatic 1 fer of ﬂuidizing compounds from the function. Above 0.3–0.5 g H2O g dw, cytoplasm into membranes (see Section other (compatible) solutes can also be 10.4). effective (see also Section 10.5.1). The question remains as to why post- Employing phosphofructokinase (PFK), an

poning or preventing the rise in Tm with extremely labile protein when dried or dehydration might be beneﬁcial. The fact freeze-dried, Carpenter and co-workers that dry pollen and seeds can be safely have shown that the disaccharides sucrose, stored below 20°C (i.e. under conditions maltose and trehalose are very effective sta- that make the gel-phase formation particu- bilizing agents, particularly in the presence larly likely) indicates that the presence of of certain divalent cations (Carpenter et al., the gel phase per se is not detrimental. A 1987a,b, 1990). FTIR spectroscopy studies phase change leads to transiently increased indicate that sugars act as a water substi- permeability, and a loss of solutes may be tute by satisfying the hydrogen-bonding expected under these conditions. The requirement of polar groups on the surface extent of this loss will depend on the vis- of the dried protein (Carpenter and Crowe, cosity of the surrounding cytoplasmic envi- 1989; Prestrelski et al., 1993; Wolkers et 1 ronment. Below 0.8 g H2Og dw, the al., 1998b). This mechanism is analogous viscosity increases exponentially and to the water replacement hypothesis men- becomes extremely high when the cyto- tioned above for membranes, although ﬁxa- plasm enters a glassy state (Leprince and tion of the protein’s secondary structure Hoekstra, 1998) (see Section 10.5.4). No also occurs via glass formation (see Section loss of solutes is to be expected when the 10.5.4) (Franks et al., 1991). Thus, protein phase change of the membranes occurs unfolding and aggregation during dehydra- while the cytoplasm is close to reaching a tion are prevented. In dry, orthodox seeds, glassy state. However, leakage may result proteins retain their native secondary when the phase change occurs when the structure for decades, long after the seeds cytoplasm is in the liquid state. Data on have died (Golovina et al., 1997). Recently, cattail pollen have indicated that, during Wolkers et al. (1998b) contributed to the dehydration at 20°C, the gel phase and the question of whether glass formation or glassy state are formed simultaneously hydrogen bonding of sugar with protein is (Hoekstra and Golovina, 1999). Any uplift involved in the stabilization during slow of the phase diagram (i.e. the curve repre- air-drying. The sugars having the best

senting the relationship between Tm and hydrogen-bonding properties give the best water content) by ineffective depression of structural protection, but have the lowest

the Tm would result in gel-phase formation glass transition temperature (Tg), which Dessication 10 18/3/02 1:58 pm Page 302

302 J. Buitink et al.

supports the hydrogen-bonding mechanism state (Franks et al., 1991). A glass is a of protection. highly viscous solid liquid. Its high viscosity has been shown to slow down severely molecular diffusion and decrease the prob- 10.5.4. Vitrification ability of chemical reactions (see Slade and Levine, 1991; Roos, 1995, for reviews). Upon drying of desiccation-tolerant tis- Glass formation has been detected in seeds sues, as the concentration of solutes (Williams and Leopold, 1989; Bruni and increases there is an increase in the viscos- Leopold, 1992; Leopold et al., 1994; ity of the cytoplasm and a decrease in mol- Leprince and Walters-Vertucci, 1995), ecular mobility of molecules. For example, pollen (Buitink et al., 1996) and the resur- 1 in embryonic tissues below 0.8 g H2Og rection plant C. plantagineum (J. Buitink, dw, the cytoplasm becomes increasingly unpublished results). Apparently, glass for- viscous (Leprince and Hoekstra, 1998; mation is a characteristic typical of all des- Buitink et al., 2000d). Drying below 0.3 g iccation-tolerant tissues. 1 H2Og dw leads to a decrease in the mol- The presence of a glassy state is depen- ecular mobility in the cytoplasm of over dent on three factors: water content, temper- five orders of magnitude (Buitink et al., ature and chemical composition. A decrease 1 1999). Finally, at around 0.1 g H2Og dw, in the water content of the tissue results in the cytoplasm vitrifies and exists in a so- an increased glass transition temperature

called glassy state. A glass is deﬁned as an (Tg), as demonstrated by a state diagram, amorphous metastable state that resembles which depicts the relationship between the

a solid, brittle material, but retains the dis- water content and the Tg (Fig. 10.2). The order and physical properties of the liquid magnitude of Tg is also dependent on the

Fast drying 60 Slow drying C)

–30 Glass transition temperature ( –60

0.00 0.08 0.16 0.24 0.32 –1 Water content (g H2O g dry weight)

Fig. 10.2. State diagram depicting the relationship between water content and glass transition temperature of developing bean axes (O. Leprince and C. Walters, unpublished data). Seeds were harvested near mass maturity. After slow drying (i.e. 3 days), isolated axes were found to elongate and grow when cultured in vitro on agar. They were considered to be desiccation-tolerant. In contrast, isolated axes failed to elongate after fast drying (5 h) and were considered to be desiccation-sensitive. The different water contents were obtained by drying isolated axes for different times. Glass transition temperatures were measured at the mid- point using a differential scanning calorimeter (DSC) during heating at a scanning rate of 10°C min1 according to Leprince and Walters-Vertucci (1995). Data points represent three to ﬁve axes in the DSC pan. Dessication 10 18/3/02 1:58 pm Page 303

Biochemistry and Biophysics of Tolerance Systems 303

composition of the amorphous state. For the glassy state can be effective. Although

one-component model systems, Tg is known glass formation is not a mechanism that to vary with Mr in a characteristic and theo- initially confers tolerance to desiccation retically predicted fashion (Slade and during drying, its formation is indispens- Levine, 1991). For instance, a sugar of a high able for surviving the dry state, as dis-

Mr (like stachyose) exhibits a higher Tg over cussed below. the entire range of water contents than a An important consequence of the forma-

small Mr sugar such as glucose. tion of the glassy state in tissues is the absence of crystallization (Leopold et al., 1994; Sun and Leopold, 1997). It has been 10.5.4.1. Role of vitriﬁcation in desiccation suggested that loss of viability could be tolerance and longevity due to time-dependent crystallization lead- Intracellular glasses were, originally, sug- ing to loss of membrane structure and cel- gested to play a role in desiccation tolerance. lular integrity (Caffrey et al., 1988; Sun and Considering the glass-forming capability of Leopold, 1993). However, so far there is no sugars, several studies have appeared experimental evidence that demonstrates attempting to link changes in sugar composi- crystallization events in desiccation- tion during the acquisition of desiccation sensitive tissues (Sun and Leopold, 1993). tolerance with the existence of glasses upon It is surmised that the complex cytoplas- drying (Koster, 1991; Williams and Leopold, mic composition is most probably respon- 1995). For instance, sugars similar to those sible for preventing crystallization found in desiccation-tolerant seeds (sucrose, (Walters, 1998; Buitink et al., 2000e). The rafﬁnose) are capable of forming glasses at major function of intracellular glasses in ambient temperatures, whereas sugar mix- dry seeds may be their contribution to the tures similar to those found in axes that do stability of macromolecular and structural not tolerate desiccation were found only to components during storage. form glasses at sub-zero temperatures One of the most studied functions of (Koster, 1991). Williams and Leopold glasses is maintenance of the structural and (1995) showed that, after 50 h of imbibition, functional integrity of macromolecules (see

the Tg of desiccation-sensitive pea embry- Slade and Levine, 1991, 1993; Roos, 1995, onic axes was remarkably lower than that for reviews). Glasses are known to slow of desiccation-tolerant axes imbibed for 14 down detrimental reactions, such as the rate h, accompanying the loss of oligosaccha- of browning reactions (Karmas et al., 1992), rides and replacement by monosaccha- to increase the stability of enzymes (Chang rides. However, in other studies, glasses et al., 1996) and to prevent conformational have also been found in desiccation-sensi- changes in proteins (Prestrelski et al., 1993). tive tissues (Sun et al., 1994; Buitink et al., It has also been shown in model systems 1996). For instance, the state diagrams of that glasses are capable of preventing the developing embryonic axes of bean after fusion of membranes. Leakage from lipo- fast and slow drying (which renders them somes can be the result of a phase change desiccation-sensitive and -tolerant, respec- (Section 10.5.3), but also because of fusion tively) were found to be identical (Fig. 10.2). between the liposomes. In the former case, This questions the exclusive role of glasses the size of the liposomes remains the same, in desiccation tolerance. It is important to but, in the latter case, the size increases realize that the water content at which glass considerably. Protection of liposomes has formation occurs during drying at room also been shown to depend on how effec- temperature in seeds (~ 10% moisture; Fig. tively sugars can form glasses (Crowe et al., 10.2) is much lower than the critical water 1998). In this respect, monosaccharides are content most desiccation-sensitive species poor protectants, despite their generally exhibit. Apparently, dehydration-induced excellent capability to interact with the damage in these seeds occurs at water con- polar head groups (Section 10.5.3). tents far above those at which protection of Monosaccharides differ from the di- and Dessication 10 18/3/02 1:58 pm Page 304

304 J. Buitink et al.

oligosaccharides in having low Tgs (below water contents (Buitink et al., 2000c). All room temperature). This means that during these arguments suggest that ageing rates drying the liposomes remain in a liquid and, consequently, life span of germplasm environment, which results in fusion- are inﬂuenced by the molecular stability of induced release of the entrapped contents. the cytoplasm, signifying the pivotal func- The importance of both the ability to form tion of intracellular glasses in conferring glasses at ambient temperature and direct such stability during storage. interaction with the polar head groups to Recently, the consequences of being in protect membranes (see Section 10.5.3) has or near the glassy state have gained further been demonstrated by drying liposomes in physiological signiﬁcance. A study on the the presence of two different compounds, molecular mobility in desiccation-tolerant hydroxy-ethyl starch and glucose (Crowe et tissues indicated that the special composi- al., 1997b). Hydroxy-ethyl starch has a tion of biological glasses might have a role

high Tg, thus forming a glass at ambient in survival (Buitink et al., 2000e). Based on temperature, but it does not depress Tm saturation transfer-electron paramagnetic because it is too large to ﬁt between the resonance (ST-EPR) spectroscopy measure- head groups to efﬁciently interact. Glucose, ments, Buitink et al. (2000e) observed a sec-

by contrast, depresses Tm in dry lipids but ond kinetic change in mobility at a deﬁnite will not easily form a glass at ambient tem- temperature above Tg, referred to as the crit- perature. Thus, hydroxy-ethyl starch pre- ical temperature (Tc). The occurrence of Tc vents the leakage associated with fusion has been coupled to the collapse tempera- 1 (below 1.5 g H2Og dw), but cannot pre- ture of sugar glasses (Tg + 15°C; Fig. 10.3), a vent the leakage associated with the phase well-documented phenomenon, which is 1 transition (below 0.25 g H2Og dw). attributed to a reduction in viscosity such Together, however, hydroxy-ethyl starch that a ﬂow on a practical time scale is and glucose protect the dry liposomes, but observed. Although the viscosity decreases

neither compound is effective alone. Both around Tg, it is not until the temperature Tc properties – timely glass formation during is reached that the viscosity abruptly drops drying and interaction with the polar head (see Fig. 10.3). This is contrary to the groups – appear to be required for preser- belief that the viscosity decreases abruptly

vation. Disaccharides combine these two at Tg, as is often assumed. The Tc in desic- essential properties within one compound. cation-tolerant organisms occurs at temper-

The effect of glasses on the stability of atures as high as 55°C above Tg (Fig. 10.3). macromolecular and structural components A high Tc implies high stability as a result 8 during storage has led to the concept that of high viscosity (> 10 Pa s) far above Tg. glasses play an essential role in the This high Tc in biological tissues has longevity of seeds and pollen. For example, important implications for the survival of the storage stability of Arabidopsis thaliana germplasm in its natural environment. seeds has been found to correlate with the Under ambient conditions, for example molecular density of the cellular matrix 20°C and 50% relative humidity (RH), seed

(Wolkers et al., 1998a). Sun and Leopold tissues are around their Tg. This means that (1994) have found that seed deterioration any environmental ﬂuctuation that results appears to be accelerated when seeds are in an increase in RH or temperature will

not in the glassy state, as estimated through bring the tissue above its Tg. However, the the viability equation of Ellis and Roberts unique properties of the intracellular glass (1980). Probably the most compelling evi- protect the tissue from dramatic changes dence to suggest that ageing rates are caused by environmental ﬂuctuations. If the affected by the viscosity of the intracellular intracellular glass were composed of glass has come from the linear relationship sucrose alone, a small increase in RH or between ageing rate and cytoplasmic mole- temperature would bring the sucrose glass

cular mobility found for many different tis- above its Tc (Fig. 10.3), resulting in crystal- sues over a wide range of temperatures and lization and loss of macromolecule function Dessication 10 18/3/02 1:58 pm Page 305

Biochemistry and Biophysics of Tolerance Systems 305

Fast

Sucrose

Poly-L-lysine Molecular mobility

Bean embryonic axis

Slow –20 0 20 40 60 80 100 120 T – Tg ( C)

Fig. 10.3. The effect of melting the glassy state on the molecular mobility of a guest molecule (spin probe) incorporated in dry glasses composed of sucrose or poly-L-lysine, and in the intracellular glass of dried embryonic axes of bean. Molecular mobility was measured by electron paramagnetic resonance spectroscopy (Buitink et al., 2000e). The indicates the critical temperature, Tc, where the dynamics of the system changed from solid-like to liquid-like (signiﬁed by an abrupt drop in viscosity).

and integrity (see Roos, 1995, for review). role in intracellular glass formation. Sun

Therefore, the characteristically high Tc of and Leopold (1993) found that the magni- intracellular glasses serves as an ecological tude of the glassy signal and Tg, both mea- and physiological advantage. sured by the thermally stimulated depolarization current (TSDC) method, decreased during accelerated ageing of soy- 10.5.4.2. Composition of glasses in bean seeds. Yet no differences were desiccation-tolerant organisms observed in sucrose, rafﬁnose and stachyose When the concept of glasses was intro- contents during the same period of time. duced in seed science, sugars were thought Despite different sugar compositions in soy- to play an important role in the composi- bean axes compared with oak cotyledons, tion of the glass. This assumption was their state diagrams are similar (Sun et al., based on the fact that sugars are present in 1994). Also, the state diagrams of immature large amounts in desiccation-tolerant and mature soybean axes are similar, tissues and are known to be excellent glass- despite the accumulation of oligosaccha- formers. The correlation between oligosac- rides during maturation. Another indication charides and longevity (Horbowicz and that sugars alone are not sufﬁcient to Obendorf, 1994; Bernal-Lugo and Leopold, explain the formation of the vitreous state in 1995; Steadman et al., 1996; Sun and seeds came from Sun and Leopold (1997), Leopold, 1997) and the knowledge that who showed that the state diagram of maize

oligosaccharides increase Tg in model sys- embryos is different from that of a represen- tems (Slade and Levine, 1991; Roos, 1995) tative carbohydrate mix. An extensive added to the notion that sugars are impor- calorimetric study on the glass transition in tant for in vivo glass formation. bean axes revealed the complexity of intra- However, several reports do not support cellular glasses. Correspondence of differ- the contention that sugars play a dominant ential scanning calorimetry (DSC) data Dessication 10 18/3/02 1:58 pm Page 306

306 J. Buitink et al.

from beans with a model that predicts the Leprince et al., 1990a; Blackman et al.,

effects of glass components on Tg has sug- 1992) and/or galactosyl cyclitols gested that intracellular glasses could be (Horbowicz and Obendorf, 1994; Obendorf, composed of a highly complex oligomeric 1997). Also accumulating during the acqui- sugar matrix, such as, for instance, malto- sition of desiccation tolerance are different dextrin (Leprince and Walters-Vertucci, members of the LEA protein family (Ingram 1995). Buitink et al. (2000a,b) observed and Bartels, 1996). The current under- that a change in sugar composition upon standing is that these molecules seem to

priming did not change Tg or the molecular act as stabilizing agents through one or mobility in the intracellular glass. All these more different mechanisms. Furthermore, a data suggest that, besides sugars, other role for HSPs in desiccation tolerance has molecules play a crucial role in intracellu- recently been put forward (Wehmeyer and lar glass formation. Vierling, 2000). The (putative) functions of In this respect, the role of proteins in these protective molecules will be dis- intracellular glass formation has received cussed in the following sections. recent attention. Wolkers et al. (1998a,b) have found that the molecular density (i.e. hydrogen-bonding strength) of dry seeds of 10.6.1. Sucrose/oligosaccharides A. thaliana was quite different from that of a sugar glass, but much more comparable to The roles of sucrose and trehalose in pref- that of a protein–sugar glass. Investigations erential exclusion, water replacement and on the glass properties in biological sys- vitrification have been implicated as dis- tems using EPR spectroscopy also point to cussed above. However, the general abun- a role for proteins in intracellular glass for- dance of oligosaccharides in anhydrobiotic mation (Buitink et al., 2000e). The temper- higher plant systems raises the question as ature dependence of molecular mobility in to why they too are preferentially accumu- intracellular glasses is much more compa- lated (see Chapters 1 and 5). rable to that in protein glasses than that in The accumulation of oligosaccharides sugar glasses (see Fig. 10.3). Although more and cyclitols during seed maturation and research is needed to elucidate what types their disappearance during germination of protein may play a role in the glass for- has led to the hypothesis that these sugars mation, LEA proteins are possible candi- are important for desiccation tolerance. dates (Sun and Leopold, 1997). Wolkers et However, it has been shown that desicca- al. (1999) suggested that LEA proteins that tion tolerance in seeds can occur in the are embedded in the glassy cellular matrix absence of oligosaccharides (Hoekstra et confer stability on slowly dried carrot al., 1994; Lin and Huang, 1994; Bochicchio somatic embryos. Indeed, LEA proteins et al., 1997; Black et al., 1999; Buitink et change the hydrogen-bonding properties of al., 2000a). Moreover, the accumulation of model sugar systems comparable to those oligosaccharides does not necessarily lead of intracellular glasses, pointing to a possi- to the establishment of desiccation toler- ble participation of LEA proteins in intra- ance (Still et al., 1994; Black et al., 1999). cellular glass formation (Wolkers et al., Although oligosaccharides do not appear to 2000). be pivotal for desiccation tolerance, there seems to be a significant correlation between the oligosaccharide:sucrose ratio 10.6. Roles of Specific Compounds in and storage longevity of dry seeds Stability (Horbowicz and Obendorf, 1994; Steadman et al., 1996; Sun and Leopold, 1997). For A specific feature of all anhydrobiotic example, Horbowicz and Obendorf (1994) organisms is the accumulation of non- calculated that orthodox seeds of species reducing sugars, particularly of the raffi- with a sucrose:oligosaccharide ratio of nose series (Koster and Leopold, 1988; < 1.0 have storability half-viability periods Dessication 10 18/3/02 1:58 pm Page 307

Biochemistry and Biophysics of Tolerance Systems 307

of > 10 years, whereas those with a ratio oligosaccharides accumulate during matu- > 1.0 have storability half-viability periods ration, it could be possible that oligosac- of < 10 years. None the less, there are charides are linked indirectly with storage exceptions where there is no relation stability, being merely an indicator of seed between the oligosaccharide content and maturity. Future research will have to longevity (Steadman et al., 1996; Buitink et focus on roles for oligosaccharides in seed al., 2000a), indicating that molecules other maturation other than cellular protection. than oligosaccharides might have a similar function and take over the role of these sugars in their absence. 10.6.2. Late embryogenesis abundant How oligosaccharides mediate the (LEA) proteins increase in longevity is not yet resolved.

Because of their intrinsic high Tg it has Generally, the presence of LEA proteins been suggested that they play a role in the correlates well with desiccation tolerance protection of cytoplasmic components dur- (see Chapters 1, 5, 11). They accumulate

ing storage by elevating the Tg of the intra- during late maturation of developing seeds cellular glass, thereby increasing viscosity (Galau et al., 1986; Bartels et al., 1988; (see Section 10.5.4). Yet there is no evidence Baker et al., 1995; Blackman et al., 1995; that oligosaccharides have an effect on the Ingram and Bartels, 1996; Kermode, 1997; Tg or viscosity of intracellular glasses Oliver and Bewley, 1997; Cuming, 1999), (Buitink et al., 2000a,b). Considering that in dehydrating vegetative tissues of the oligosaccharides often make up only 4% of desiccation-tolerant grass Sporobolus stap- the dry weight in seeds like Impatiens or ﬁanus (Kuang et al., 1995), and in the res- bell pepper (Buitink et al., 2000a), it is per- urrection plant C. plantagineum haps unsurprising that no effect on intra- (Piatkowski et al., 1990). However, lea cellular glass properties could be transcripts have also been detected in measured. An obvious role for sugars in recalcitrant seeds and in desiccation-sensi- seeds is the protection of macromolecular tive tissues submitted to water and/or tem- structures in the dry state, especially mem- perature stress (Kermode, 1997). LEA branes. Hydrogen bonding of sugar mole- proteins represent a broad class of highly cules with macromolecules will increase conserved genes expressed in a wide range their stability (see Crowe et al., 1998, for a of plants. Comparisons of the deduced review; see Section 10.5.3). However, this polypeptide sequences of the various lea explanation does not provide a clue to the genes have led to the establishment of ﬁve role of oligosaccharides in particular. In subclasses of LEA proteins, simply desig- fact, there are indications that oligosaccha- nated Group 1, Group 2 (dehydrins), Group rides are less effective than disaccharides 3, Group 4 and Group 5 LEA proteins (see at hydrogen bonding with the polar head Cuming, 1999, for a review, and Chapters groups, particularly in the case of saturated 1, 5 and 11). Spatial patterns of LEA pro- phospholipids (Crowe et al., 1986, 1996). tein accumulation indicate that they are Also, oligosaccharides in seeds have been primarily localized in the cytosol and suggested to be able to prevent crystalliza- nucleus (Asghar et al., 1994; Goday et al., tion. Notwithstanding the argument that 1994; Blackman et al., 1995; Egerton- oligosaccharides are indeed capable of pre- Warburton et al., 1997). In the leaves of the venting crystallization in model sugar resurrection plant C. plantagineum, LEA glasses (Caffrey et al., 1988), this character- proteins were found to be both cytosolic istic would most probably not be required and present in the chloroplasts of the in vivo because of the complex mixture of leaves (Schneider et al., 1993). all the different compounds in the cyto- Furthermore, the presence of LEA proteins plasm, as mentioned before (see Section has been detected at the membranes of pro- 10.5.4). Because the maturation stage of tein and lipid bodies of Zea mays kernels seeds correlates with storage stability, and (Egerton-Warburton et al., 1997) and at the Dessication 10 18/3/02 1:58 pm Page 308

308 J. Buitink et al.

plasmalemma of Saccharomyces cerevisiae lar macromolecules, coating these macro- (Sales et al., 2000). Whereas the molecular molecules with a cohesive water layer and genetics of LEAs will be discussed else- preventing their coagulation during desic- where (Chapter 11), this section will be cation (Section 10.5.1; Close, 1996). Upon limited to the hypothetical mechanisms of removal of their own hydration shell, these how LEA proteins act in stabilization and proteins would still be capable of playing a protection during desiccation. role in stabilizing macromolecular struc- The strongest support for a primary des- tures, as they could provide a layer of their iccation-protecting role comes from the own hydroxylated residues to interact with observation that the accumulation of the the surface groups of other proteins, acting proteins coincides with the acquisition of as ‘replacement water’ (see Section 10.5.3; desiccation tolerance. Bartels et al. (1988) Cuming, 1999). It has been shown that demonstrated that desiccation tolerance purified maize dehydrin has potent cryo- could be induced precociously in imma- protective activity in vitro in a rabbit lac- ture barley embryos by the application of tate dehydrogenase freeze–thaw assay, abscisic acid (ABA), coinciding with the especially in combination with solutes accumulation of LEA proteins. Similarly, including compatible solutes such as Blackman et al. (1995) used exogenous sucrose, proline and glycine-betaine (Close, ABA to elevate the level of heat-soluble 1996). Rinne et al. (1999) suggested that, in LEA-like proteins in axes from immature cold-acclimatized apices of birch, dehy- seeds of soybean. As the LEA-like proteins drins might create local pools of water in accumulated in response to ABA, solute otherwise dehydrated cells, thereby main- leakage from the dried soybean embryos taining enzyme function. Under conditions upon rehydration markedly declined. Both of low water activity (20% polyethylene factors were apparently dependent on the glycol (PEG), corresponding to approxi- presence of ABA. These data are consistent mately –0.5 MPa), the activity of -amylase with the hypothesis that the LEA-like pro- was greater in the presence of a partially teins contribute to the increase in desicca- purified dehydrin fraction than in the pres- tion tolerance in response to ABA. ence of bovine serum albumin (BSA) as a Since the earliest identification of LEA control (Rinne et al., 1999). proteins, hypotheses regarding their func- For the Group 2 and 3 LEA proteins, tions in desiccation tolerance have hypotheses concerning their biological revolved around physiological and bio- function are based on their potential for chemical experiments and predictions on amphipathic helix formation. It has been the structure–function relationship that suggested that the amphipathic helices of can be deduced from the amino acid the Group 3 LEA proteins could form bun- sequence. The fact that LEA proteins accu- dles through hydrophobic interactions, mulate to a much higher cellular concen- thereby exposing a highly charged surface tration than is typically the case for to the exterior to which ions can bind enzymes, together with their predicted (Dure, 1993). Thus, sequestration of ions structural flexibility (of Group 2 and 3 LEA can take place, whose intracellular concen- proteins), appears to rule out an enzymatic trations might otherwise become unaccept- role. For the Group 1 LEA proteins, it has ably high within the dehydrating cell. been calculated that these polypeptides The nuclear localization of dehydrins have a tremendously high potential for raises the possibility of a dehydrin– hydration, several times greater than that chromatin alliance or an association with for ‘normal’ cellular proteins (McCubbin et the nuclear matrix, since the matrix is the al., 1985). This is a function of the remark- chromatin-organizing structure (Nickerson ably high number of charged and et al., 1989). In this regard, it is interesting uncharged polar residues within the struc- to note that many dehydrins contain a tract ture. Because of these specific properties, of serine residues (the S segment). In maize LEA proteins potentially bind to intracellu- RAB17 and tomato TAS14, it has been Dessication 10 18/3/02 1:58 pm Page 309

Biochemistry and Biophysics of Tolerance Systems 309

demonstrated that the serine residues in Savage et al., 1994). It seems that the sole the S segment can be phosphorylated, and ability, or lack thereof, to express LEAs or it has been proposed that phosphorylation dehydrin-like proteins cannot be taken as is related to the binding of nuclear localiza- an indication that the seeds of a particular tion signal peptides and, therefore, to species can or cannot withstand dehydra- nuclear transport (Goday et al., 1994; tion, reflecting the earlier views of Godoy et al., 1994). Because the matrix is Blackman et al. (1991, 1992) and Leprince associated with regulatory processes, dehy- et al. (1993) that desiccation tolerance drins may protect genetically sensitive must be the outcome of the interplay of areas. As in the nucleus, nucleolar dehy- more than one (and probably many) mech- drins may have a structural role via an anisms or processes. association with the nucleolar filament matrix, or a functional role in protecting transcriptionally active regions (Godoy et 10.6.3. Heat-shock proteins (HSPs) al., 1994). It has also been speculated that LEA Another class of proteins that have recently proteins might play a role in glass forma- been associated with desiccation tolerance tion (Sun and Leopold, 1997; Wolkers et are the HSPs (see also Chapters 1 and 5). In al., 1999; Buitink et al., 2000e). Wolkers et contrast to those of other eukaryotes, the al. (1999) suggested that LEA proteins that most prominent HSPs of plants are small are embedded in the glassy matrix might heat-shock proteins (sHSPs). They have confer stability on slowly dried carrot monomeric molecular masses of 15–42 somatic embryos. Indeed, LEA proteins kDa, but assemble into oligomers of nine to changed the hydrogen-bonding properties over 20 subunits, depending on the protein of model sugar systems toward those of (Waters et al., 1996, and references intracellular glasses, pointing to a possible therein). Their implication in desiccation participation of LEA proteins in intracellu- tolerance has been inferred from studies on lar glass formation (Wolkers et al., 2000; gene expression in developing seeds and in see Section 10.5.4). resurrection plants. In pea, Arabidopsis Despite numerous studies on the gene and sunflower seeds, sHSP expression is expression of LEA proteins in plants, at always observed significantly before dis- present, their biological function remains cernible seed desiccation, and sHSPs are to be assessed in vivo. None the less, it abundant in the dry seeds (Coca et al., appears that, based on their observed local- 1994; DeRocher and Vierling, 1994; ization at a number of sites, coupled with Wehmeyer et al., 1996). During germina- their accumulation in response to desiccation, the developmentally regulated sHSPs tion stress, dehydrins have a general role in are relatively abundant for the first few protection during drought and/or desicca- days and then decline quickly. However, tion stress. Recently, Black et al. (1999) the precise timing corresponding to the found that, in wheat embryos, dehydrin acquisition and loss of desiccation toler- accumulation is not regulated by factors ance was not assessed in the above studies. that specifically control the induction of In vegetative tissues of C. plantagineum, tolerance. Instead, it would appear that the constitutive expression of sHSPs has been dehydrin-like protein is produced in detected (Alamillo et al., 1995). response to grain detachment, even when Further evidence that HSPs may be impli- this is not followed by dehydration, as was cated in desiccation tolerance comes from also concluded for soybean (Blackman et the observation that there appears to be a al., 1991). Remarkably, recalcitrant seeds coordinated expression of lea and sHSP tran- were also found to accumulate dehydrins scripts during embryo development in during seed development and in response response to ABA, indicating the existence of to dehydration or to ABA treatment common regulatory elements of both LEA (Bradford and Chandler, 1992; Finch- proteins, sHSPs and desiccation tolerance Dessication 10 18/3/02 1:58 pm Page 310

310 J. Buitink et al.

(Almoguera and Jordano, 1992; Wehmeyer et HSPs are thought to offer a general protec- al., 1996). Also, in desiccation-sensitive cal- tive role in dry anhydrobiotes, based on the lus tissue of Craterostigma, no sHSP-related observation that HSPs are molecular chap- polypeptides could be detected, but sHSP erones (i.e. they interact with other pro- expression and the concurrent acquisition of teins and, in doing so, minimize the desiccation tolerance in the callus were initi- probability that these other proteins will ated by exogenous ABA treatment (Alamillo interact inappropriately with one another et al., 1995). Recently, a reporter-gene tran- (Waters et al., 1996; Gething, 1997; Feder scription assay showed that sHSP expression and Hofmann, 1999)). exhibits little tissue specificity, but instead The function and working mechanisms spreads throughout the embryo during of HSPs are well investigated in mam- development until essentially all cells are malian systems, although direct evidence stained in the mature seeds prior to com- originates only from in vitro studies (see plete desiccation (Wehmeyer and Vierling, Feder and Hofmann, 1999, for a review). It 2000). The activity of the reporter gene was is currently known that HSPs recognize strongly reduced in fus3-3, lec1-2 and was and bind to other proteins when these pro- almost nil in abi3-6, all desiccation- teins are in non-native conformations, intolerant mutants of Arabidopsis seeds. whether because of protein-denaturing This would suggest an overall protective stress or because the peptides they com- effect of HSPs in the cells during drying. As prise have not yet been fully synthesized, for all protective mechanisms reviewed so folded, assembled or localized to an appro- far, sHSPs may be necessary for desiccation priate cellular compartment. Binding tolerance, but they are unlikely to be suffi- and/or release of these other proteins is cient. often regulated by association with and/or In contrast to other seeds, which typi- hydrolysis of nucleotides. Typically, HSPs cally accumulate low to moderate levels of function as oligomers, or as complexes of sHSPs, recalcitrant chestnut (Castanea several different chaperones, co-chaper- sativa) seed cotyledons contain a highly ones, and/or nucleotide exchange factors. abundant sHSP (Collada et al., 1997). This Interaction with chaperones is variously sHSP was shown to exhibit molecular responsible for: (i) maintaining HSP part- chaperone activity in vitro, as demon- ner proteins in a folding-competent, folded strated by the ability of the sHSP to main- or unfolded state; (ii) organellar localiza- tain soluble cytosolic proteins in their tion, import and/or export; (iii) minimizing native formation during both heat and cold the aggregation of non-native proteins; and stress (Soto et al., 1999). A model liposome (iv) targeting non-native or aggregated pro- system with the encapsulated fluorescent teins for degradation and removal from the dye calcein was used to investigate the pro- cell. Presumably, the last two functions are tection of membranes by the LEA-like pro- the most important in coping with environ- tein HSP 12 from S. cerevisiae during mental stress (Feder and Hofmann, 1999). desiccation (Sales et al., 2000). This LEA- like HSP was found to act in an analogous manner to trehalose and protect liposomal 10.7. Conclusion and Outlook membrane integrity against desiccation. The interaction between HSP 12 and the From the above evidence amassed so far it is liposomal membrane was judged to be clear that more than one mechanism acts in electrostatic, as membrane protection was conferring desiccation tolerance on plant only observed with positively charged lipo- organisms. Often, the involvement of a spe- somes and not with either neutral or nega- cific substance in desiccation tolerance is tively charged liposomes. difficult to establish, because different sub- So far, there is no direct experimental stances may substitute for one another. evidence that points to a specific role of Decreasing water potential appears to neces- sHSPs in desiccation tolerance. Small sitate successive mechanisms of protection Dessication 10 18/3/02 1:58 pm Page 311

Biochemistry and Biophysics of Tolerance Systems 311

during drying. In the early stages of dehy- as preferential hydration and enzymatic dration, partitioning of amphiphiles into antioxidant activity ineffective. Then, membranes might cause signalling of the immobilization in a glassy matrix via stress, leading to a variety of responses. hydrogen bonding with water-replacing Among these responses are the production substances gains importance. This prevents of antioxidants, compatible solutes, dehy- excessive ordering, e.g. crystallization, and dration proteins and, probably, sHSPs. protects the structure of macromolecules. Whereas in seeds these substances are pro- There is an important role for sugars in this duced as a part of the developmental pro- hydrogen-bonding process – from interac- gramme, in vegetative plants that are tion with proteins to interaction with other sensitive to complete dehydration these sugar molecules in the formation of a responses often occur upon exposure to glassy matrix. Some of the dehydration moderate levels of water loss (= drought). proteins may help improve the stability of Since these substances appear to improve the glassy matrix. Glasses are considered as tolerance to drought, it can be implied that particularly important in slowing molecu- the mechanisms involved give protection lar mobility and chemical reaction rates. under conditions where there is still bulk Consequently, they are important in water left. Among these mechanisms could depressing the rate of ageing in the dry be depression of metabolism, improved free- state. radical scavenging and preferential hydra- The majority of investigations concern- tion of macromolecules. Oxidative damage, ing anhydrobiosis in plants have been membrane fusion and protein denaturation focused on phenomena in the dry state. are thus prevented. It has to be realized that Considering the fact that desiccation-sensi- it is often in these high water content tive organisms usually die at relatively ranges, i.e. when bulk water is present, that high water contents of, for example, 2–0.5 1 desiccation-sensitive organisms die. g H2Og dw, future research should be When most of the bulk water has disap- addressed more towards mechanisms of peared, the interaction between molecules protection that operate in this particular increases, which renders mechanisms such range of water contents.

10.8. References

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11 Molecular Genetics of Desiccation and Tolerant Systems

Jonathan R. Phillips,1 Melvin J. Oliver2 and Dorothea Bartels3 1Max-Planck-Institute for Plant Breeding Research, Carl-von-Linné-Weg 10, D-550829 Köln, Germany; 2USDA-ARS Plant Stress and Germplasm Development Unit, 3810 4th Street, Lubbock, Texas 79415, USA; 3Institute of Botany, University of Bonn, Kirschallee 1, D-53115 Bonn, Germany

11.1. Deﬁnition of Desiccation Tolerance 320 11.2. Resurrection Plants: Deﬁnition and Distribution 320 11.3. Metabolic Changes During the Dehydration–Rehydration Cycle in Resurrection Plants 321 11.4. Molecular Studies with Resurrection Plants 321 11.4.1. LEA proteins 323 11.4.2. Carbohydrate metabolism 324 11.5. Regulation of Gene Expression During the Desiccation Process in Resurrection Plants 324 11.6. Desiccation-tolerant Bryophytes 326 11.7. Constitutive Cellular Protection 327 11.8. Cellular Damage and Recovery Following Rehydration 328 11.9. Gene Expression During Recovery 329 11.9.1. Rehydrins 330 11.10. Transgenic Approaches towards Improving Plant Dehydration/ Desiccation Tolerance 330 11.10.1. Compatible solutes or osmolytes 331 11.10.2. Oxygen-scavenging proteins 333 11.10.3. LEA proteins 334 11.10.4. Regulatory genes 334 11.11. Conclusions and Perspectives 335 11.12. Acknowledgements 336 11.13. References 336

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11.1. Definition of Desiccation tive tissues during drying. By using differen- Tolerance tial screening approaches, cDNAs corresponding to transcripts expressed only in Desiccation is the drying out of an organ- response to water deficit have been isolated ism that is exposed to air. Under these con- and characterized (Ingram and Bartels, ditions, most of the protoplasmic water is 1996; Bockel et al., 1998). However, it lost and a very low amount of tightly should be emphasized that, due to the bound water remains in the cell. purely descriptive nature of the data, the Desiccation tolerance apparently depends functional role of many gene products is on the ability of the cells to maintain the unclear. Using examples from resurrection integrity of the cell membranes and to pre- plants and the moss Tortula ruralis, this vent denaturation of proteins. Tolerance in chapter will review what is currently organs such as seeds and pollen is wide- known about the molecular responses that spread among higher plants and in fact par- occur during the acquisition of desiccation tial desiccation is a prerequisite for the tolerance and how this knowledge may be completion of the life cycle in most species applied to improve plant tolerance to limit- producing seeds. In contrast, only a few ing water conditions. plants possess mature foliage or vegetative tissue that is desiccation-tolerant. These include a small group of angiosperms, 11.2. Resurrection Plants: Definition termed resurrection plants (Gaff, 1971), and Distribution some ferns and fern allies (Bewley and Krochko, 1982), and several species of Desiccation tolerance in vegetative tissues of algae, lichens and bryophytes (Oliver, higher plants has been most studied in the 1996; Oliver and Bewley, 1997; Oliver et so-called resurrection plants, which possess al., 2000; see Chapters 1 and 7). the unique ability to revive from an air-dried Desiccation has to be distinguished from state (Gaff, 1971). Such plants are often a mild water deficit, which is a condition poikilohydrous and their water content cor- where the water status of plants undergoes relates with fluctuations in the relative relatively small changes (Bray, 1997). Many humidity (RH) of the local environment. plants are able to cope with this challenge Broadly two types of desiccation-tolerant either by reducing water flux through the plants have been reported: those that lose plant or by increasing their water uptake. chlorophyll during dehydration and those Water loss can be avoided by various that retain chlorophyll (see Chapters 1 mechanisms such as stomatal closure, and 7). reduction of leaf growth or production of Resurrection plants colonize ecological specialized leaf surfaces to avoid transpira- niches with restricted seasonal water avail- tion, whereas water uptake can only be ability. Most species are found preferen- increased by the development of specialized tially on rock outcrops at low to moderate root structures. Many of these mechanisms elevations below 2000 m in tropical and are common to both desiccation-tolerant subtropical zones and to a lesser extent in and non-tolerant plants; however, little is temperate climates (Porembski and known about those mechanisms that, for Barthlott, 2000). The geographic locations example, allow a resurrection plant to sur- where resurrection plants have been identi- vive equilibrium with 0% air humidity. fied are southern Africa (including Desiccation tolerance has been studied at Madagascar), Australia, India and parts of the molecular level by examining tolerant South America (Gaff, 1977, 1987; Gaff and systems such as seeds, resurrection plants Bole, 1986). Although rarely found in and mosses. As a result of these studies, it Europe, a few species have been observed has become apparent that tightly regulated in the western Balkan mountains (Stefanov programmes of gene expression, both at the et al., 1992). spatial and temporal levels, occur in vegeta- Desiccation-tolerant plants comprise Dessication 11 18/3/02 1:58 pm Page 321

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monocotyledonous and dicotyledonous physiological and morphological studies, species within the angiosperms. To date, has been recently reviewed in some detail however, no desiccation-tolerant plants (Oliver and Bewley, 1997; Hartung et al., have been reported that belong to the gym- 1998; Scott, 2000). The emphasis in this nosperms. The desiccation-tolerant chapter will be on summarizing the results dicotyledonous species appear to be repre- of biochemical and molecular studies. sented mainly in the Gesneriaceae, Myrothamnaceae and Scrophulariaceae plant families, in contrast to their mono- 11.4. Molecular Studies with cotyledonous counterparts, which are more Resurrection Plants widely distributed throughout evolution. Many of the described dicotyledonous res- To date, molecular studies of resurrection urrection plants belong to the plants are limited to several species: the Scrophulariaceae, which include ten dicotyledonous species Craterostigma Craterostigma and 15 Lindernia species plantagineum (Bartels et al., 1990; Frank et that are indigenous to Africa (Fischer, al., 2000), the monocotyledonous species 1992) (see Chapter 7). Sporobulus stapfianus (Neale et al., 2000) The acquisition of desiccation tolerance and Xerophyta villosa (Mundree et al., in resurrection plants is complex. This is 2000), and the moss T. ruralis (Wood et al., due to the multiple stresses that are 1999). In C. plantagineum and S. stapfi- imposed on plant tissues during severe anus the majority of changes in gene dehydration. Consequently, tolerant plants expression occur during dehydration, not must overcome several problems, including during the rehydration phase of the resur- minimization of mechanical damage asso- rection process, which in turn leads to a ciated with turgor loss, maintenance of the very efficient protection system against functional integrity of macromolecules desiccation. This contrasts with what is such as proteins and nucleic acids, mini- observed for T. ruralis and may be linked to mization of toxin accumulation and free- differing mechanisms of desiccation toler- radical damage, and initiation of repair ance that exist in vascular higher plants mechanisms upon rehydration. The speed versus bryophytes. of water loss and the events before dehy- Dehydration induces the expression of a dration appear to be critical for the sur- large number of transcripts in both C. plan- vival, such that, if the speed of dehydration tagineum and S. stapfianus (see Table is too fast, plants do not acquire tolerance 11.1). Homology analyses reveal a broad to desiccation. This observation suggests spectrum of differentially regulated genes that the acquisition of desiccation toler- with diverse putative functional identities, ance is an active process and requires spe- which underlines the fact that desiccation cific biochemical changes and the tolerance is the result of a complex interac- synthesis of desiccation-related molecules. tion of different cellular processes. It has The nature of these molecules has recently been suggested that the dehydration- been described for some species by molec- induced gene products can be associated ular and biochemical studies. with signal transduction pathways and regulation of stress-specific transcription, with carbohydrate metabolism or with cellular 11.3. Metabolic Changes During the protection (Phillips and Bartels, 2000). Dehydration–Rehydration Cycle in Comparison between the dehydration- Resurrection Plants induced genes identified in C. plantagineum and S. stapfianus has revealed Physiological, morphological, biochemical that several genes encode homologous pro- and molecular studies have been per- teins and thus belong to the same func- formed with several resurrection plants. tional group. These include gene products This information, largely derived from with a putative protective function such as Dessication 11 18/3/02 1:58 pm Page 322

322 J.R. Phillips et al. AJ005833 U33915 AJ132000 Z46647 Y11795 AJ001293 AJ001294 AJ242805 X69883 AJ242806 Phospholipase DSerine/threonine protein kinaseSerine/threonine phosphatase type 2CVP-1/ABI3Homoeodomain leucine zipper proteinsmyb-related proteins AJ242803 eIF1 protein AJ005373 AJ005820 Sucrose synthase AJ133000 TransketolaseSucrose-phosphate synthaseCytosolic glyceraldehyde-3-phosphate dehydrogenase X78307 U33917 AJ000552 Major intrinsic proteins AJ242801 AJ131999 Y11821 Late embryogenesis abundant proteinsEarly light inducible protein Z46648 X74067 AJ001292 X66598 CpPLD-2 Craterostigma CpPK1SDG37c Craterostigma CpVP1 Sporobolus CpHB-1/2 Craterostigma Craterostigma CpM-7/10 Craterostigma SDG134c Sporobolus CpSS-1/2 Craterostigma CpTKT-7/10 Craterostigma CpSPS-1/2 Craterostigma CpGAPDH Craterostigma CpPIP Craterostigma SDG50cCp6-19 Sporobolus Cp27-45 Craterostigma DSP22 Craterostigma Craterostigma SDG69c Sporobolus Desiccation-related genes from resurrection plants. phospholipid cleavage Regulation of gene expression via phosphorylation/dephosphorylation activator Transcriptional regulators Transcriptional initiation factor Translation Carbohydrate metabolism Conversion of sucrose uridine-diphosphate into fructose and UDP-glucose Synthesis of sugar phosphate intermediates Synthesis of sucrose 6-phosphate from fructose 6-phosphate and uridine 5’-diphosphate-glucose Reversible oxidation and phosphorylation of glyceraldehyde-phosphate to 1,3-bisphosphoglycerate Cellular protection Small molecule/water transporters Protection of macromolecular structures Protection of photosystem II Table 11.1. Table Putative function Signal transduction Secondary messenger production via Gene Origin Gene product Acc. No. Dessication 11 18/3/02 1:58 pm Page 323

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late embryogenesis abundant (LEA) pro- tolerant and also in desiccation-sensitive teins, the thylakoid membrane-associated plants (Close, 1997; Bartels, 1999; Cuming, early light-induced protein (ELIP) or 1999; see Chapters 1, 5 and 10). LEA pro- tonoplast intrinsic protein (TIP). Homology teins comprise a large family of plant pro- also extends to cDNAs isolated from T. teins that accumulate to high levels during ruralis (Wood et al., 1999). Expression pat- late stages of embryo development (Galau terns of the isolated genes have been et al., 1986). Expression studies show that described at the mRNA level. Three basic LEA proteins are generally associated with patterns of gene expression are observed: cellular dehydration in seeds and in class 1 transcripts accumulate to high lev- response to water deficit in vegetative tis- els from an initial low level during dehy- sues. Treating plant tissues with the plant dration and disappear during rehydration; hormone ABA can also induce the expres- class 2 transcripts accumulate transiently sion of lea genes. A common feature of during low-level dehydration; class 3 tran- most LEA proteins is their high scripts are down-regulated during dehydra- hydrophilicity, which permits solubility tion. Most of the class 1 transcripts and after boiling. Correlative studies and bio- some of the class 2 transcripts also accu- chemical features strongly suggest a protec- mulate following the application of exoge- tive role in the plant cell during nous abscisic acid (ABA), which indicates dehydration. a central role for ABA in mediating gene LEA proteins from different plant species expression during dehydration. have been divided into groups based on pre- It has been hypothesized that the tempo- dicted biochemical properties and sequence ral and spatial expression patterns would similarities (Dure et al., 1989; Ingram and provide information concerning the func- Bartels, 1996; Cuming, 1999). The strong tion of a gene. Data on spatial expression conservation of motifs in LEA proteins dur- patterns and subcellular localization as ing evolution points to domains with func- investigated by RNA in situ hybridization tional constraints. One such motif, which is and cell fractionation are only available characteristic for group 1 LEA proteins, is 20 from C. plantagineum. The analyses amino acids in length and was first found in revealed that RNAs and proteins show a the wheat Em protein. Group 2 LEA pro- specific tissue and cellular distribution teins, also referred to as dehydrins, are the (Schneider et al., 1993). LEA-like proteins, most widely studied LEA proteins (Close, which may have a general protective func- 1997). Many homologues have been isolated tion, are found in most tissues and cell from species ranging from gymnosperms to types, and accumulate in the cytoplasm or dicotyledonous and monocotyledonous in chloroplasts. Water channel-like pro- angiosperms. Dehydrins are characterized by teins, which are predicted to be involved a lysine-rich 15-amino-acid motif (termed in water flux or transport of small mole- the K-segment), which is predicted to form cules, are associated with membranes an amphipathic -helix, a tract of contiguous (Mariaux et al., 1998). Transcripts encoding serine residues and a conserved motif con- sucrose synthase are only detected in the taining the consensus sequence DEYGNP, external phloem of vascular bundles, sug- which is found close to the N- gesting an association with transport terminus of the protein. Group 3 LEA pro- processes within the plant (Kleines et al., teins share a characteristic repeat motif of 11 1999). amino acids, which appears to have undergone duplication and some substitution events. Dure et al. (1989) predicted that the 11.4.1. LEA proteins 11-amino-acid peptide forms an amphipathic -helix with possibilities for intra- LEA proteins represent one major group of and intermolecular interactions. In relation proteins that are reported to be expressed to the others, LEA proteins belonging in response to dehydration in desiccation- to groups 4 and 5 are less frequently Dessication 11 18/3/02 1:58 pm Page 324

324 J.R. Phillips et al.

represented in the literature. Group 4 is function may be to protect the cell via glass characterized by a conserved N-terminus formation rather than solutes crystallizing predicted to form -helices and a diverse C- (see Chapter 10). Through the presence of terminal part with a random coil structure. sugars, a supersaturated liquid is produced Group 5 LEA proteins contain more at desiccation with the mechanical proper- hydrophobic residues than groups 1 to 4 and ties of a solid. Secondly, sucrose may main- consequently are not soluble after boiling, tain hydrogen bonds within and between leading to the suggestion that they probably macromolecules and maintain the structure. adopt a globular conformation. The lea genes This property has been shown in in vitro from all five groups have been isolated and experiments for trehalose, a non-reducing characterized at the molecular level from the disaccharide of glucose (-D-glucopyranosyl resurrection plant C. plantagineum, and a (1-1) -D-glucopyranoside) (Crowe et al., remarkably high level of LEA proteins has 1992). Trehalose is found in desiccation-tol- been found to accumulate in desiccated veg- erant lower organisms such as yeast or etative tissues. This observation leads to the Selaginella, but only in small amounts in the hypothesis that cellular desiccation toler- resurrection plants Myrothamnus flabelli- ance depends on a relatively high concentra- folia and S. stapfianus (Bianchi et al., 1993; tion of a number of different LEA proteins, Drennan et al., 1993; Albini et al., 1994). which are simultaneously expressed in When the carbohydrate levels in hydrated response to water deficit. To test this, a quan- and dehydrated tissues in resurrection titative comparison of LEA protein accumu- plants are compared, it is clear that in some lation in tolerant and sensitive vegetative species large qualitative and quantitative tissues of two closely related species may be changes occur during the dehydration– informative. rehydration cycle but in others only small Despite extensive studies, biochemical differences are observed (see Table 11.2). knowledge of the function of LEA proteins One well-documented change in drying is scarce. Functional studies have used two leaves is the conversion of the highly abun- approaches: in vitro protection assays with dant C8-sugar 2-octulose into sucrose, purified proteins, and in vivo studies over- which comprises up to 40% of dry weight expressing LEA proteins in plants or yeast. in desiccated leaves, as first reported for C. Results from both types of experiments plantagineum (Bianchi et al., 1991a). support a role for LEA proteins in the acquisition of desiccation tolerance. 11.5. Regulation of Gene Expression During the Desiccation Process in 11.4.2. Carbohydrate metabolism Resurrection Plants

In addition to de novo synthesis of proteins, Knowledge of regulatory pathways is of par- major changes in carbohydrate metabolism ticular importance because they determine take place during the resurrection process. the expression of a set of genes in a multi- While the nature of dehydration-induced genic trait. Despite the large body of infor- proteins is broadly similar among different mation concerning genes that are induced resurrection species, the abundant carbo- during dehydration, knowledge of the regu- hydrate molecules in the hydrated tissues latory network(s) is scarce. Most informa- appear to be diverse. The accumulation of tion concerning the regulation of gene sucrose in dehydrated tissues is, however, expression is derived from promoter analy- a common theme (see Table 11.2). Taken ses and comes from desiccation-sensitive together, resurrection plant species appear species, in particular Arabidopsis. Mutants to possess different metabolic pathways have yet to be fully exploited as a potential that result in the synthesis of sucrose. This tool for dissecting regulatory pathways. supports a role for sucrose in desiccation This is mainly due to the facts that desicca- tolerance, which may be twofold. One tion tolerance is a polygenic character and Dessication 11 18/3/02 1:58 pm Page 325

Molecular Genetics of Desiccation and Tolerant Systems 325 ., 1998 et al ., 1993 ., 1993 ., 1991b ., 1991a ., 1997 ., 1997 ., 1994 et al et al et al et al et al et al et al Bianchi Bianchi a a dry weight) 1 Glucose-glycerol 14 Sucrose 90 mol g a a Sugar content ( Trehalose 29 Trehalose 17.5 Trehalose 148 Trehalose 136 Drennan Sucrose 383 Sucrose 463 ) Octulose 620 Sucrose 520 Bianchi ) Octulose 1120 Sucrose 304 A. Richter and D. Bartels, unpublished data ) Glucose-glycerol 8 ) Octulose 810 Sucrose 59 A. Richter and D. Bartels, unpublished data ) Octulose 373 Sucrose 198 A. Richter and D. Bartels, unpublished data ) Octulose 914 Sucrose 195 A. Richter and D. Bartels, unpublished data ) Sucrose 59.7 Sucrose 165 Müller ) Sucrose 14 ) Sucrose 96 Sucrose 204 Müller ) Sucrose 236 Sucrose 772 Albini ) Sucrose 116 Sucrose 156 A. Richter and D. Bartels, unpublished data ) Sucrose 43 Sucrose 83 Ghasempour Scorophulariaceae ( Scorophulariaceae ( Myrothamnaceae ( Scorophulariaceae ( Poaceae Gesneriaceae ( Poaceae ( ( Scorophulariaceae Scorophulariaceae ( Gesneriaceae ( ( Gesneriaceae Velociaceae ( ( Contents of abundant carbohydrates in hydrated and dehydrated leaves some resurrection plants. These values are expressed as % sugar in aqueous extracts. Oropetium thomaeum Sporobulus stapﬁanus Haberlea rhodopensis Ramonda myconi Boea hygroscopica Craterostigma lanceolatum Craterostigma hirsutum Lindernia acecularis Table 11.2. Table SpeciesCraterostigma plantagineum Lindernia brevidens Hydrated leavesXerophyta villosa a Dehydrated leaves Reference Myrothamnus ﬂabellifolia Dessication 11 18/3/02 1:58 pm Page 326

326 J.R. Phillips et al.

that many resurrection plants are polyploid whereas pcC27-45 and pcC11-24 promoters and consequently do not lend themselves are not active in vegetative tissues. The to mutational studies. Here, regulation of ectopic expression of the Arabidopsis gene expression in C. plantagineum will be ABI-3 gene product did, however, lead to discussed with reference to the general ABA-inducible promoter activity in leaves field, and some recent discoveries concern- of Arabidopsis, suggesting that the activity ing Arabidopsis will be described later (see of transcription factors in the leaves of C. Section 11.10). plantagineum is absent from the leaves of The plant hormone ABA is associated Arabidopsis or tobacco (Furini et al., 1996). with dehydration-regulated gene expres- The promoter analysis approach led to the sion. Exposure to exogenous ABA causes identification of cis-elements that have the induction of genes that otherwise are since been used to isolate DNA-binding activated by dehydration. Mutants with proteins. altered sensitivity to ABA or a modified Molecules with putative transcriptional- ABA biosynthesis pathway provide evi- activating activities or putative signalling dence for the role of ABA in mediating molecules have been obtained from differ- gene expression in response to water ential screening experiments. These deficit (Leung and Giraudat, 1998). It has, include members of the myb transcription however, also become apparent that ABA- factor family (Iturriaga et al., 1996), a heat- independent regulatory systems function shock transcription factor (Bockel et al., in gene expression under the same stress 1998), members of the homoeodomain (Frank et al., 1998; Shinozaki and leucine zipper family (Frank et al., 1998), Yamaguchi-Shinozaki, 2000). phospholipase D (Frank et al., 2000) and a The regulation of gene expression by novel C. plantagineum gene cDT-1 (Furini dehydration and ABA in vegetative tissues et al., 1997). Transcripts encoding these of C. plantagineum involves several sig- molecules are induced by dehydration, nalling pathways. Different types of cis- suggesting an involvement of the gene acting elements are required for stress- products in the dehydration process. A dif- responsive, coordinated gene expression. ficult challenge is to identify the target Promoters from different groups of stress- genes of these putative regulatory mole- inducible genes have been analysed and cules. The findings from C. plantagineum compared. The most extensively studied are extended mainly by studies of the promoters from C. plantagineum regulate response to dehydration in Arabidopsis, the expression of three ABA-responsive which involves transcription factors lea-like genes, pcC6-19, pcC27-45 and belonging to the Myb, Myc, AP2/EREBP pcC11-24 (Michel et al., 1993, 1994; and bZip classes, protein kinases and pro- Velasco et al., 1998). Despite the fact that tein phosphatases. Although the exact role dehydration and ABA regulate all three of most factors in gene regulation is genes, no common sequence motifs were unknown, it is interesting to note that apparent in the promoter sequences. members of different regulator families are Promoter analyses were performed in part of the regulatory dehydration network. transgenic tobacco and Arabidopsis plants to determine the functional cis elements. All three promoters were found to be 11.6. Desiccation-tolerant Bryophytes highly active in seeds and pollen. However, the pcC6-19 promoter differs Desiccation-tolerant bryophytes are found from pcC27-45 and pcC11-24 since no worldwide and inhabit a variety of habi- protein synthesis is required for ABA- tats, most of which could, during some mediated transcription. A second differ- period of the year, be considered as ence is that the pcC6-19 promoter is extreme, either on a macro or micro level. inducible by dehydration or ABA in vege- In most cases, the extremes that these tative tissues of tobacco or Arabidopsis, plants experience are both in water avail- Dessication 11 18/3/02 1:58 pm Page 327

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ability and temperature (Clausen, 1952; Lee the first hour or two following rehydration. and Stewart, 1971; Norr, 1974; Dilks and This has led to the suggestion that a major Proctor, 1976; Alpert and Oechel, 1987; see component of the mechanism of desiccation Chapter 7). tolerance in bryophytes is a rehydration- Desiccation-tolerant bryophytes, because induced cellular repair response (see of their simple architecture, have little or Bewley and Oliver, 1992; Oliver and no morphological (or indeed physiological) Bewley, 1997, for reviews). The implication characteristics or adaptations that can limit is that, although cellular protection and water loss or regulate plant temperature. hence desiccation tolerance are constitutive, As a result of this, the internal water con- it is not sufficient to prevent some damage tent of their photosynthetic tissues rapidly from occurring (or being manifested) upon equilibrates to the water potential of the rehydration, and thus repair processes are environment once free water is lost from needed and induced when water returns to the surface of the plant. This in turn means the protoplasm of the cells. Much of the evi- that these plants experience drying rates dence for these hypotheses comes from the that are much faster than those experi- study of a family of desiccation-tolerant enced by their more complex pteridophyte mosses, the Tortula complex, and in partic- or angiosperm counterparts. In fact, the ular the species T. ruralis (synonymous with drying rates that desiccation-tolerant Syntrichia ruralis). bryophytes experience are in the main lethal to desiccation-tolerant ferns and flowering plants (Bewley and Krochko, 11.7. Constitutive Cellular Protection 1982). The fact that bryophyte tissues rapidly equilibrate to the water potential of Observational experiments confirm a pro- the environment means that, in the major- tective component to the mechanism of ity of cases where temperatures become desiccation tolerance in bryophytes. extreme, hot or cold, these plants are dry. It Freeze–fracture studies of dried T. ruralis is in the dried state that desiccation-toler- cells (both rapidly and slowly dried) ant bryophytes (and most desiccation-toler- demonstrate that cellular integrity is ant plants of the less complex clades) maintained during drying (Platt et al., tolerate temperature extremes (Norr, 1974; 1994) and plasma membranes, internal Malek and Bewley, 1978). membranes and structures remain The rapid equilibration of protoplasmic undamaged. Physiological experiments water potential with that of the environ- designed to elucidate the effect of desic- ment in bryophyte tissues appears to cation on photosynthesis suggest that the demand a type of desiccation tolerance that protection mechanisms that are operating is significantly different from that exhib- in Tortula are very effective in protecting ited by the resurrection plants so far the photosynthetic apparatus and in described (Oliver and Bewley, 1997). allowing for the rapid recovery of photo- Rather than acquiring desiccation tolerance synthetic activity (Tuba et al., 1996; in response to a dehydration event as seen Proctor et al., 1998; Csintalan et al., 1999; in Craterostigma, Sporobolus and other Proctor, 2000). The almost instantaneous desiccation-tolerant angiosperms, desicca- photosystem recovery and the relatively tion-tolerant bryophytes appear to express short time needed (20 min) to reach a this trait constitutively (Bewley and Oliver, positive carbon balance, as described by 1992; Oliver and Bewley, 1997). This form these authors, occur at a time when of desiccation tolerance is considered the chloroplast structure is substantially dis- most primitive of those that have received rupted (see below). How this is achieved attention so far (Oliver et al., 2000). In this is enigmatic but ecologically it makes type of tolerance, the major genetic response sense for these opportunistic bryophytes to a desiccation event, at least at the level of selectively to protect their photosynthetic gene expression, occurs after the fact, during capability. Dessication 11 18/3/02 1:58 pm Page 328

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Metabolically, desiccation of gameto- recently been reported. Protein analyses phytic tissues of T. ruralis results in a rapid using purified antibodies raised against the decline in protein synthesis, as in all desic- common C-terminus of maize seedling cation-tolerant and sensitive mosses tested dehydrins (Close et al., 1993) show that T. so far (see Bewley and Oliver, 1992; Oliver ruralis produces two major dehydrins and Bewley, 1997, for reviews). In T. ruralis, (80–90 kDa and 35 kDa). These are present this loss of protein synthetic capacity is in the hydrated state and do not appear to manifested by a loss of polysomes resulting increase during rapid or slow drying from the run-off of ribosomes from mRNAs, (Bewley et al., 1993). A similar result was concomitant with their failure to reinitiate obtained with the desiccation-tolerant protein synthesis (see Bewley, 1979; moss Thuidium delicatulum (T.L. Bewley and Oliver, 1992, for reviews). The Reynolds, M.J. Oliver and J.D. Bewley, rapid loss of polysomes during drying and unpublished data). the apparent sensitivity of the initiation step of protein synthesis to protoplasmic drying lead to the conclusion that the pro- 11.8. Cellular Damage and Recovery tection component of the mechanism of Following Rehydration tolerance for these plants does not involve the synthesis of proteins induced by the Following rehydration, gametophytic cells onset of a water deficit. This is borne out of desiccation-tolerant mosses undergo by the observation that no new mRNAs are substantial and universal disruption of cel- recruited into the protein synthetic com- lular integrity including breaches of all plex, even if the rate of water loss is slow membrane systems (see Oliver and Bewley, (Oliver, 1991, 1996). The fact that the moss 1984a, for review). Internal organelles survives rapid desiccation (even when des- swell and distort and their internal mem- iccation is achieved in a few minutes in a brane systems become dispersed. lyophilizer) also indicates that an Nevertheless, the cells do not die, as do inducible protection mechanism is not cells of sensitive species, but return to a necessary for survival. normal appearance within 12–24 h. The As discussed above, LEA proteins and amount of cellular disruption that occurs carbohydrates are important components of during rehydration clearly depends upon protective mechanisms in desiccation- the rate at which water was lost during tolerant plants and plant tissues. In desiccation. Chloroplasts of T. ruralis dried desiccation-tolerant mosses, sucrose is the to air dryness over 4–6 h (a natural rate) are only free sugar available for cellular protec- swollen when rehydrated but retain more tion (Bewley et al., 1978; Smirnoff, 1992). of their normal internal structure and The amount of this sugar in gametophytic exhibit fewer clefts in their membranes cells of T. ruralis is approximately 10% of than do the chloroplasts in rehydrated cells dry mass, which is sufficient to offer mem- of gametophytes dried within an hour brane protection during drying, at least in (Tucker et al., 1975). The greater retention vitro (Strauss and Hauser, 1986). Moreover, of chloroplast structure allows slow-dried neither drying nor rehydration in the dark T. ruralis to effect a more rapid recovery of or light results in a change in sucrose con- photosynthesis, achieving a positive car- centration, suggesting that it is important bon balance within 20 min following rehy- for cells to maintain sufficient amounts of dration (Bewley, 1979; Tuba et al., 1996). this sugar (Bewley et al., 1978). The lack of The time required for full photosynthetic an increase in soluble sugars during drying recovery upon rehydration, however, varies appears to be a common feature of desicca- considerably among species, depending on tion-tolerant mosses (Smirnoff, 1992). The their degree of desiccation tolerance existence of dehydrin-type LEA proteins in (Proctor et al., 1998). Electrolyte leakage desiccation-tolerant vegetative tissues of upon rehydration, a measure of membrane desiccation-tolerant bryophytes has only damage, is also affected by the speed at Dessication 11 18/3/02 1:58 pm Page 329

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which desiccation occurs. After slow dry- in T. ruralis, Oliver (1991) demonstrated ing, leakage in moss is less than half as that during the first 2 h of hydration the great as after rapid desiccation and is simi- synthesis of 25 proteins is terminated, or lar to leakage of hydrated controls, indicat- substantially decreased, and the synthesis ing minimal membrane damage (Bewley of 74 proteins is initiated, or substantially and Krochko, 1982). It is these observa- increased. Controls over changes in synthe- tional studies that first led to the hypothesis of these two groups of proteins, the for- sis that the mechanism for tolerance in mer termed hydrins and the latter desiccation-tolerant bryophytes includes a rehydrins, are not mechanistically linked. repair-based strategy to recover from the It takes a certain amount of prior water loss damage manifested upon rehydration. to fully activate the synthesis of rehydrins upon rehydration. This may in turn indicate that there is also a mechanism by 11.9. Gene Expression During Recovery which the amount of water loss is ‘sensed’ and ‘translated’ into a protein synthetic The repair aspect of the mechanism of des- response upon rehydration. Such a sce- iccation tolerance in these plants, although nario was also proposed for the novel pat- demonstrated to be a major component of tern of protein synthesis associated with tolerance, is difficult to detail and charac- the drying of S. stapfianus (Kuang et al., terize. Most work has focused on the pro- 1995). Perhaps this is a strategy that has teins whose synthesis is induced evolved to link the amount of energy immediately upon rehydration of desic- expended in repair to the amount of dam- cated gametophytic tissue. Early work (see age potentiated by differing extents of dry- Bewley, 1979, for review) established the ing. ability of T. ruralis and other mosses Since rehydrins appear to be synthe- rapidly to recover synthetic metabolism sized from a transcript pool that is qualita- when rehydrated. The speed of this recov- tively unaffected by desiccation or ery was dependent upon the rate of prior rehydration (Oliver, 1991), it was of inter- desiccation: the faster the rate of desicca- est to determine what are the translational tion, the slower the recovery. In addition, controls of gene expression that operate although the pattern of protein synthesis in during the initial phases of recovery in T. the first 2 h of rehydration of T. ruralis is ruralis. A partial answer was gained from distinctly different from that of hydrated the use of rehydrin cDNA clones isolated controls, novel transcripts were not made from differential screening of a T. ruralis in response to desiccation (Oliver and rehydration cDNA library (Scott and Bewley, 1984b; Oliver, 1991). Hence it was Oliver, 1994). RNA blots revealed that sev- suggested that T. ruralis responds to desic- eral rehydrin transcripts accumulate dur- cation by an alteration in protein synthesis ing slow drying (Oliver and Wood, 1997; upon rehydration that is in large measure Wood and Oliver, 1999) at a time when it is the result of a change in translational con- assumed that transcriptional activity is trol. Changes in transcriptional activity rapidly declining. These transcripts do not were observed for nearly all transcripts accumulate during rapid desiccation, nor is studied (Scott and Oliver, 1994) but did not their accumulation during slow drying result in a qualitative change in the tran- associated with an increase in endogenous script population during desiccation or ABA accumulation. ABA is undetectable in rehydration. It thus appears that T. ruralis this moss (Bewley et al., 1993; M.J. Oliver, relies more upon the activation of pre- unpublished data), and T. ruralis does not existing repair mechanisms for desiccation synthesize specific proteins in response to tolerance than it does on either pre-estab- applied ABA. The accumulation of these lished or activated protection systems. transcripts was postulated to be the result In a detailed study of the changes in of an increase in mRNA stability brought protein synthesis initiated by rehydration about by the removal of water from the Dessication 11 18/3/02 1:58 pm Page 330

330 J.R. Phillips et al.

cells (Scott and Oliver, 1994). Recent stud- protein contains 15 15-amino-acid repeats ies clearly demonstrate that these tran- predicted to form amphipathic helices scripts are sequestered in the dried (Velten and Oliver, 2001), which is very gametophytes in messenger ribonucleopro- reminiscent of the predicted structural tein particles (mRNPs) (Wood and Oliver, characteristics of group 3 LEA proteins 1999) and that this results in the change in (Dure et al., 1989). The interesting aspect their stability. The implication from this of this protein is that it appears to be syn- work is that the sequestration of mRNAs thesized during the rehydration event and required for recovery hastens the repair of not at all during drying, indicating that damage induced by desiccation or rehydra- LEA-like proteins may play a role in dam- tion and thus minimizes the time needed age repair as well as cellular protection. It to restart growth upon rehydration. These is also possible that some LEA proteins findings may also explain the ability of T. play a role in protecting the cell from dam- ruralis to ‘harden’ during recurring desic- age initiated during rehydration. cation events in the absence of inducible More recently, Wood et al. (1999) dehydrin or sugar responses (Schonbeck reported the establishment of a small and Bewley, 1981a,b). Expressed Sequence Tag (EST) database from a cDNA library constructed from slow-dried gametophyte polysomal RNA 11.9.1. Rehydrins (in an attempt to target sequences sequestered in mRNPs – see above). Of 152 Eighteen rehydrin cDNAs, isolated by Scott ESTs that were generated and partially and Oliver (1994), have been sequenced sequenced, only 30% showed significant (Oliver et al., 1997; Wood et al., 1999). homology to previously identified nucleic Only three exhibit significant sequence acid and/or polypeptide sequences. homology to known genes in the Genbank Interestingly, several ESTs showed signifi- databases. Tr155 has a strong sequence cant similarity to unidentified desiccation- similarity to an alkyl hydroperoxidase tolerance genes isolated from C. linked to seed dormancy in barley (Aalen plantagineum (Bockel et al., 1998). et al., 1994) and Arabidopsis embryos (Haslekas et al., 1998), and in rehydrated but dormant Bromus secalinas L. seeds 11.10. Transgenic Approaches towards (Goldmark et al., 1992). Tr213 exhibits a Improving Plant Dehydration/ high degree of similarity to polyubiquitins Desiccation Tolerance from several plant sources. The finding that polyubiquitin is a rehydrin is indicative of The relevance of desiccation tolerance in an increased need for protein turnover dur- determining productivity under moisture- ing recovery from desiccation, an idea that limited environments is debatable as, agri- is not new (Ingram and Bartels, 1996). In culturally, desiccation represents a small Tortula there are three detectable ubiquitin proportion of the total instances of drought transcripts, two appear to be constitutively (Subbarao et al., 1995). Furthermore, yield expressed but the third is responsive to reduction due to water deficit becomes desiccation and rehydration (O’Mahony important before desiccation occurs. and Oliver, 1999). This is in contrast to However, improvement in yield in relation Sporobolus, where only two classes of to limiting water supply is agronomically ubiquitin transcripts are evident and both desirable. In environments where water respond to desiccation and rehydration deficits can occur at any stage of growth, (O’Mahony and Oliver, 1999). Tr288 has a knowledge of the mechanisms of desiccation dehydrin-like K box sequence at the tolerance should play a role in survival of C-terminus of the predicted protein but lit- the crop until soil moisture levels improve. tle other sequence similarity to known Transgenic approaches offer a powerful dehydrins. However, the predicted Tr288 means of gaining valuable information Dessication 11 18/3/02 1:58 pm Page 331

Molecular Genetics of Desiccation and Tolerant Systems 331

towards a better understanding of the Overexpression of a gene encoding for mechanisms that govern stress tolerance. moth bean P5CS in transgenic tobacco They also open up new opportunities to plants resulted in accumulation of proline improve stress tolerance by incorporating up to 10–18-fold over control plants and genes involved in stress protection from better growth under dehydration stress any source into agriculturally important (Kavi-Kishor et al., 1995). The transgenic crop plants. To date, the ‘transgenic plants demonstrated enhanced biomass approach’ has been to transfer a single gene production and flower development, as into plants and then observe the pheno- determined by increased root length, root typic and biochemical changes before and dry weight, capsule number and seed num- after a specific stress treatment. A limita- ber per capsule. The same gene was also tion of this strategy is that the functions of introduced into rice under the control of an very few genes involved in desiccation tol- ABA-responsive promoter (Zhu et al., erance have been established, or not 1998). The transgenic plants accumulated enough is known about the regulatory up to 2.5-fold more proline than control mechanisms. Molecular marker analysis plants under stress conditions. This study has been used to study dehydration toler- showed that the stress-inducible expres- ance, principally in cereals (Quarrie, 1996). sion of the p5cs transgene in rice plants Based on results from this type of resulted in an increase in biomass as approach, it is mainly viewed that toler- reflected by higher fresh shoot and root ance to water deficit is a complex quantita- weight under salt- and water-stress condi- tive trait, since no single diagnostic marker tions compared with untransformed plants. for tolerance has been found. However, As previously mentioned, trehalose transgenic plants with improved tolerance accumulates in a large number of organ- to water deficit have been produced using isms in response to different stress condi- various genes (see Table 11.3) and will be tions. With the exception of two described in the following text. resurrection plants (see Section 11.4.2.), trehalose is generally not accumulated in plants. However, genes for trehalose metab- 11.10.1. Compatible solutes or osmolytes olism have been identified in higher plants and characterized by expression studies Many plants respond to water deficit by and functional complementations of corre- accumulating organic compounds of low sponding yeast mutants (Müller et al., molecular weight, known as compatible 1999). The data obtained from plants exter- solutes or osmolytes. Therefore, engineer- nally supplied with trehalose or from ing-increased osmolyte content in trans- transgenic plants expressing trehalose genic plants is a rational strategy for biosynthesis genes from microorganisms protecting plants against dehydration point towards a role for trehalose in dehy- stress. Transgenic plants harbouring genes dration tolerance. For example, the yeast encoding enzymes involved in the produc- trehalose 6-phosphate synthetase gene tion of proline, fructans and trehalose (TPS1) was introduced into tobacco and show a reduction of dehydration stress. the resulting trehalose-accumulating plants Most of these studies have been carried out showed improved dehydration tolerance, in tobacco and Arabidopsis because the although a decrease of 30–50% in growth transformation technology is very well rate in conditions optimal for growth was established; however, an improvement in also reported (Holmström et al., 1996). In a stress tolerance has also been reported in second study, Pilon-Smits et al. (1998) other species such as rice and sugar beet. introduced bacterial trehalose 6-phosphate The enzyme 1-pyrroline-5-carboxylate synthase (otsA) and trehalose 6-phosphate synthetase (P5CS) catalyses the conversion phosphatase (otsB) into tobacco. The leaves of glutamate to pyrroline-5-carboxylate, of the transgenic plants were larger and which is then reduced to proline. showed better growth, in terms of dry Dessication 11 18/3/02 1:58 pm Page 332

332 J.R. Phillips et al. ., 1995 ., 1999 ., 1998 ., 1995 ., ., 1996 ., 1997 ., 1996 ., 2000 ., 1999 et al et al et al et al et al ., 1998 et al ., 1997 et al et al ., 1998 et al ., 1996 et al et al et al et al Survival of callus without ABASurvival of callus without pretreatment Furini Higher survival rate Kasuga Rice development Zhu Craterostigma Arabidopsis Wheat symptoms Sivamani -factor -methyltransferase Tobacco Higher photosynthetic rate Sheveleva trans O -pyrroline-5-carboxylate synthetase Tobacco Enhanced root biomass and ﬂower Kavi-Kishor 1 Fructosyl transferase Tobacco Higher growth rate Pilon-Smits Fructosyl transferase synthetaseTrehalose-6-phosphate Tobacco Increase in leaf survival Sugarbeet Holmström Pilon-Smits Myo-inositol Manganese-superoxide dismutase Lucerne Higher survival rate McKersie Regulatory RNA or short polypeptide DRE-binding Trehalose-6-phosphate synthaseTrehalose-6-phosphate phosphataseTrehalose-6-phosphate Tobacco Tobacco dry weight Higher photosynthetic rate and increased Pilon-Smits Transgenic plants with altered tolerance to water deﬁcit. Transgenic cerevisiae crystallinum plumbaginifolia Oat Arginine decarboxylase Rice Lower chlorophyll loss Capell BarleyMoth bean Group 3 LEA protein Rice Higher growth rate and delay in damage Xu sacB Bacillus subtilis TPS1 Saccharomyces IMT1 Mesembryanthemum Sod Nicotiana cdt-1 Craterostigma Table 11.3. Table GeneAdc Origin Gene product Host phenotype Tolerance Reference DREB1a Arabidopsis HVA1 otsAotsB Escherichiap5cs coli E. coli Dessication 11 4/4/02 2:25 pm Page 333

Molecular Genetics of Desiccation and Tolerant Systems 333

weight, under dehydration stress. Detached transgenic plants was inhibited less during leaves from young, well-watered transgenic dehydration and salt stress, and the plants plants showed better capacity to retain recovered faster than wild type. water when air-dried than the wild-type Furthermore, preconditioning of plants plants. These transgenic plants also had expressing the imt1 cDNA in low-salt more efficient photosynthetic activity. media increased D-ononitol amounts and Although the trehalose protective effect is resulted in increased protection when the unclear at the molecular level, correlative plants were stressed subsequently with a evidence suggests that trehalose stabilizes higher salt concentration. Unlike solutes proteins and membrane structures under such as proline and sucrose, D-ononitol stress (Colaco et al., 1995; Iwahashi et al., levels did not show significant diurnal 1995). fluctuations This led the authors to suggest Fructans are polyfructose molecules that that stress-inducible solute accumulation are produced by many plants and bacteria. may provide better protection under Owing to their high solubility, they may drought conditions than do strategies using help plants survive periods of osmotic osmotic adjustment by metabolites that are stress. Pilon-Smits et al. (1995) introduced constitutively present. a gene encoding a bacterial fructan syn- Polyamines are small nitrogenous cellu- thase (sacB) isolated from Bacillus subtilis lar compounds that have being implicated into tobacco. Under unstressed conditions, in a variety of stress responses in plants. the presence of fructans had no significant Polyamines accumulate under several abi- effect on growth rate and yield. The trans- otic stress conditions including drought. genic plants performed significantly better Cultivars demonstrating a higher degree of under osmotic stress than wild-type salt tolerance contain higher levels of tobacco, and the stress resistance correlated polyamines. Furthermore, exogenous with the amount of fructan accumulated. application of polyamines results in pro- The same sacB gene was introduced into tection against osmotic stress. Transgenic sugarbeet to produce bacterial fructans rice cell lines and plants have been pro- (Pilon-Smits et al., 1999). The transgenic duced that express an oat arginine decar- sugarbeets accumulated fructans to low lev- boxylase cDNA, the gene product of which els in both roots and shoots. Two indepen- converts ornithine to the diamine dent transgenic lines of fructan-producing putrescine, under the control of the cauli- sugarbeets showed significantly better flower mosaic virus (CaMV) 35S promoter growth under dehydration stress than did (Capell et al., 1998). A four- to sevenfold untransformed beets. Dehydration-stressed increase in arginine decarboxylase activity fructan-producing plants attained higher was observed in transformed plants com- total dry weights than wild-type sugarbeet, pared with wild-type controls. Biochemical due to higher biomass production of analysis of cellular polyamines indicated leaves, storage roots and fibrous roots. up to a fourfold increase in putrescine lev- Again, no significant differences were els in transgenic plants. Although the observed between the transgenic and wild- plants had improved drought tolerance in type plants under well-watered conditions. terms of chlorophyll loss under drought The introduction of fructan biosynthesis in stress, constitutive expression of this gene transgenic plants is therefore a promising slowed down growth severely. approach to improving crop productivity under dehydration stress. Expression of a cDNA encoding myo- 11.10.2. Oxygen-scavenging proteins inositol O-methyltransferase (imt1) in tobacco during salt and dehydration stress One consequence of dehydration and many resulted in the accumulation of methylated other stresses is the production of activated inositol D-ononitol (Sheveleva et al., 1997). oxygen molecules that cause cellular injury Photosynthetic carbon dioxide fixation in (see Chapters 9 and 10). The protection of Dessication 11 18/3/02 1:58 pm Page 334

334 J.R. Phillips et al.

sensitive metabolic reactions by stabiliza- expressing transgenic and non-transgenic tion of protein complexes or membrane controls under moderate water-deficit con- structures by increasing the capacity for ditions. The two homozygous transgenic hydroxyl radical scavenging in plants should plant lines also had significantly greater provoke improved performances under non- total dry mass, root fresh and dry weights, lethal stress conditions. Transgenic lucerne and shoot dry weight compared with the expressing Mn-superoxide dismutase cDNA two controls under soil water-deficit condi- tended to have reduced injury from water- tions. As is the case for all LEAs, the pre- deficit stress as determined by chlorophyll cise mode of action of HVA1 under drought fluorescence, electrolyte leakage and conditions remains unclear. regrowth from crowns (McKersie et al., In contrast, attempts to introduce three 1996). A 3-year field trial indicated that lea-like genes from the resurrection plant both yield and survival of transgenic plants C. plantagineum into tobacco did not was significantly improved, supporting the result in a drought-tolerant phenotype hypothesis that tolerance of oxidative (Iturriaga et al., 1992). However, this result stress is important in adaptation to field is perhaps less surprising considering that environments. drought stress does induce an array of different LEA-related proteins in plants (see Section 11.4.1). It is also likely that other 11.10.3. LEA proteins factors are required for the expression of tolerance where LEA-type proteins are A barley group 3 LEA protein termed involved. HVA1 is specifically expressed in the aleurone layer and the embryo during the late stages of seed development, correlating 11.10.4. Regulatory genes with the acquisition of seed desiccation tolerance. ABA and several stress condi- The machinery leading to the expression of tions including dehydration also induce dehydration-responsive genes is expected HVA1 expression in young seedlings. Xu et to conform to a general cellular model. In al. (1996) produced transgenic rice plants general, signal transduction cascades can expressing the barley HVA1 gene, driven be divided into the following basic steps: by a constitutive promoter. This led to the perception of stimulus; processing, includ- constitutive accumulation of HVA1 protein ing amplification and integration of the sig- in both leaves and roots of transgenic rice nal; and a response reaction in the form of plants. The second-generation transgenic de novo gene expression. As mentioned rice plants showed increased tolerance to earlier (see Section 11.5), studies of dehy- water deficit and salinity. In a second dration-activated signalling cascades have study, HVA1 was introduced into spring resulted in the identification of potential wheat (Sivamani et al., 2000). High levels regulatory genes, such as transcription fac- of expression of the HVA1 gene, regulated tors. The transformation of plants using by a maize ubiquitin promoter, were regulatory genes is an attractive approach observed in leaves and roots of indepen- for producing dehydration-tolerant plants. dent transgenic wheat plants. Progenies of Since the products of these genes regulate four selected transgenic wheat lines were gene expression and signal transduction tested under greenhouse conditions for tol- under stress conditions, the overexpression erance of soil water deficit. Potted plants of these genes can activate the expression were grown under moderate water deficit of many stress-tolerance genes simultane- and well-watered conditions, respectively. ously. Two homozygous and one heterozygous Expression of many Arabidopsis dehy- transgenic lines expressing the HVA1 gene dration-responsive and cold-regulated had significantly higher water-use effi- (COR) genes is mediated by a DNA regula- ciency values as compared with the non- tory element termed the dehydration- Dessication 11 18/3/02 1:58 pm Page 335

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responsive element/C-repeat (DRE/CRT) contrast, calluses transformed with control (Yamaguchi-Shinozaki and Shinozaki, vectors did not survive dehydration. The 1994). A major breakthrough was made desiccation-tolerant transformants accumu- when a transcriptional activator, CBF1, that lated anthocyanins and their phenotype binds to the DRE/CRT was identified was indistinguishable from the original (Stockinger et al., 1997). In a second report transfer DNA (T-DNA)-tagged mutant line. it was demonstrated that overexpression of RNA hybridization analysis confirmed that CBF1 in transgenic Arabidopsis plants at the desiccation-tolerant transformants con- non-acclimatizing temperatures induces tained high levels of the 0.9 kb transcript COR gene expression and increases plant and constitutively expressed the ABA- freezing tolerance (Jaglo-Ottosen et al., responsive marker genes. This regulatory 1998). More recently, Kasuga et al. (1999) gene has a unique structure, but does share transformed Arabidopsis with a cDNA some features with mammalian retrotrans- encoding DREB1a, a homologue of CBF1, posons. The function of the cDT-1 gene driven by either the constitutive CaMV 35S product is not immediately obvious promoter or an abiotic stress-inducible pro- because it encodes a transcript with no moter. The overexpression of this gene acti- large open reading frame. It is possible that vated the expression of many stress- the biologically active product of cDT-1 is a tolerance genes such as lea genes and P5CS. regulatory RNA or a short polypeptide. In all cases, the transgenic plants were more tolerant to drought, salt and freezing stresses. However, the constitutive overex- 11.11. Conclusions and Perspectives pression of DREB1a also resulted in severe growth retardation under normal growth Molecular analyses of desiccation-tolerant conditions. In contrast, the stress-inducible systems use a variety of strategies and expression of this gene had minimal effects involve different plant species. Initial on plant growth and provided greater toler- research has been largely descriptive and ance to stress conditions than genes driven many genes have been isolated that play a by a strong constitutive promoter. potential role in desiccation tolerance. Although C. plantagineum can tolerate Furthermore, major themes in the molecu- extreme dehydration, in vitro-propagated lar response have been established such as callus derived from this plant has a strict changes in sugar metabolism and the requirement for exogenously applied ABA expression of lea genes. Studies have in order to survive a severe dehydration. begun to examine mechanisms that control This property has been exploited for isola- gene expression and regulatory pathways tion of dominant mutants by activation tag- are being established. Attempts to under- ging, in which high expression of resident stand gene function have used transgenic genes activated by insertion of a foreign plants, the results of which are of clear promoter would confer desiccation toler- biotechnological importance. ance to the transformed cells without prior In order to address further the question ABA treatments. One gene was identified of gene function and ultimately to under- (cDT-1), whose high expression did confer stand the molecular basis of desiccation the expected phenotype in calli and led to tolerance, other experimental approaches constitutive expression of several ABA- are required. One strategy is the develop- and dehydration-inducible genes (Furini et ment of a genetic model system to study al., 1997). In a second experiment, trans- desiccation tolerance in vegetative tissue. genic calluses that constitutively express Insertional mutagenesis via T-DNA or cDT-1 under the control of two different transposon tagging could then be employed promoters were produced. When calluses – both are proven methods to deduce the from both lines were dehydrated, transfor- function of genes in genetic model systems mants from both lines were able to with- such as Arabidopsis. Secondly, natural stand desiccation in the absence of ABA. In allelic variation has proved successful for Dessication 11 18/3/02 1:58 pm Page 336

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identifying genes involved in plant devel- habit can occur via minor gene alterations opment (Swarup et al., 1999). Quantitative (Doebley and Stec, 1991). trait locus (QTL) analysis of plant acces- The existence of ‘common genomes’ sions that exhibit extensive variation for suggests that genes required for any path- desiccation tolerance may be a means of way are present in all plant species. identifying genes in complex regulatory However, the genes involved in pathways networks. may differ in their spatial expression pat- In mosses, where desiccation-tolerant terns and locations, due to minor changes tissues are haploid, the possible use of in regulatory regions. For example, differ- gene replacement, either directed or by ences in tissue specificity and/or control of random tagging, utilizing efficient homolo- gene expression among members of a tran- gous recombination techniques, offers a scription factor gene family in desiccation- novel and powerful technology for func- tolerant and desiccation-sensitive species tional gene analysis (Puchta, 1998; Reski, may account for differences in desiccation 1999). tolerance. Therefore, it may be possible to Comparative mapping studies have alter significantly multigenic traits such as demonstrated that closely related plant desiccation/dehydration tolerance, by the species have highly conserved gene con- identification and transfer of single genes tent and chromosomal positions of genes that account for the physiological differ- are also maintained, even though chromo- ence between the species. somal rearrangements differ. The similarity in gene content of, for example, grasses indicates that genes are rarely created 11.12. Acknowledgements within individual species and variation between species is likely to arise from gene The work in the laboratory of D. Bartels duplication and/or minor sequence modifi- was supported by the DFG Schwerpunkt cations of existing genes (Bennetzen and ‘Molekulare Analyse der Phytohormon- Freeling, 1993). This hypothesis is sup- wirkung’. We thank A. Richter, Vienna, for ported by evidence that major modifica- communicating data on sugar analysis tions in plant morphology and growth before publication.

11.13. References

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12 Rehydration of Dried Systems: Membranes and the Nuclear Genome

Daphne J. Osborne,1 Ivan Boubriak1 and Olivier Leprince2 1The Oxford Research Unit, Open University, Foxcombe Hall, Boars Hill OX1 5HR, UK; 2UMR Physiologie Moléculaire des Semences, Institut National d’Horticulture, 16 Bd Lavoisier, F49045 Angers, France

12.1. Introduction 344 12.2. The Dangers of Rehydration and Membrane Changes 344 12.2.1. Factors governing imbibitional injury 344 12.2.2. Causes for solute leakage as mechanisms of imbibitional injury: the fate of plasma membranes during rehydration 346 12.2.2.1. Conformational properties of plasma membranes 346 12.2.2.2. Mechanical stress 347 12.2.3. Protections against imbibitional injury 347 12.2.3.1. Seed coat 347 12.2.3.2. Protection at the molecular level 349 12.3. Maintaining Integrity of the Genome 350 12.3.1. Hydration-determined changes in DNA 350 12.3.2. Seeds 350 12.3.2.1. Survival after rehydration reﬂects the dry state experience 351 12.3.2.2. First events of seed rehydration 352 12.3.2.3. Partial rehydration: priming 353 12.3.2.4. The recalcitrant seeds 354 12.3.3. Pollen 354 12.3.3.1. First events of pollen rehydration 355 12.3.4. Whole plants 356 12.3.5. Requirements for successful rehydration of a genome 356 12.4. Acknowledgements 359 12.5. References 359

344 D.J. Osborne et al.

12.1. Introduction erance has not always been fully appreci- ated. For example, Kovach and Bradford The period of water uptake by a dry anhy- (1992) showed that the loss of viability in drobiote is one of hazard and sensitivity to wild rice was due to imbibitional damage stresses. Imbibitional injury can occur in a that had previously been interpreted as wide range of desiccated organisms includ- desiccation intolerance. A similar conclu- ing seeds (Vertucci, 1989; Hoekstra and sion was reached by Ellis et al. (1990) with Golovina, 1999), pollens (Hoekstra et al., pea seeds and by Sacandé et al. (1998) with 1992, 1999), algae, mosses and ferns seeds of neem, a tropical recalcitrant (Bewley, 1979), and yeasts (van Stevenick species, which exhibits an orthodox behav- and Ledeboer, 1974). iour when stored seeds are rehydrated The embryos of most seeds, certain pol- above 20–25°C. Vegetative anhydrobiotic lens and spores, many lower plants and a tissues (mosses, lichens and resurrection diverse, but limited, selection of whole plants) also appear to be sensitive to the vascular plants – the so-called resurrection rehydration phase (Oliver et al., 1997, plants (Oliver et al., 1998) – have devel- 1998; Quartacci et al., 1997). oped remarkable mechanisms to permit Essential components for this mainte- water loss without irreparable damage to nance of cellular integrity throughout the their cytoplasmic integrity and without processes of drying and rehydration are the imperilling their future survival. But those structural organization and composition of cell types that successfully pass through intracellular membranes and the ﬁdelity the hazard of dehydration must undergo and conformation of the DNA in nuclear preparation for this event ahead of time, chromatin and probably also that in mito- and it is critical to the success of dehydra- chondria and plastids. tion and to the subsequent orderly accom- This chapter addresses what is currently plishment of rehydration that both cellular known and speculated upon for these two compartmentation and genetic information major determining factors in cell survival are maintained unimpaired. The restora- under the temporal and successive condition of cytoplasmic organization and functions of changing water availability. tion within minutes of water being readmitted to these same dehydrated cells remains to be properly understood, partic- 12.2. The Dangers of Rehydration and ularly since all dry tissues show some leak- Membrane Changes age of cytoplasmic contents on their ﬁrst impact with free water. 12.2.1. Factors governing imbibitional injury Several important crops, particularly those of tropical and subtropical origins, The sensitivity of seeds and pollen to imbi- are known to suffer when the seeds take up bitional stress is controlled by at least three water at chilling temperatures. These crops factors: the initial moisture content of the include soybean, maize, sorghum, cotton, tissues, the temperature of the imbibition bean, cowpea and rice. Injury may also medium and the rate of water uptake. occur at room temperature in species such Chilling temperatures, very low moisture as pea and Brassica sp. when the seeds are contents and rapid water uptake generally very dry before imbibition. Upon imbibi- result in greater injury (Fig. 12.1). Slowing tional stress, dry anhydrobiotes leak down the rate of water uptake by soaking solutes and macromolecules, leading to seeds in polyethylene glycol solutions faulty metabolism, loss of cellular integrity improves the quality of seedlings of vari- and/or infection by opportunistic ous species (Woodstock and Taylorson, pathogens, which consequently lead to the 1981; Vertucci and Leopold, 1983; Priestley death of the tissues. Despite its widespread and Leopold, 1986; Chern and Sung, 1991). occurrence, the impact of imbibitional While the low water potential of dry anhy- stress on the expression of desiccation tol- drobiotes is the driving force for imbibi- Dessication 12 18/3/02 1:58 pm Page 345

Rehydration of Dried Systems 345

100

25C 80

40 % Germinated seeds

0 0 5 10 15 20 25

Initial water content (% dw)

Fig. 12.1. The effect of initial water content of axes on the ﬁnal percentage of germination of cowpea (Vigna unguiculata). After equilibration at various relative humidities, seeds were soaked in water at 5°C () or 25°C () for 1 h, then rolled in ﬁlter papers and incubated at 20°C for 7 days. At both temperatures, there is a threshold water content below which seeds suffer from imbibitional injury. Least-squares linear regressions were determined for the data below the threshold water content. Water contents are expressed on a dry weight (dw) basis.

tion, the permeability of the seed regulates ture content at which the seed is most sen- the rate of water uptake. The permeability sitive to imbibition (Vertucci and Leopold, of seed tissues to water is a complex func- 1983). Wolk et al. (1989) suggested that the tion of the seed morphology, structure, wettability depends on the water-binding composition and water content. It is note- characteristics of the dry tissues. In dry tis- worthy that, during ageing, seeds and sues a glassy state is presumed to prevail pollen become increasingly sensitive to (Chapter 10), which has certain peculiar imbibition (Woodstock and Taylorson, physical properties. Whether the condition 1981; Bruggink et al., 1991; van Bilsen et of the glassy state plays a role in the initial al., 1994; Sacandé et al., 1998). wettability of the tissues remains to be Experimental evidence (Vertucci and ascertained. Large increases in seed vol- Leopold, 1983; Wolk et al., 1989) and theo- ume are usually observed during imbibi- retical considerations about water penetration (Vertucci, 1989), and these changes in tion in dry materials (reviewed by Vertucci, volume have been interpreted as an unfold- 1989) suggest that the water uptake occurs ing of biopolymers of unknown nature as in two phases: an initial wetting phase and the seed takes up water (Vertucci, 1989). a subsequent hydraulic ﬂow. It has been This author calculated that the difference suggested that for seeds imbibitional dam- between the rate of water uptake and rate age is imposed by the initial wetting of volume change during imbibition is because the moisture content at which this greatest at the time when the seed is most phase is observed corresponds to the mois- sensitive to imbibitional damage, suggest- Dessication 12 18/3/02 1:58 pm Page 346

346 D.J. Osborne et al.

ing that structural factors may contribute to rehydration, a transition from a gel to a imbibitional damage. It is likely that the liquid phase occurs under those conditions volume increase during imbibition also of temperature and moisture content that interferes with the wettability of the tissues promote solute leakage and loss of viability (Vertucci and Leopold, 1983). upon imbibition (Hoekstra et al., 1992, The considerable volume of literature on 1999; Hoekstra and Golovina, 1999). Earlier imbibitional injury points to plasma mem- experiments on liposomes had shown that branes as the target of the rehydration during a thermotropic phase transition stress. Indeed, imbibitional injury interferes from gel to liquid crystalline, membranes with the rapid re-establishment of mem- become leaky (Crowe et al., 1989). Leakage branes, as has been shown from the exten- is thought to occur because of the struc- sive leakage of cytoplasmic solutes and the tural defects at the boundary between the disorganization and ruptured appearance of coexisting phases. Analogous to model sys- membranes following rehydration tems, it was suggested that the transiently (Vertucci, 1989; Bedi and Basra, 1993; coexisting phases allow leakage of solutes Hoekstra et al., 1999). One can argue that on the penetration of liquid water in the plasma membranes are the ﬁrst macromole- dry plant tissues (Crowe et al., 1989; cular structures to be affected during imbi- Hoekstra et al., 1992). This hypothesis bition because solute leakage may occur received support from pollen experiments within a few seconds to minutes, which is showing that treatments prior to imbibition before the resumption of metabolism. that reduce leakage (such as exposure to humid air or an increase in soaking temperature) also promote the return of the 12.2.2. Causes for solute leakage as phospholipid bilayer to the liquid- mechanisms of imbibitional injury: the fate of crystalline state (Crowe et al., 1992; plasma membranes during rehydration Hoekstra et al., 1992). Impatiens pollen is comparatively tolerant to imbibition at low In the past, several hypotheses have been temperature (Hoekstra and Golovina, promoted to explain the mechanisms of 1999). It appears that the phase transition leakage during the imbibition of seeds and temperature of plasma membranes in dry pollen. Successive hypotheses (e.g. the dis- Impatiens pollen is very low, suggesting ruption of membranes in the dry state, or that a gel phase barely forms, thus prevent- the formation of hexagonal phases), which ing leakage during imbibition. Thus, these ﬁrst prevailed and were then abandoned, data would imply that the transition tem-

have been recently reviewed by Crowe et perature (Tm) at which membranes undergo al. (1997), Hoekstra and Golovina (1999) a phase change can determine imbibitional and Hoekstra et al. (1999). Our current injury. understanding points to two causes that Recent evidence, however, now suggests lead to solute leakage during rehydration that changes in membrane phase per se are as a result of imbibitional injury: the physi- insufﬁcient to explain the permanent loss of cal and/or conformational properties of membrane integrity in rehydrating tissues. membranes in the dry state (see Chapter 9; In Typha latifolia pollen, the permeability Crowe et al., 1992) and mechanical stresses after a membrane-phase change during rehy- imposed during rehydration (Spaeth, 1987; dration appears to exhibit both a transient Vertucci, 1989; Hoekstra et al., 1999). and permanent character (Hoekstra et al., 1999). Using an electron paramagnetic resonance (EPR) spin-probe technique that per- 12.2.2.1. Conformational properties of mits a detailed analysis of the kinetics of plasma membranes leakage during imbibition of pollen, it was Imbibitional injury in anhydrobiotes has shown that plasma membranes are highly been linked to the occurrence of mem- permeable within the ﬁrst 10 s of imbibi- branes in gel phase in the dry state. During tion. Furthermore, this permeability persists Dessication 12 18/3/02 1:58 pm Page 347

Rehydration of Dried Systems 347

in conditions that permit plasma mem- membranes of dry mosses, pollen and seeds branes to pass through a phase transition appear intact (Platt et al., 1994, 1997; (Hoekstra et al., 1999). To explain this obser- Hoekstra et al., 1999; Claessens et al., 2000). vation, another mechanism leading to increased permeability has been proposed. 12.2.2.2. Mechanical stress Golovina et al. (1998) showed that amphipathic compounds such as flavonoids, Whether the ultrastructural damage to which are present in the cytoplasm, parti- membranes upon imbibition results from tion into the lipid phase during drying and mechanical stresses and/or changes in vice versa during rehydration. Endogenous physical properties of the membrane phase amphipathic compounds extracted from remains to be fully ascertained. Several various dry anhydrobiotes were found to lines of evidence suggest that membranes fluidize membranes isolated from these will undergo considerable mechanical organisms and also from prepared lipo- stress during imbibition. Vertucci (1989) somes (Hoekstra et al., 1997; Golovina et al., estimated that the rate of volume increase 1998). Results on liposomes and in situ on imbibition is larger than the rate of experiments on pollen using an amphiphilic water uptake. Thus, the resulting cellular spin probe suggest that an anhydrobiote expansion may stretch the plasma mem- would leak as long as the amphiphilic com- brane beyond its extensibility limit and pounds resided in the plasma membrane. induce lesions, as demonstrated for cucum- Since the increase in water volume during ber cotyledons (Willing and Leopold, imbibition eventually induces the partition- 1983). Spaeth (1987) suggested that inter- ing back into the cytoplasm (Golovina et al., nal pressure during imbibition may be a 1998), leakage should then cease. The tran- driving force for membrane damage. His sient nature of such leakage was confirmed suggestion was based on the observations with leakage experiments using a fluores- of proteins and starch grains that were cent dye (Hoekstra et al., 1999). The tran- extruded through blisters formed on the sient leakage is thought to be responsible for surface of imbibed cotyledons of bean and some loss of germinative vigour of the pea. The process of extrusion resembled a pollen but not of viability. The above obser- process in which viscous fluids are forced vations lead to the conclusion that the per- by internal pressure through irregular ori- manent nature of the leakage that leads to fices (Spaeth, 1987). Further evidence sup- loss of viability must originate from an irre- porting the role of internal pressure in versible damage that is other than an imbibitional injury comes from studies on amphipath partition phenomenon. In con- the formation of cracks during imbibition. trast to liposome systems, where the transi- Cracks in cotyledons originate from tensile tion from the gel to liquid crystalline phase stresses in the dry interior of partially during rehydration is accompanied by a hydrated tissues (Spaeth, 1987, and refer- transient leakage, it is possible that a phase ences therein). Since tensile stresses are transition in anhydrobiotes may induce due to compressive strains (i.e. a form of much more severe and irreversible changes pressure), it implies that there is an inter- during rehydration (Hoekstra et al., 1999). nal pressure that is applied on partially Indeed, electron microscope observations of imbibed tissues. However, this contention freeze–fracture images of imbibitionally has not received further attention. injured pollen and seeds show folding irreg- ularities and holes in the plasma mem- 12.2.3. Protections against imbibitional branes (Hoekstra et al., 1999; Claessens et injury al., 2000). Similar damage has been widely reported in earlier electron microscope 12.2.3.1. Seed coat studies in which dry tissues were fixed in cold aqueous fixatives (Buttrose, 1973). The seed coat or testa is extremely impor- When anhydrous fixation is used, plasma tant in protecting the seed from imbibi- Dessication 12 18/3/02 1:58 pm Page 348

348 D.J. Osborne et al.

tional injury. Differences in vigour and via- In certain crops, the seed coat semi-per- bility of seeds have been associated with meability or impermeability to water has the pigmentation of the testa. been attributed to the presence of waxy Characteristically, seeds of the same materials embedded in the epidermis species with a light-coloured or translucent (McDonald et al., 1988) and high levels of testa are more sensitive to imbibition than phenolics or hydroxyphenolics that are seeds with a darker testa. The relationship oxidized (Marbach and Mayer, 1974) between the presence of a coloured testa and/or polymerized into insoluble lignin and the absence of imbibition injury has polymers (Marbach and Mayer, 1974; Egley been established in a wide range of crops et al., 1983, and references therein). In soy- (e.g. pea, dwarf French bean, faba bean, bean, hydroxyproline-rich glycoproteins cowpea; Legesse and Powell, 1992; Kantar accumulate in large amounts in the seed et al., 1994; Demir, 1996) and weeds (proso coat during late maturation. They could be millet, Khan et al., 1996). A cause–effect involved in regulating the water entry in relationship between seed-coat pigmenta- the embryonic tissues (Cassab et al., 1985). tion and imbibition stress has been con- It is not yet understood how these proteins firmed by comparing the imbibitional or polymeric substances interact with damage in five pairs of isogenic lines in water molecules to influence the rate of pea, differing only in the A gene for seed imbibition. According to Powell (1989), the colour (Powell, 1989). presence of anthocyanins in coloured seed Several lines of evidence suggest that the coat is thought to decrease the wettability role of the seed coat against imbibitional of the inner surface of the seed coat. injury is to act as a physical barrier that reg- However, in pea, this cannot explain why ulates water movement both temporally coloured seeds suffered less imbibitional and spatially. The kinetics of water uptake damage than white seeds. Indeed, the dif- (Powell, 1989) in pea seeds indicated that ferences in imbibition kinetics between pigmented seeds imbibed more slowly than coloured and white seeds were maintained those with completely or partially light- when the seed coat was removed (Powell, coloured or white testas. Harvesting prac- 1989). In resurrection plants, there is no tices that damage the seed coat such as the direct evidence indicating that protective formation of epidermal cracks (Duke et al., layers may regulate the rehydration of 1986; McCormac and Keefe, 1990; Bruggink dried tissues. However, several studies et al., 1991) or the actual removing of the report that leaves of Craterostigma sp., testa (Abdel Samad and Pierce, 1978; Duke Xerophyta viscosa and Sporobolus stapfi- and Kakefuda, 1981; McDonald et al., 1988) anus are covered with waxes or cuticular resulted in a higher rate of water uptake by coatings or hairy structures, which are the dry seeds and higher leakage rate dur- thought to control the loss of water during ing imbibition. Even imbibitional damage- drying (Dalla Vecchia et al., 1998; Sherwin resistant seeds become sensitive to chilling and Farrant, 1998). Thus, it may be possi- stress after testa damage (Tully et al., 1981; ble that these epicuticular waxes and other Prusinski and Borowska, 1996). These epidermal features may also act to regulate seeds also showed increased hydration water entry during rehydration. rates and solute leakage. In the particular Decoated seeds suffer more from imbibi- case of Arabidopsis thaliana, the rate of tional damage than do intact seeds. water uptake is likely to be mainly con- However, in groundnut, the seed coat does trolled by a mucilage layer surrounding the not appear to pose a physical barrier to seed coat without the interference of the rehydration of the embryonic tissues seed coat tannins and anthocyanins (Albert (Abdel Samad and Pierce, 1978). This et al., 1997; Debeaujon et al., 2000). This observation leads to the suggestion that fac- may explain the lack of correlation between tors other than regulating the water uptake imbibitional stress and testa colour in may be involved in protecting the seed tis- Arabidopsis seeds. sues from imbibitional stress. Considering Dessication 12 18/3/02 1:58 pm Page 349

Rehydration of Dried Systems 349

that the seed coat contains phenolics, and/or water uptake is repaired by mole- which are amphiphilic substances cules that are synthesized during drying (Marbach and Mayer, 1974), and that these and/or rehydration. Work on Tortula ruralis substances can alter the conformational and two resurrection plants (Craterostigma state of the membranes (Hoekstra et al., plantagineum and Sporobolus stapfianus) 1997; Golovina et al., 1998), it would be has identified a series of transcripts, which interesting to investigate whether are referred to as rehydrins (Ingram and amphiphilic substances migrate from the Bartels, 1996; Oliver et al., 1997; see seed coat to the surface cells of the embryo Chapters 1 and 11). So far, three of the rehy- with the water front, thereby modifying the drin transcripts (Tr155, Tr213 and Tr288) physical properties of membranes con- have been studied. Tr213 shows a high comitant with the water entry. The degree of similarity to polyubiquitins. The swelling properties of the seed coat during main function of ubiquitin is to eliminate imbibition may also contribute to the regu- undesirable proteins that are damaged or lation of water uptake. For example, the being recycled. The degradation involves testa of coloured pea seeds were found to the conjugation of ubiquitin with targeted remain closely associated with the cotyle- proteins and degradation via a reaction cas- dons whereas the white seed coats were cade involving the proteosome (Jentsch and loosened and water was held between the Schlenker, 1995). The presence of polyubiq- seed tissues and the testa (Powell, 1989). uitins during rehydration points to an increased removal of proteins during imbibitional stress. Thus, repair machinery 12.2.3.2. Protection at the molecular level appears to play an active role during rehy- Have anhydrobiotes evolved protecting dration of vegetative tissues in eliminating strategies at the molecular level against the damaged proteins, which are accumulated dangers of rehydration? Studies on pollen during drying. The mechanisms that regu- membranes during rehydration have late the synthesis of this machinery appear demonstrated that depressing the phase to be complex. Rehydrins can be constitu- transition temperature of dry tissues may tively expressed in the plant or both quali- be beneficial in reducing the risk of leakage tatively and quantitatively transcribed during water uptake. This depression is during dehydration and/or rehydration thought to be achieved by altering the (Ingram and Bartels, 1996; Oliver et al., phospholipid composition, as in Impatiens 1997; O’Mahony and Oliver, 1999). The pollen, and/or synthesis of fluidizing question remains as to whether a ubiquitin- amphipaths that partition into the mem- based mechanism of repair is also essential brane during drying (Hoekstra and to alleviate imbibitional stress in seeds. In Golovina, 1999). From results of in vitro this respect, Tr288 is an interesting tran- experiments using liposomes, it has been script because it has similarity to a tran- suggested that disaccharides (sucrose and script specifically expressed during trehalose) can reduce the risk of imbibi- rehydration of dormant embryos of barley tional injury by suppressing the transition and Bromus secalinas (Oliver et al., 1997). temperature of the membrane during dry- In addition to repair mechanisms, dry ing and increasing fluidity (Crowe et al., anhydrobiotes appear to be endowed with 1997). However, the concentrations of sug- specific proteins that protect macromolecu- ars are not sufficient to protect fully in vivo lar structures from the rapid entry of water, membranes of dry anhydrobiotes, even if although data supporting this idea are all sugar molecules would form hydrogen scant. Dry oily seeds contain large amounts bonds with the phospholipid head groups of oleosins, the interfacial proteins that (Hoekstra et al., 1997). surround the oil bodies. The main function Another strategy has evolved in mosses of oleosins is thought to be to maintain the and resurrection plants. In these tissues, the integrity of the oil bodies as discrete extensive damage that occurs during drying organelles during rehydration (Leprince et Dessication 12 18/3/02 1:58 pm Page 350

350 D.J. Osborne et al.

al., 1998). In those recalcitrant seeds that Currently we have no information as to are devoid of oleosins (cocoa), the oil bod- whether such hydration-driven changes ies remain relatively stable after slow or occur during the successful desiccation fast drying, but, during rehydration, they and rehydration of any plant cell or how fuse to form large droplets, resulting in the far chromatin topology might be altered by loss of cellular integrity. So far, no struc- even small changes in the overall water sta- tural proteins other than the oleosins have tus. However, the variable and decreasing been detected in dry anhydrobiotes that longevity of seeds held under different could similarly protect cytoplasmic conditions of even small levels of increas- organelles during imbibition. ing humidity tells us at once that the cytoplasm of embryo cells is in a physically dynamic state with intracellular molecular 12.3. Maintaining Integrity of the interactions undergoing constant change. Genome Since so little is known of DNA integrity in the mitochondria or plastids in seeds or 12.3.1. Hydration-determined changes pollen, this part of the rehydration chapter in DNA is essentially confined to what is known of nuclear DNA. Cells in the dry state are never wholly dry. Molecules of water are closely associated with specific chemical groups on the 12.3.2. Seeds charged proteins and nucleic acids within the cytoplasmic matrix. In the nuclear Perhaps the earliest evidence for nuclear DNA, water molecules are intrinsic to the DNA changes in stored dry seeds has come phosphate backbone of the fully hydrated from microscopic studies showing the B-form DNA that exists in most living cells. increase in chromosomal aberrations that Loss of water molecules from the DNA appear in nuclei of embryo cells (Navashin, backbone can, in vitro, successively con- 1933). That embryos could still germinate vert B-form to A-form and Z-form confor- after such DNA damage and that cells did mations and there is evidence for such not necessarily perpetuate the initial chro- conformational changes during dehydra- mosomal aberrations through subsequent tion in prokaryotes (Setlow, 1992a) and meristematic cell divisions were the first during differentiation in specialized clear evidence for DNA repair processes eukaryote cells (Nordheim et al., 1986). As operating early during the biochemical Setlow (1992a) has shown for Bacillus events of rehydration (Nichols, 1941). subtilis, the A-form is maintained by the However, the extent of change during stor- binding of small acidic soluble proteins age also depends upon the condition of the (SASPs) synthesized during dehydration, seed when it is shed from the parent plant. and the dry spores, with their A-form DNA, The maternal history during seed develop- are remarkably resilient to high or low tem- ment and maturation is partly determined peratures and to chemical stress. genetically and partly determined by the Furthermore, the A-form is converted back environmental conditions (temperature, to the normal B-form when the spores are humidity, sunlight) that the mother plant rehydrated through the action of an SASP- experiences. Thus, when the seed is har- specific protease, at which point the spores vested or shed, factors such as potential lose their tolerance to desiccation and dormancy, seed-coat restrictions and other stresses (Setlow, 1992b). This indi- embryo vigour are already predetermined cates the critical part played by available variables. Given the signal inputs that an water and DNA conformation in determin- embryo receives during development, the ing the survival of the bacterial spore uncertainties of the maturation and final through both dehydration and rehydration desiccation phases, and the continuous processes. molecular changes that progress through- Dessication 12 18/3/02 1:58 pm Page 351

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out the period of seed storage, it can be 1986 and later in plant mitochondria and seen that the biochemical processes of plastids (for review, see Benne, 1996) and rehydration and germination do not repre- involves the insertion or deletion of uri- sent a single or simple system. dine residues in a complex of several enzymes (the editosome), or direct conver- sions of cytosine to uridine (Smith et al., 12.3.2.1. Survival after rehydration reflects 1997). The conversion of L-aspartyl the dry state experience residues in ageing proteins into abnormal Continued storage, even under optimum L-isoaspartyl groups can also be achieved conditions, leads to a progressive deteriora- in many plant and animal tissues by the tion, and all the events listed timewise for action of L-isoaspartyl methyl transferase rye (see following section) are reduced in without total protein degradation, and this activity and extended in time when enzyme has been shown to be present in retested as the period of storage becomes seeds (Mudgett and Clarke, 1993). All the longer. The progressive lessening of the other nucleic acids, proteins, lipids and ability to incorporate amino acids into pro- polysaccharides are subject to synthesis tein in the early hours of imbibition is a and degradation and are not, as far as we measure of an embryo’s being no longer know, subject to an enzymic, energy- able to germinate quickly. The time to root requiring process of molecular repair. This emergence, the first critical round of S- makes DNA and the genomic information it phase DNA synthesis and the first cytoki- carries of special importance in embryo nesis can be delayed for hours or even days cell survival in the dry state and critical to (Osborne, 1983). Eventually, the embryo the competence for DNA repair when the can no longer synthesize protein at all on cells first imbibe water on rehydration. rehydration, but, interestingly, there is a There is no precise moisture content late stage in the detrimental programme of that determines what we call the ‘dry state’ change in the dry state when transcription of a seed. When embryos reach maturation of short oligonucleotides can still take dehydration to moisture contents below place in the nucleus; however, neither 10%, the cytoplasm of the embryo enters stored messages nor these new small tran- into a glassy state in which molecular scripts are translated by the embryo (Bray movement is strictly limited (Williams and and Dasgupta, 1976; Sen and Osborne, Leopold, 1989; Sun and Leopold, 1993; 1977). Currently, we do not know what Buitink et al., 2000; see Chapter 10). But as these small transcripts might code for. different parts of a seed can be at slightly They might, of course, just represent the different levels of hydration and the attain- most stable sequences in the heterochro- ment of the glassy state in different species matin of genomic DNA and do not there- depends upon the molecular composition fore code for anything that is critical to of the cells (Leopold et al., 1994), the levels embryo survival. of hydration that provide a glassy state for The detrimental changes in an ageing all the different tissues of a seed can differ. embryo are multiple and much work has As far as current detection methods can been directed towards determining the bio- reveal, there is no respiration or ATP gen- chemical and physical nature of these eration, no transcription and no translation events. Of all the macromolecules of the in dry seeds; synthetic events of all kinds living cell, only one, DNA, is known to be are therefore excluded (Bewley, 1979). routinely repaired. A caveat may be added Non-energy-requiring processes are not, here that the post-transcriptional editing of however, excluded, so free radicals can be mRNA has also been considered as a repair generated as local events and non-energy- process as the molecule is not degraded requiring enzyme activities such as those before bases are removed and replaced. of nucleases and proteases can occur where Such editing was first detected in mito- they are in close molecular juxtaposition chondrial transcripts of trypanosomes in with their substrates. The progressive loss Dessication 12 18/3/02 1:58 pm Page 352

352 D.J. Osborne et al.

of DNA integrity to increasing levels of ran- and, using autoradiography, incorporation dom-sized small-molecular-weight frag- of tritium(3H)-labelled precursors into ments without loss of total DNA (Cheah nucleoli was evidence of rRNA synthesis and Osborne, 1978) and the loss of activity within 10 min. Incorporation of 3H-thymi- in many enzymes without loss of the actual dine into nuclei of embryo cells has shown protein molecules are evidence of this that DNA repair (but not replication) is also (Osborne, 1983). But, whereas some activated within minutes of imbibition in hydrolytic enzymic proteins are remark- rye, and incorporation of labelled amino ably stable (DNases, RNases) and long out- acids into protein is apparent in the cyto- live in function the life span of the embryo plasm within 15 min (Osborne et al., 1977). (Osborne et al., 1977), others start to lose The initiation of S-phase DNA synthesis enzymatic activity soon after harvest and and DNA replication from 2C to 4C levels, on storage they become progressively less first evident in nuclei of root-tip meris- efficient with time. In this group are the tems, occurs late in the process of rehydra- enzymes concerned with DNA repair: tion and the first cytokinesis may be hours nuclear DNA polymerases (Yamaguchi et or days after RNA transcription and protein al., 1978), -type polymerase (Castroviejo synthesis are fully established. Whether et al., 1990), DNA polymerase- (Coello- the start of replication requires a particular Coutiño et al., 1994) and DNA ligase (Elder physical state of hydration of the nucleus et al., 1987), so that, when a fresh or a is not known. Embryos become highly stored seed is rehydrated from the dry metabolically active on imbibition even state, the extended time required to repair though they may remain dormant; those of fragmentation lesions in the DNA and to Avena fatua can reach a moisture content synthesize new repair enzymes is immedi- as high as 40% without a sign of growth. ately evident (Elder et al., 1987). This can These embryos may defer amplification of account for the delays in germination that nuclear DNA from 2C to 4C levels for are observed between freshly harvested months or years before initiating the entry seeds and those that have been stored. to cell cycling and attendant germination. The control factors that hold the cells of imbibed embryos in this homoeostatic but 12.3.2.2. First events of seed rehydration metabolic state, while also preventing pro- Assuming that seeds have been matured gression into cell cycling, cell expansion under satisfactory conditions on the and cell division, have still to be deter- mother plant and that they have not been mined, and this is still a key question in held after harvest in a way that would seed biology. It is of special interest to impair their ability to germinate, then the studies of desiccation tolerance because, events of rehydration from the dry state fol- throughout their dormancy, imbibed seeds low an essentially similar pathway of can be dehydrated back to their original nuclear reactivation for all the embryos weight without loss of viability or cell that have been studied in detail. death. A clue to the survival of dormant On imbibition in water, there is first a embryos is their ability to maintain an active physical hydration of the cytoplasm, which DNA repair from the time of rehydration and is usually completed by 60 min throughout the duration of imbibition. (Obroucheva, 1999). This will occur in Although replicative DNA synthesis is embryos of dead as well as living seeds blocked, DNA repair synthesis is as effective (Hallam et al., 1972, 1973) but in living in dormant as in germinating embryos. As seeds further uptake then proceeds contin- experiments with A. fatua have shown, even uously. The transcription of all classes of the repair of -irradiation-induced single- RNA within 20–40 min has been demon- and double-strand breaks occurs as effi- strated by electrophoretic fractionation of ciently and in the same time in dormant and newly synthesized radiolabelled nucleic non-dormant imbibed material (Elder and acids in rye embryos (Sen et al., 1975), Osborne, 1993). Dessication 12 18/3/02 1:58 pm Page 353

Rehydration of Dried Systems 353

12.3.2.3. Partial rehydration: priming visible cell division has not started and the

embryo has not been held at G2M for a pro- It might be concluded that even a short longed period, imbibed or primed seed can period of imbibition that is then followed be dried back safely and the seedling will by dehydration back to the dry state with- emerge rapidly on planting (Fig. 12.2). out damage or loss of any of the newly syn- The secret of successful priming, there- thesized components might lead to an fore, is to establish the stage before the first improvement in subsequent seed germina- cell cycle division when the nucleus has tion when water is again available. In fact, reinitiated transcription, has fully repaired this is generally true and forms the basis of any DNA damage from the dry state and priming seed (Heydecker and Coolbear, the optimal spectrum of newly translated 1977; van Pijlen et al., 1996). Successful but desiccation-stable proteins has been priming achieves and conserves both DNA translated and they are present in active repair and the early events of germination, form in the cytoplasm. including a significant level of mitochon- However, this is not necessarily the drial DNA synthesis. These are events that whole story, for, although primed seeds are stable to desiccation and take place will germinate quickly, they frequently before the embryo cells pass into the condi- store less well (Tarquis and Bradford, 1992; tion of desiccation sensitivity, when Nascimento and West, 2000), and in some embryo cells will die if dehydrated (Ashraf species they lose viability more readily and Bray, 1993). The conversion from a than those of unprimed seeds. The reasons state of tolerance to cytoplasmic water loss for this are uncertain, but would appear to to one of intolerance is still not fully be linked to the changes that take place understood, but it coincides with the stage during priming in the physical state of the in embryo cells, particularly those of the nucleus and cytoplasm and to the progres- root tip, when they approach the first sion in the stage of the cell cycle at which

cytokinesis at G2M and are on the border of the nuclei are dried back. A low level of undergoing the ﬁrst cell division. Provided replicative DNA synthesis continues

Desiccation- Desiccation- sensitive sensitive 90 Desiccation- tolerant

M Replication 2 Cytokinesis % Moisture content arrest G →

1 G

G1 10

Seed Dry Rehydration maturation state

Fig. 12.2. Progression of hydration-linked cell cycle events in a seed embryo during water restriction at maturation and the renewal of free water at rehydration, showing the relation with desiccation tolerance. Dessication 12 18/3/02 1:58 pm Page 354

354 D.J. Osborne et al.

throughout priming, progressing from 2C ther low molecular weight DNA disintegra- towards 4C though not to cytokinesis. tion (Fig. 12.3d). There is evidence that cells will accumu-

late at G2M and this may represent a topology for nuclear DNA that is particularly 12.3.3. Pollen accessible to nuclease fragmentation. Not all imbibed or primed seeds from different Certain pollens can stay dry and alive for species will reach this same condition of prolonged periods of time, depending on cytoplasmic or nuclear organization after the ambient relative humidity. The level of the same duration of rehydration, and moisture in dry pollen can vary and is low- therefore on drying back they will not est (c. 8%) for wind-dispersed pollen. reach similar stages of vulnerability to stor- Damage to these dry pollen cells can be age. A comparison of primed seeds (ach- more severe than that for the embryos of enes) of Ranunculus sceleratus and seeds. This is because the individual wind- Ranunculus arvensis is one such example. blown pollen cells are unprotected from With R. sceleratus, longevity of the dried- ultraviolet light (UV) and other environ- back seeds is increased, whilst that of R. mental stresses from the moment they are arvensis is reduced (Probert et al., 1991). shed from the anther. Since these pollen cells are haploid and lack the resource that doubled (diploid) genetic information pro- 12.3.2.4. The recalcitrant seeds vides, damage to the single genome can Many seeds, particular those of tropical result in serious genetic consequences to species, and many large-seeded species of the next generation. In these special cir- temperate climates maintain a high per- cumstances, DNA repair in pollen plays a centage of water in both axes and cotyle- crucial role and both photoreactivation and dons. Although they can accommodate a dark DNA repair systems operate together small amount of water loss without harm, in a germinating pollen grain (Ikenaga and desiccation to levels of 10–26% are lethal Mabuchi, 1966; Jackson and Linskens, and embryos do not restore synthetic activ- 1978). Incorporation of 3H-thymidine in ity when rehydrated. A feature that has germinating pollen grains of Petunia starts now been explored is whether or not the immediately water becomes available and DNA repair function is lost on dehydration continued incorporation in the presence of of these seeds below a certain critical level. hydroxyurea (which blocks replication) In Avicennia marina, a mangrove species confirmed active DNA repair synthesis in indigenous to the tropics and subtropics, a germinating pollen. Hydration of birch water loss from the seed that exceeds pollen at 100% humidity is not of itself 20–30% normally leads to seed death. sufficient to permit full excision repair, Experimental samples of the excised axes although the germination rate is improved, were dehydrated under a cool air stream to but, on transfer to free water, fully imbibed different levels of water loss, then irradi- pollen will then complete repair within 2 h ated from a -source to introduce a similar (Grodzinsky and Bubryak, 1985). During level of single- and double-strand breaks in rehydration, the excision DNA repair sys- each, and then rehydrated for a period of tem of pollen can remove a number of dif- 2 h (Boubriak et al., 2000). Results have ferently induced lesions incurred in the shown that fully hydrated embryos (Fig. dry state, including those of chemical 12.3a and b) permit almost full recovery of mutagenesis (Jackson and Linskens, 1978, fragmented DNA to levels of that in unirra- 1979), heavy metal damage and -irradia- diated controls. However, generating a 22% tion (Jackson and Linskens, 1982; Bubryak dehydration of the axes prior to irradiation et al., 1991). Efficiency of such repair can led to the complete failure of DNA repair differ in pollens depending upon the dif- (Fig. 12.3c) and, by 46% dehydration, the ferent levels of environmental stress under fragmentation of DNA continued into fur- which they were formed, i.e. lowland or Dessication 12 18/3/02 1:59 pm Page 355

Rehydration of Dried Systems 355

1.2 750 Gy (fully hydrated) 1.2 750 Gy (fully hydrated) 2 h Imbibed (a) (b)

1.0 1.0

1.2 750 Gy (22% dehydrated) 750 Gy (46% dehydrated) 2 h Rehydrated 1.2 2 h Rehydrated

1.0 1.0

Low Mol. Wt. High Mol. Wt. Migration

Fig. 12.3. Loss of DNA integrity with loss of DNA repair capability in embryo axes of Avicennia marina seeds following different levels of dehydration, shown by competence to repair a -irradiation damage of 750 Gy during 2 h rehydration. Molecular weight proﬁles of scans of DNA fractionated by electrophoresis on neutral agarose gels: (a) fully hydrated axes after -irradiation ((Low Mol. Wt.) DNA strand breaks induced); (b) fully hydrated axes, -irradiated, then rehydrated 2 h ((Low Mol. Wt.) DNA repaired); (c) 22% dehydrated axes, -irradiated, then rehydrated 2 h (no DNA repair); and (d) 46% dehydrated axes, - irradiated, then rehydrated 2 h (further DNA disintegration).

high-altitude mountains (Bubryak and essential to retain genetic ﬁdelity. This is Grodzinsky, 1985; Grodzinsky and not so if pollen is shed as a three-nuclei Bubryak, 1988). cell in which the generative nucleus has already divided to produce the two haploid sperm cells (as in maize). For a long time it 12.3.3.1. First events of pollen rehydration has been puzzling that there was no evi- Efﬁcient DNA repair during the imbibition dence for excision repair during rehydra- of pollen grains is an essential physiologi- tion of maize pollen even though this is cal event only for two-nuclei pollen grains also wind-distributed and can therefore be (e.g. birch) where, at shedding, each damaged by UV. Maize pollen is viable for

nucleus is held at G2 (2C DNA values). a few days only, so it is possible that no During germination, the generative nucleus DNA repair system is maintained in these of the pollen grain divides to produce two haploid sperm cells since they have

haploid sperm cells, so it is at this G2 already passed their last G2 checkpoint. checkpoint that DNA repair is absolutely An efﬁcient DNA repair system is not Dessication 12 18/3/02 1:59 pm Page 356

356 D.J. Osborne et al.

confined to wind-pollinating pollens but now known of the genes induced during has been shown to occur also in the two- dehydration processes in these desiccation- cell pollens of certain insect-pollinated tolerant plants (Neale et al., 2000; see plants such as Petunia and Amaryllis. Chapter 11) and of mechanisms involved These pollens can repair induced UV and in transcription and translation (Oliver et -irradiation damage despite the fact that al., 1997, 1998). However, we have little they rarely receive a high radiation dose evidence yet that links the successful rehy- during the pollination process. Although dration of these plants to their competence such repair can occur, it is less efficient either to sustain fidelity of the nuclear, than in wind-pollinated species (Bubryak mitochondrial or plastid genomes on dehy- and Grodzinsky, 1985). dration or, perhaps more importantly, to It is quite possible that, in pollen, check- the restoration the overall fidelity of the

point repair (in G2) for generative nuclei different genomes on rehydration. requires time and, therefore, a repair capa- Current knowledge indicates that pro- bility to withstand DNA damaging factors in gressive changing states of gene expression the environment is maintained for longer. occur at distinct thresholds of hydration This may include desiccation tolerance of throughout a dehydration process (Neale et the two-nuclear pollen both for wind-polli- al., 2000; Chapter 11). The possibility then nated and insect-pollinated species. Before arises that there could be alterations in the shedding from the hydrated enclosure of the conformational state of DNA as it becomes catkin, pollen is killed if it is dehydrated, progressively dehydrated, so perhaps this but at shedding (when the moisture content area should be investigated for evidence of is reduced to 8–14%) pollen becomes stable a transition to A-form DNA. What occurs to dry storage for long periods of time (years on rehydration is even less sure, and how for hazel and birch). Such pollen can be far cell survival depends upon the mainte- hydrated in moist air and dehydrated back nance of genomic fidelity during the period several times without losing the capability of desiccation or upon the repair of DNA for excision repair and for germination. lesions at rehydration, or both, remains to However, if exposed to liquid water, be discovered. hydrated pollen grains germinate and then, At least it now seems sure that water like the embryos of seeds, the germinated deficits can lead to a rise in abscisic acid pollen loses tolerance to drying and (ABA) levels and thence to the induction of becomes, once again, desiccation-sensitive ABA-directed new gene expressions. It is (Osborne and Boubriak, 1994). What we do also now sure, however, that desiccation not know is the topological organization of tolerance is not always controlled by ABA pollen nuclear DNA throughout these in all plants. In certain mosses and ferns, changing levels in water status. for example, ABA is either not detected or the levels may actually fall during desiccation (Reynolds and Bewley, 1993). 12.3.4. Whole plants Considering the diverse groups of plants and tissues that have acquired desiccation Much interest is currently directed towards tolerance, it seems likely that more than the survival mechanisms of vascular plants one survival mechanism will have evolved. under drought conditions, and a relatively For all, however, preservation of genetic small group of plants including mono- information will be paramount. cotyledon grasses, dicotyledon ‘resurrection plants’, many liverworts, mosses and ferns are sufficiently drought-tolerant to 12.3.5. Requirements for successful survive a 95% water loss for months or rehydration of a genome years, then recover and become metabolically active when free water is again avail- There would appear to be two aspects of able (Bewley, 1979; Gaff, 1980). Much is the rehydration of a dry but living cell that Dessication 12 18/3/02 1:59 pm Page 357

Rehydration of Dried Systems 357

are essential for the successful re-establish- (Zarain et al., 1987), an early synthesis of ment of organized metabolic activity. One DNA that did not correspond in character is the physical rehydration of dry to either repair or replication was found. organelles and the rehydration of a dry One possibility was that this represented cytoplasm. This involves the molecular mitochondrial DNA synthesis (Vazquez- reorganization of the membranes defining Ramos and Osborne, 1986) but further each region of compartmentation, the rehy- studies by Bucholc and Buchowicz (1992) dration of the protein–polysaccharide using oligonucleotide-hybridizing tech- structures of the cell walls and the free- niques showed that part of the DNA repair water access to folded macromolecules of synthesis that occurs after imbibition of the proteins and nucleic acids (see Section embryo can be attributed to a new synthe- 12.2). Before information exchange can sis of telomeric DNA. Furthermore, they take place between nucleus and cytoplasm, found that one of the degradative changes the nuclear chromatin must itself be rehy- in DNA of stored wheat embryos is the pro- drated and the DNA made available for reg- gressive cleavage and removal of the telom- ulated transcription. For accurate transfer eric repeats. of genomic information to the cytoplasm, This shortening of telomeric DNA cap- any damage present in DNA must first be ping repeats on storage of seed embryos is, replaced. Only if this second and metabolic like the loss that occurs in mammalian aspect of rehydration is achieved can a cell cells, a measure of their age and may repre- re-establish directed metabolic activity sent a commonality in the progress to with an opportunity for renewed cell senescence and death. Delay or failure to growth and development. Only if the mito- reinitiate these DNA end groupings may chondrial genome is also fully restored can therefore be another critical factor in deter- an integrated informational exchange prop- mining success in the rehydration that pre- erly operate. cedes germination. The stability of the Another aspect of DNA degradation, the telomere polymerase may be found to play recovery from which could be considered a key role in restoring the overall integrity as a DNA repair operation, is the loss of of the embryo genome. repetitive telomeric sequences from the ter- Although dry seeds appear to accumu- mini of DNA molecules within the double late most DNA damage in the form of single- helix. Ageing human fibroblasts show a strand DNA breaks, presumably from shortening of these telomeric regions enzymatic endonuclease cleavage or possi- (Harley et al., 1990) and convincing exten- bly also from free-radical attack and by sions of the life span in retinal pigment spontaneous base loss (Dandoy et al., epithelial cells has been achieved by trans- 1987), the effects of the accumulation of all fecting cells obtained from telomerase- types of damage before rehydration can be negative cell types with vectors encoding linked to the progressive failure of DNA the human telomerase catalytic subunit repair functions when water is again avail- (Bodnar et al., 1998); telomere-expressing able. Such events have led to the proposi- clones have elongated telomeres, contin- tion that loss of DNA repair is a major ued cell divisions and an absence of senes- factor in determining the loss of genomic cence. integrity and poor survival of an embryo on Telomeres and telomerases are present rehydration (Elder and Osborne, 1993; on the chromosomes in nearly all plant Osborne and Boubriak, 1994; Boubriak et cells (Adams et al., 2000; Leitch, 2000). al., 1997). The role of telomerases in seeds is there- Evidence that competent DNA repair is fore of considerable interest with respect to indeed an essential factor for successful ageing and the events of early rehydration. rehydration comes from experiments with In studies of DNA repair in embryos of rye the embryos of rye (Boubriak et al., 1997). (Vazquez-Ramos and Osborne, 1986), of If isolated embryos are -irradiated from a wheat (Marciniak et al., 1987) and of maize caesium source (750 Gy) whilst in the dry Dessication 12 18/3/02 1:59 pm Page 358

358 D.J. Osborne et al.

state in order to introduce DNA damage, still in the desiccation-tolerant period (Fig. 12.4a,b) subsequent rehydration in when rehydrated (Boubriak et al., 1997). It water for 2.5 h leads to complete restora- would be of considerable interest now to tion of the fragmented DNA to high molec- assess if there is a similar requirement for ular weight (Fig. 12.4c). These embryos functional DNA repair during the rehydra- survive. However, if DNA repair is blocked tion of other desiccation-tolerant and leafy by inhibitors of both - and -polymerase species such as mosses and resurrection (aphidicolin and dideoxythymidine tri- plants. phosphate, respectively), DNA integrity is Although nuclear transcription in the not restored, DNA becomes even more frag- embryos of fresh, dry seeds starts almost mented over 2.5 h and the embryos die (see immediately on imbibition, it is unclear at DNA scans in Fig. 12.4d). The inhibition of what level of hydration the mitochondria DNA repair has prevented the successful ﬁrst become active and ATP ﬁrst becomes rehydration and survival of these DNA- available for synthetic processes (Attucci et damaged embryos, even though they were al., 1991). It may be as low as 14% (apple

Control (dry) 750 Gy (dry) 1.5 1.6

(a) (b)

1.1 1.0

1.5 750 Gy (rehydrated 2.5 h) 750 Gy (rehydrated 2.5 h 1.5 in AP + dTTP) (c) (d) Absorbance at 260 nm (relative units)

1.1 1.0

Low Mol. Wt. Migration High Mol. Wt.

Fig. 12.4. Restoration of DNA integrity on rehydration requires DNA repair, shown by the effect of blocking DNA repair in -irradiated (750 Gy) embryos of rye, Secale cereale. Molecular weight proﬁles of scans of DNA fractionated by electrophoresis on neutral agarose gels from: (a) untreated dry embryos (controls); (b) dry embryos -irradiated ((Low Mol. Wt.) DNA strand breaks induced); (c) irradiated embryos rehydrated for 2.5 h (DNA repaired); and (d) irradiated embryos rehydrated for 2.5 h in presence of aphidicolin (AP) -polymerase inhibitor and dideoxythymidine triphosphate (dTTP) β-polymerase inhibitor (DNA repair blocked). Dessication 12 18/3/02 1:59 pm Page 359

Rehydration of Dried Systems 359

embryos) or as high as 25% in pea seeds tion of these positions and re-establishment (Leopold and Vertucci, 1989). Because of the of a fully functional genome are early and low levels of early ATP synthesis at rehydra- necessary events upon rehydration of both tion, an alternative non-mitochondrial embryo and pollen cells. The speed and cytosolic source has been sought to fuel the fidelity at which these processes are earliest synthetic events through a glycolic accomplished dictate the lag period to the fermentation of starch or lipid (Raymond et initiation of the first cell cycle after the dry al., 1985; Perl, 1986). How important this state and hence determine the eventual might be and at what levels of hydration it success of germination. Not only does DNA might operate are not yet known. Certainly, repair in seeds have the essential role of continued mitochondrial ATP generation restoring fidelity to DNA fragmented dur- has a critical function in providing the ing dry storage, it also plays a critical part essential energy generation for new protein in maintaining desiccation tolerance synthesis, including that for new DNA throughout the early hours of rehydration. repair enzymes and for the mitochondrial Efficient DNA repair is thus a critical dehydrogenases, both of which lose activity component of the cell machinery, which with time in a stored seed (Throneberry and may act immediately on rehydration of all Smith, 1955; Elder et al., 1987). The extent desiccation-tolerant dry cells, and the of the DNA repair function when an embryo extent of the success of DNA repair can be first becomes hydrated from the dry state seen as a major factor determining the fate depends not only upon the remaining activ- of plant cells following a dehydration/ ity of the repair enzymes stored within the rehydration cycle. It will therefore be of dry cells but also upon the available ATP for much interest to discover the facts that will this repair to take place in restoring integrity be revealed when resurrection plants are to fragmented nuclear DNA molecules and scrutinized for the status of genomic, plas- to the DNA of the mitochondria. Only on tid or mitochondrial DNAs following desic- the re-establishment of intact coding cation and rehydration, and to learn sequences in both nucleus and mitochon- whether telomere lengths are subject to dria can new repair-enzyme mRNAs be tran- change in similar circumstances. scribed in either organelle. These experiments raise intriguing questions in seed and pollen survival, which 12.4. Acknowledgements remain to be properly resolved. How stable are the circles of mitochondrial DNA? Are O. Leprince is grateful to the Netherlands the DNA sequences that code for the DNA Organization for Scientific Research, the repair enzymes specially labile to nuclease Netherlands Technological Foundation for or free-radical assault? Is the conforma- Scientific Research and the French Ministry tional folding of DNA, the state of methyla- of Agriculture and Fisheries for support. Dr tion or acetylation and the nature of F.A. Hoekstra is acknowledged for his con- DNA-binding proteins at the specific DNA structive comments on Section 12.2. repair sites on these genomes critical to D.J. Osborne and I. Boubriak acknowl- successful DNA repair on rehydration? edge support from Hortlink MAFF (Scottish Whether or not the sites of DNA cleav- Office), Framework IV FAIR5–3711 and the age are specific hypersensitive sites, religa- Royal Society, London.

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Part V

Retrospect and Prospect Dessication 13 18/3/02 2:00 pm Page 366 Dessication 13 18/3/02 2:00 pm Page 367

13 Damage and Tolerance in Retrospect and Prospect

Michael Black,1 Ralph L. Obendorf2 and Hugh W. Pritchard3 1Division of Life Sciences, King’s College, Franklin Wilkins Building, 150 Stamford Street, London SE1 6NN, UK; 2Seed Biology, Department of Crop and Soil Sciences, Cornell University, Ithaca, New York, USA; 3Seed Conservation Department, Royal Botanic Gardens Kew, Wakehurst Place, Ardingly, West Sussex RH17 6TN, UK

The preceding chapters have provided a ability to complete germination is often wide perspective on desiccation in plants. employed as the indicator of desiccation tol- In this brief, concluding chapter we will erance in seeds. Since the completion of select some information from these germination, i.e. expansion of the axis to accounts, which point to generalizations rupture the enclosing structures, normally that can be made. This is not intended as a occurs before cells begin to divide, germina- comprehensive overview or evaluation but tion as an index of tolerance may not reflect rather an attempt to identify some areas the integrity of cell division. Some authori- that offer prospects for further research and ties therefore insist that unequivocal desic- increase in our understanding. cation tolerance is shown only by seeds that The ability to tolerate desiccation obvi- germinate to produce normal seedlings, ously lies at the heart of the desiccation and where the capacity for cell division has not survival scenario. But there are problems been compromised. Moreover, since germi- implicit in this statement: how do we define nation in the strict sense is a response spe- and measure tolerance and survival? We can cific to the axis, the elongation ability of the see from foregoing chapters in this book that previously desiccated axis may not neces- tolerance, as considered by authorities, cov- sarily indicate the level of tolerance in the ers a wide spectrum of properties from, for remainder of the embryo, i.e. the cotyledons example, a rigorously defined, single bio- or scutellum. Considerations of a similar physical event (Chapters 4 and 10), such as type might also apply to recalcitrance, the partitioning of an amphiphile into a which is often taken as the ‘archetype’ of membrane, to the ability of a system (such desiccation sensitivity in seeds. Yet such as a seed or a whole plant) to resume nor- seeds possess or exhibit many features of mally the whole gamut of its growth and tolerant seeds, such as the occurrence of metabolic abilities. Between these two abscisic acid (ABA) and late embryogenesis extremes there is a range of parameters abundant (LEA) proteins, and degrees of taken by researchers to indicate tolerance, subcellular integrity maintained after dehy- and, in some cases, where a high degree of dration. The characteristics that are critical tolerance exists, some measure of intoler- to intolerance in recalcitrant seeds indeed ance nevertheless remains. For example, the prove difficult to identify.

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Similar complications exist in respect of requires further study. An interesting find- vegetative tissues. For example, some resur- ing is that the majority of desiccation-toler- rection plants (the homoiochlorophyllous ant bryophytes age most rapidly in the types) exhibit a high degree of desiccation range 9 to 22 MPa and have greatest tolerance in that the chloroplasts retain longevity at between 150 and 300 MPa their organization and chlorophyll after (Chapter 7); this is also generally true for dehydration, whereas the poikilochloro- orthodox seeds and pollen (Chapters 5 and phyllous types, though in almost all other 6). Thus, the water relations of survival and respects desiccation-tolerant, are intolerant longevity appear to be similar in material so far as chloroplast and chlorophyll that is physiologically, biochemically and integrity are concerned. These few exam- morphologically diverse. Moreover, develop- ples illustrate that desiccation tolerance ing seeds that are detached from the parent must be carefully defined and that indica- plant readily become desiccation-tolerant tors of tolerance and intolerance can exist when held at the upper range, whereas side by side in a single biological system. here most recalcitrant seeds succumb to This complexity might arise partly irreversible damage. These approximate because of the multiplicity of damaging water-potential ranges thus appear to be events that occur as cells dry out (Chapter 9) crucial for tolerance and longevity, and and the relative readiness for different kinds research aimed at understanding these of damage to be repaired (Chapter 12). In processes should, perhaps, be focused there. respect of the former, it is clear that different At the same time, we must address the prob- levels of arrest or damage occur as lem in mechanistic terms of why seeds of decreases, though the biophysical or molecu- various species show such differences in lar reasons for this are not clear in all cases. longevity around two orders of magnitude It is interesting to speculate that a similar under identical storage conditions. The scale of response to might also occur in the practical benefits with regard to storage and repair processes during rehydration but conservation arising from such knowledge more information on this is needed. are obviously extremely important. Another factor that might contribute to The multiplicity of factors participating the coexistence of tolerance and intoler- in desiccation tolerance have been exten- ance indicators as mentioned above is the sively discussed in several chapters. As bio- simultaneous occurrence, as dehydration physical, biochemical, cell biological and progresses, of protective mechanisms (i.e. molecular technologies have become more conferring tolerance) as well as damage sophisticated and adapted for use in seeds, processes. The inception of the protective pollen, spores and vegetative tissues, our mechanisms occurs in response to initial understanding of the processes involved in dehydration and develops to completion tolerance has gathered momentum. But provided that water loss is not too precipi- uncertainty (verging on controversy) has tous, while damage increases progressively developed in respect of some topics. For as falls The rate of drying (Chapter 3) example, while the evidence shows that therefore determines the relative progress glass formation is an important element in of protective and damaging events, i.e. the the protective mechanisms in ‘dry’ cells, it is development of both tolerance and intoler- not clear what the components of the vitrifi- ance characteristics and their presence in cation system are (Chapter 10). Sugars, espe- the final desiccated system. cially sucrose, are certainly involved, and One important point that has been possibly also proteins (e.g. LEA proteins) but touched on in the preceding chapters (e.g. the role of oligosaccharides, which once Chapter 10) is the relationship between exercised a strong claim for participation desiccation tolerance and longevity in the (in longevity also), has been questioned dry state: are the same or similar regulatory (Chapter 10; Bentsink et al., 2000). Most mechanisms involved? We are far from of the evidence for the participation of resolving this question, which clearly oligosaccharides in tolerance and longevity Dessication 13 18/3/02 2:00 pm Page 369

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comes from studies of correlations between therefore have a spurious appearance of a the oligosaccharide content and the appro- causal relationship. Longevity is measured priate physiological property. But, since des- by the ability to germinate, which might iccation tolerance and presumably longevity also reflect reserve oligosaccharide content. are likely to depend on multi-component Both drought stress and desiccation in action (e.g. carbohydrates, LEA proteins, plants have been widely studied at a molec- antioxidants, free-radical removal, etc.), cor- ular level, especially in vegetative tissues. relative evidence may be difficult to inter- In the former, numerous genes have been pret if one or more components other than, identified whose expression is regulated by for example, oligosaccharides become limit- water stress. Much has also been learned ing: in such a case, the oligosaccharide con- about signal transduction mechanisms tent would be scarcely relevant. In addition operating in the stress syndrome, and, in to the raffinose-series oligosaccharides the case of many, but not all genes, ABA (galactosides of sucrose), other galactosides – participates at an early stage in signalling. of cyclitols – are commonly present in seeds. In the desiccation phenomenon too, expres- Maturing seeds of some species accumulate sion of many genes occurs in response to fagopyritols, for example, which have been early water loss from vegetative tissues, suggested to substitute for the role of some of which are, again, ABA-regulated oligosaccharides, so knowledge restricted to (Chapter 11). But, in contrast, our under- the content of the latter might not provide a standing of molecular events involved in full picture (e.g. Steadman et al., 1996). seed desiccation is less advanced. But, although there is persuasive evi- Expression of several genes in developing dence against a determinative role for seeds is certainly affected by drying, the oligosaccharides in desiccation tolerance most intensively studied being those for and longevity, including on genetic grounds several reserve proteins in which the capac- (Bentsink et al., 2000), the accumulation of ity for expression is ‘switched off’ by the these compounds during seed maturation, dehydration experience (Jiang et al., 1995), apparently in response to the incipient dry- an event that is unlikely to contribute to ing signal (Blackman et al., 1992; Black et desiccation tolerance. There is some evi- al., 1999) (and similarly stimulated in dence, though, that certain reserve proteins hypocotyls (Brenac et al., 2002)), demands or close relatives might be involved in cell an explanation. After its maturation, two desiccation processes (Chapter 5). Several major developmental steps await the seed – positive effects of drying on gene expres- quiescence and then germination. For the sion or enzyme production have been completion of germination, easily utilizable recorded; but many of these are connected reserves should be immediately available in with reserve mobilization (e.g. Cornford et the extending organ itself, usually the radial., 1986) and are therefore unlikely to be cle, and oligosaccharides such as raffinose involved in desiccation tolerance. The pro- often fulfil this need (see review by motive effects of incipient dehydration on Peterbauer and Richter, 2001). It might be, oligosaccharide accumulation in maturing therefore, that the maturing seed uses the seeds (see above) may possibly operate same signal, incipient drying, to register the through positive action on expression of imminence of two different processes, the genes involved in their biosynthesis (see first being quiescence in the ‘dry’ state, for review by Peterbauer and Richter, 2001), which desiccation tolerance is necessary, such as galactinol synthase or stachyose and the second being germination, for synthase, but this has not been determined. which readily utilizable reserves (relatively The loss of water can also convert a seed low-molecular-weight carbohydrates such from the developmental to the germinative as the raffinose-series oligosaccharides) mode in respect of patterns of synthesized must be prepared. The coincidence proteins, an effect that has been confirmed between oligosaccharide accumulation and as operating at gene level (for review, see the onset of desiccation toleration may Kermode, 1995), but again it is not likely Dessication 13 18/3/02 2:00 pm Page 370

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that these observed changes are related to course, desiccation-sensitive (Chapter 5). desiccation tolerance. The essential point In general, then, there is a paucity of is, however, that gene expression in devel- information about gene expression associ- oping seeds can be modified by water status, ated with desiccation in seeds, especially so this effect could participate in the estab- as compared with what is known in respect lishment of desiccation tolerance. The of vegetative parts. Another aspect of the major relevant genes that might be affected desiccation scenario for which we lack a by drying are those encoding the LEA pro- balanced perspective concerns post-drying teins and certain heat-shock proteins, both rehydration. During this process a discrete of which may aid in the protection against set of genes and proteins is expressed in some of the damage that desiccation would mosses that are likely to be involved in var- inflict. None the less, it is not certain that ious repair and protective phenomena. We expression of these genes is universally up- know little about this so far as seeds (and regulated by the hydration state of develop- vegetative tissues) of angiosperms are con- ing seeds; on the contrary, certain lea genes cerned, and it seems important to explore do not appear to respond to desiccation this area if we are to advance our knowl- while others are down-regulated, at least in edge of survival mechanisms. developing seeds that have been dried pre- One problem inherent in the study of maturely (Han et al., 1996). gene activity during seed desiccation is that ABA and LEA proteins are almost cer- several processes are occurring during mat- tainly involved in the medium to long term uration/drying when tolerance is expressed in the desiccation scenario in seeds and veg- (induced) in planta. Some of these have etative tissues. In the latter, concentrations been mentioned above and in addition there of hormone increase in response to drought are those associated with the establishment stress and impending desiccation. In seeds, of dormancy and with the alterations in pro- however, the highest concentrations of ABA duction, destruction and sensitivity to hor- are normally reached in mid-development mones, for example ABA. It may be difficult and are declining at the time when dehydra- to identify those changes in gene expression tion commences; there is no evidence that that are specifically associated with the syn- loss of water generally provokes ABA syndrome of desiccation damage and/or toler- thesis, as it does in leaves, for example. ance but, none the less, experimental None the less, application of ABA can con- protocols could be designed to minimize fer desiccation tolerance on developing such extraneous complications. For exam- embryos (Chapter 5), so it is conceivable ple, one way to separate operationally the that the hormone participates in the in specific desiccation-related processes from planta situation. In vegetative tissues, ABA others is to use very young embryos (prior up-regulates the expression of several lea to dormancy inception and reserve accumu- genes, including the dehydrins. There is cir- lation), as was done in early experiments cumstantial evidence that some LEA pro- with barley (Bartels et al., 1988) and more teins are also up-regulated by ABA in recently in the examination of amphiphile developing seeds but the association among partitioning in wheat (Golovina et al., 2001). dehydration, ABA and LEA proteins at the There is mounting evidence that the stage of seed development when desiccation responses to drying and the early events in tolerance is initiated is actually fairly the desiccation scenario in seeds and vegeta- obscure. Though the implication of LEA tive tissues are initiated by the first changes proteins and ABA in the desiccation phe- in water content at the beginning of dehydra- nomena of seeds may be undeniable, the tion (see above). But what actually fires the precise details of their involvement remain starting signal of the dehydration transduc- unresolved. One confusing point is that the tion chain remains to be clarified. A plausi- developing embryos of several types of ble assumption is that the detection of early recalcitrant seeds are relatively rich in both water loss in vegetative tissues is the same LEA proteins and ABA and yet they are, of whether only relatively mild water stress is Dessication 13 18/3/02 2:00 pm Page 371

Damage and Tolerance in Retrospect and Prospect 371

suffered or whether the loss continues into tions where water loss can be strictly regu- severe desiccation. Detection of changes in lated (such as by osmotic means) might be turgor are likely to be responsible, involving an approach that will allow the application trans-membrane sensors such as the putative of advanced biological techniques, but cau- histidine kinase osmosensor in Arabidopsis tion must be exercised to minimize (Urao et al., 1999). An intriguing point, how- mechanical damage by rapid dehydration ever, is that the release of the initial signal or rehydration of tissues not protected by a sets in motion, in one case, a series of events testa or pericarp. that culminates in protection against In summary, then, there is still much to extreme dehydration (e.g. in resurrection be learned about the extent to which the plants) but, in others, only the ability to cope very early, medium- and longer-term events with, in comparison, mild drought stress. are shared by seeds, pollen and vegetative One of the earliest events in drought tissues. stress is the generation of transient calcium How do we take our understanding for- signals (for review, see Knight and Knight, ward? Clearly, identifying marker molecules 2001), and these are also likely to occur even for desiccation stress tolerance is highly desir- when water loss continues to desiccation. able; and quantifying the products of gene Whether or not calcium signalling partici- expression and how they interact or comple- pates in all the multiple aspects of the desic- ment each other’s effect needs to be cation syndrome has to be resolved. In seeds addressed. As has been noted in Chapter 11, a and pollen almost nothing is known about quantitative comparison of LEA proteins in the early signalling events set in motion by tolerant and sensitive tissues would be partic- the incipient dehydration that initiates the ularly informative; in the same context, heat- acquisition of desiccation tolerance. Is turgor shock proteins also qualify for further loss by the embryo the first perception of investigation. Ultimately, we should know the drying and is this, too, followed shortly by intracellular location of these proteins and transient calcium signalling? examine the possibility of a redistribution of Recent research indicates that in pollen these molecules and others as part of the tol- and seeds an early detectable intracellular erance scenario (Chapter 10). Quantification response to dehydration is the partitioning and identification of free radicals in biological of amphiphiles from the aqueous cytoplasm systems is a similar challenge, as short half- to the membrane lipid phase (Chapter 10). lives means that determination by electron This has been suggested to occur in the paramagnetic resonance is difficult. There is a establishment of desiccation tolerance, pos- need to identify new techniques and methods sibly exerting a protective effect on mem- in pursuit of the causes of desiccation sensi- branes by virtue of the properties (e.g. tivity as, until then, we may well run the risk antioxidant) of certain endogenous of measuring mainly the consequences of des- amphiphiles. It is important to determine iccation stress. The other side of the coin is, of what the endogenous amphiphiles are and if course, recovery from such stress upon rehy- the same partitioning phenomenon occurs dration and the repair processes involved in desiccation-tolerant vegetative tissues. therein. This must surely be an area meriting Investigation of the cell and molecular great attention. Desiccation damage, protec- biology of signal perception and transduction against it, and repair all involve a multi- tion in seeds and the early events that plicity of components but we might ask if all closely follow will require the utilization play equally important roles or if the effects of experimental systems that are rigorously and actions of some are more critical than controllable. It is doubtful if the methods others. that have been employed hitherto – drying Work on lower plants (Chapters 1, 7 and whole seeds, either in planta or in vitro at 11) and angiosperm seeds (Chapters 5 and different rates and relative humidities, etc. 8) has revealed some interesting general – will be satisfactory. The use of isolated associations with habitat, i.e. likely adapta- embryos (or parts thereof) under conditions to local conditions. Continuing to Dessication 13 19/3/02 2:08 pm Page 372

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screen biodiversity will undoubtedly sug- Looking to the not-too-distant future, gest some interesting paradigms on which to one can see the use of our knowledge to work. As the power of geographical infor- manipulate desiccation tolerance and seed mation systems increases, in terms of layers longevity. Rapid advances in genomics and of information that can be analysed and the related sciences will increasingly provide precision of the data, it will be easier in the opportunities for the development of future to consider biological data in an eco- unique tools and reporter systems to logical context. Such a holistic approach to understand and regulate the mechanisms the issue of desiccation sensitivity will be by which seeds and other organs respond important if we are to capitalize on the to internal and external signals. The appli- effort made since the 1980s in genomics and cation of our knowledge to enhance or con- to gain the most from emerging work in this fer tolerance of desiccation and to increase area. Biomolecules contribute to the ‘body longevity has important potential in agri- plan’ of organisms, and form has an impact culture for the improvement of the quantity on function in speciﬁc desiccating environ- and quality of the food supply and will add ments. Proteomics will have an important to the success with which we can con- part to play in this context. tribute to plant conservation.

References

Bartels, D., Singh, M. and Salamini, F. (1988) Onset of desiccation tolerance during development of the barley embryo. Planta 175, 485–492. Bentsink, L., Alonso-Bianco, C., Vreugdenhil, D., Tesnier, K., Groot, S.P.C. and Koornneef, M. (2000) Genetic analysis of seed-soluble oligosaccharides in relation to seed storability of Arabidopsis. Plant Physiology 124, 1595–1604. Black, M., Corbineau, F., Gee, H. and Côme, D. (1999) Water content, raffinose, and dehydrins in the induction of desiccation tolerance in immature wheat embryos. Plant Physiology 120, 463–471. Blackman, S.A., Obendorf, R.L. and Leopold, A.C. (1992) Maturation proteins and sugars in desiccation tolerance of developing soybean seeds. Plant Physiology 100, 225–230. Brenac, P., Horbowicz, M., Dickerman, A.M., Miseray, F., Smith, M.E. and Obendorf, R.L. (2002) Up- regulation of raffinose and stachyose accumulation in buckwheat (Fagopyrum esculentum Moench) seedling hypocotyls during drying. Planta (submitted). Cornford, C.A., Black, M., Chapman, J.M. and Baulcombe, D.C. (1986) Expression of -amylase and other gibberellin-regulated genes in aleurone tissue of developing wheat grains. Planta 169, 420–428. Golovina, E., Hoekstra, F.A. and van Aelst, A.C. (2001) The competence to acquire cellular desiccation tolerance is not dependent on seed morphological development. Journal of Experimental Botany 52. 1015–1027. Han, B., Hughes, D.W., Galau, G.A., Bewley, J.D. and Kermode, A.R. (1996) Changes in late-embryogenesis abundant (LEA) messenger RNAs and dehydrins during maturation and premature drying of Ricinus communis L. seeds. Planta 201, 27–35. Jiang, L., Downing, W.L., Baszczynski, C.L. and Kermode, A.R. (1995) The 5 flanking regions of vicilin and napin storage protein genes are down-regulated by desiccation in transgenic tobacco. Plant Physiology 107, 1439–1449. Kermode, A.R. (1995) Regulatory mechanisms in the transition from seed development to germination: interactions between the embryo and the seed environment. In: Kigel, J. and Galili, G. (eds) Seed Development and Germination. Marcel Dekker, New York, pp. 273–332. Knight, H. and Knight, M.R. (2001) Abiotic stress signalling pathways: specificity and cross talk. Trends in Plant Science 6, 262–267 Peterbauer, T. and Richter, A. (2001) Biochemistry and physiology of raffinose family oligosaccharides and galactosyl cyclitols in seeds. Seed Science Research 11, 185–197. Steadman, K.J., Pritchard, H.W. and Dey, P.M. (1996) Tissue-specific soluble sugars in seeds as indicators of storage category. Annals of Botany 77, 667–674. Urao, T., Yakubov, B., Satoh, R., Yamaguchi-Shinozaki, K., Seki, M., Hiroyami, T. and Shinozaki, K. (1999) A transmembrane hybrid-type histidine kinase in Arabidopsis functions as an osmosensor. Plant Cell 11, 1743–1754. Dessication Glossary 18/3/02 2:00 pm Page 373

Glossary

ABA: Abscisic acid a sesquiterpenoid plant hormone. adsorption: The attraction of gas or liquid molecules to active surfaces. The rehydration of dry biological tissues in humidified atmosphere is through water adsorption. ageing: Time-dependent deterioration of cells and biomolecules. Ageing reactions are often water-content-dependent. algae: An informal term covering photosynthetic organisms (largely aquatic) other than the green land plants (bryophytes, pteridophytes, gymnosperms, flowering plants). Algae embrace red, green and brown seaweeds and unicellular, colonial and filamen- tous organisms from a taxonomically diverse range of groups, variously defined by different authors. amphiphilic: Having an affinity for both aqueous and non-aqueous phases. Typically used to describe the behaviour of a molecule with a polar group that can interact with the cytoplasm and a non-polar, hydrophobic group that interacts with the membrane. Molecules with two such different groups are amphipaths. angiosperms: Flowering plants, consisting of two groups, the monocotyledons and the dicotyledons, characterized by having one or two seedling leaves, respectively. anhydrobiosis: Suspended life in the dried state. anthophyte hypothesis: This hypothesis refers to the evolutionary relationships of seed plants. It proposed that the gnetophytes are the sister group (closest living relatives) to the angiosperms and thus they shared a common ancestor. Though formerly strongly supported, recent molecular data have generated considerable doubt about the idea, and there is strong evidence for the gnetophytes being most closely related to the conifers. antioxidants: Molecules that remove activated oxygen species from cells by serving as catalysts (enzymes such as catalase, superoxide dismutase and glutathione reductase) or substrates (ascorbate or tocopherol) in reactions that donate unpaired electrons. ascospore: A meiospore borne in an ascus (saclike structure formed by the Ascomycota). bicellular (also binucleate): Refers to the developmental stage of the male gametophyte of higher plants that has undergone one nuclear division after meiosis. One cell (the vegetative cell) is involved in directing pollen tube growth; the other cell later divides into two sperm cells and is located inside the vegetative cell. After division, the tricellular (or trinucleate) pollen consist of one vegetative cell with two sperm embedded in it. Depending on whether anther dehiscence occurs after the first or the second mitosis, mature pollen is designated bicellular or tricellular.

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374 Glossary

bitegmic: Of ovules (cf. unitegmic or ategmic). Ovules having both outer and inner integuments prior to fertilization and seed development. Where both persist in the mature seed, the inner integument becomes the tegmen, and the outer integument becomes the testa sensu stricto. blastospore: A spore arising by budding such as a conidium arising from a narrow region of the conidiogenous cell with elongation and swelling and then delimitation of the conidium by a septum. Boltzman distribution law: The expression for the ratio of populations of molecules at any two levels of energy. bound water: A concept to describe hydration of macromolecules based on interactions of water molecules with a macromolecular surface and with other water molecules. Bound or vicinal water has sufficient interactions with the macromolecular surface to cause changes in its thermodynamic properties and molecular mobility compared with water in a dilute solution. The concept of bound water was originally based on simple sorption theory, but has evolved to consider adsorption sites with different characteristics and relationships between bound water and the structure and activity of macromolecules. brachycytes: Spherical, thick-walled cells that are formed on moss protonemata after extended periods of culture. Bryophyta (bryophytes): Green land plants with an alternation of haploid and diploid generations, in which the diploid sporophyte (capsule) remains dependent on the haploid gametophyte for water and at least in part for nutrition (mosses, liverworts – q.v.). C3 plant: Plant in which the first detectable photosynthetic products are three-carbon molecules, e.g. 3-phosphoglyceric acid. Includes most plants of wet or mesic habitats at all latitudes. C4 plant: Plant in which the first detectable photosynthetic products are four-carbon organic acids, e.g. oxaloacetic acid, malic acid. Most characteristically plants of semiarid situations in warm climates. callose: 1-4 glucan; substance often formed in pollen upon stress. carbon balance: The cumulative net uptake of carbon by a plant; cumulative gross photo- sythesis minus respiration. cavitate : To form an air-filled space in water-filled xylem. chalaza: The chalaza is the tissue at the base of the ovule, from which the integuments arise, and is probably involved in nutrient transfer from the funicle to the developing embryo and endosperm. In some species it becomes heavily developed, surrounding and overtopping the integuments, a state known as pachychalazy. chaperones: Proteins that modify folding of other (newly formed) proteins and assist assembly of protein oligomers. chemical shift in NMR: The shift in the position of the resonance line of the nucleus because of the local electronic structure, which makes the local magnetic field slightly different from the external static field. chlorophyll fluorescence: Excitation energy absorbed by chlorophyll may: (i) bring about photochemical processes; (ii) be dissipated as heat; or (iii) be re-emitted as red fluorescence, measurement of which provides a basis for non-invasive measurements of various aspects of photosynthesis. compatible solutes (osmolytes): Osmotically active, non-toxic, low-molecular-mass molecules (such as quaternary amines, amino acids or sugar alcohols) which may accumulate in cells in response to water or freezing stress. conidium: In fungi a non-motile asexual spore usually formed at tips or sides of a sporoge- nous cell; in some cases a pre-existing hyphal cell may be converted into a conidium. constitutive: Occurring constantly rather than being induced in response to a particular stimulus. Dessication Glossary 18/3/02 2:00 pm Page 375

Glossary 375

continuous-wave NMR: A field-swept technique, when the field is swept through the resonance frequency and a frequency domain absorption spectrum is obtained. cooperative stress: Stress that is not associated with water loss per se, but exacerbates the damaging effects of water loss, possibly because it has a similar mechanism of damage. crassinucellate: Of ovules (cf. tenuinucellate). Ovules in which the nucellus is massively developed, rather than scantily so. The nucellus is the megasporangial tissue surrounding the embryo sac, and it persists only relatively rarely in the mature seed, where it forms the perisperm. critical minimum surface area: The minimum membrane surface area that can be lost from the plasmalemma or tonoplast upon contraction during water stress without causing cells to burst upon rehydration. critical minimum volume: The minimum volume to which cells or vacuoles can contract during water stress without bursting upon rehydration. Most non-acclimatized cells can contract no more than to 50% of their original volume. critical water content: The minimum water content to which cells or macromolecules can be dried without imposing irreparable damage. This water content reflects the critical water potential. critical water potential: The minimum water potential to which cells or macromolecules can be dried without imposing irreparable damage. This water potential corresponds to the critical water content. cryopreservation: The technique to preserve living organisms or cells at subzero temperatures, usually below –100°C (e.g. in liquid nitrogen –196°C), which includes pretreatment in cryoprotective solutes (if required), cooling to below 0°C, storage at low temperature, thawing and preparation for resumption of growth. dehiscence: Opening of anthers in higher plants, which allows pollen to be dispersed: or, of some fruits, to allow seeds to be dispersed. dehydrins: Group 2 LEA proteins synthesized in association with dehydration, characterized by a lysine-rich 15-amino-acid motif (K-segment), contiguous serine residues and the consensus sequence DEYGNP. demixing: Rearrangement and consolidation of similar-type molecules that increases packing efficiency in severely dehydrated systems. Demixing will disturb the original configuration of proteins and lipids within bilayers of membranes and aggregation of molecules can result in irreversible structural changes. desiccation: The extreme form of water loss, in which most of the protoplasmic water is lost and a very low amount of tightly bound water remains in the cell. desiccation avoidance: A protective strategy against drought stress where cells remain hydrated using adaptive structures that scavenge water. This strategy prevents water loss. See also desiccation resistance. desiccation damage (sensu stricto): Mechanical or structural damage to cells and cellular constituents directly resulting from water removal. desiccation resistance: A protective strategy against drought stress where cells reduce the rate of water loss by using adaptive structures that form barriers to water loss or by accumulating solutes that lower the water potential difference between the cell and the environment. This strategy prevents water loss. desiccation tolerance: The ability to recover biological functions after drying to a point at which no liquid phase remains in the cells (e.g. water content down to 5% or less of dry weight, in equilibrium with water potential down to 200 MPa or less). desorption: Opposite of adsorption; the dehydration of biological tissues in dry atmosphere is water desorption. diaspore: Any spore or other plant part able to form a new organism, e.g. the haploid structure formed out of a part of the moss gametophyte. Dessication Glossary 4/4/02 2:26 pm Page 376

376 Glossary

diffusional correlation time: The average time between jumps in position for water molecules in the system. Dollo’s law: Dollo’s law or rule suggests that, for complex characters or traits, parallel or multiple origin is unlikely, but that reversal or loss may be easy. The assumption is that many genes must change to create a morphological structure or physiological trait, but only one of them needs to change in order to lose it. dormancy: An endogenous mechanism that prevents viable hydrated plant parts (e.g. seeds, spores, buds) from resuming growth and full metabolic activity. drought: In plants, the partial limitation of water content, often for a prolonged period; ≤ usually when cell water potentials ( w) are 3 MPa in non-transpiring cells. Water contents may reach 20–25% (fresh weight basis), 0.25–0.33 g water g1 dry weight. drought tolerance: The ability to survive drought. dry: To remove water (verb) or without water (adjective). Since achieving and measuring truly dry material is logistically difficult, ‘dry’ is often used as a relative term to describe material that is drier than its undried counterpart. electron paramagnetic resonance (EPR): The absorption of electromagnetic energy during transition of electrons between two energy levels of Zeeman splitting (q.v.). endocytotic vesicles: Invaginations of plasma membrane into the cytoplasm observed during osmotic contraction of protoplasts from non-acclimatized plants. These are believed to be deleted from the membrane surface area. enthalpy (H): A defined thermodynamic variable of state that consists of internal energy of the system (E), specified pressure (P) and volume (V). H = E +PV, where the PV units are converted to calories, ergs or joules. Different enthalpy, H, describes the change of energy status. entomopathogenic: Pathogens (e.g. fungi, bacteria) that feed on insects. entropy (S): A thermodynamic term that quantifies the randomness or disorder of the system. epilithic: Growing on the surface of rocks. EPR imaging: Visualizing the distribution of paramagnetic centres in a sample. eukaryotic: Of cells having a nucleus. exocytotic extrusions: Folding of the plasma membrane to the cell exterior observed during osmotic contraction of protoplasts from acclimatized plants. These are reincorporated into the plasma membrane upon expansion and so are believed to be a mechanism to retain overall membrane surface area in contracting cells. ferns: The largest group of pteridophytes, having leaves with branching veins (mega- phylls). flash drying: Drying by rapid flow of dry air over excised embryonic axes, somatic embryos or small tissue pieces. Fourier self-deconvolution: Transformation of the absorption bands in the Fourier transform infrared spectrum to line shapes with narrower peaks (resolution enhancement technique). Fourier transformation: The mathematical method to convert the time dependence of the NMR signal (FID) into the frequency dependence of the NMR signal. free energy (Gibbs free energy, G): A quantity that is used to describe the energetics of chemical, physical and biological processes. Gibbs free energy in a system is the maximum amount of energy for work. It decreases for a spontaneous process such as rehydration of dry tissues, in which free energy of water decreases. free induction (FID) signal: The measure of the NMR signal over time. freeze–fracture: A method of preparing specimens for electron microscopy by freezing and then splitting them. freezing point depression: One of the four colligative properties of solutions in which the freezing point is depressed below that of the pure solvent. frequency: Number of cycles per second. Dessication Glossary 4/4/02 2:27 pm Page 377

Glossary 377

gametophyte: The structure that forms the haploid, gamete-forming part of a plant’s life cycle. gas exchange: Usually refers in plant-physiological contexts primarily to uptake or output

of CO2 and O2 in the course of photosynthesis and respiration. Gauss: The unit of strength of the magnetic field. g-factor: The ratio of the magnetic moment to the spin angular moment of an electron (determines the position of the EPR spectrum). glass (glassy state): A fluid that is so viscous that it acquires mechanical properties (strength) of a solid. The viscosity results from numerous intermolecular interactions in a random array forming an irregular matrix of pores. Because there is no regular pattern to the arrangement of molecules in this fluid (similar to a liquid), the structure is considered amorphous relative to the defined arrangement of molecules in a crystalline solid. Because molecular mobility of molecules within the glass is restricted, physical and chemical reactions are slowed but not stopped. Thus, the glass is considered kinetically, but not thermodynamically, stable. Glass transitions are considered second-order state changes (as opposed to phase transitions, which are first-order) because of a continuous change in enthalpy, entropy and volume (first derivative of chemical potential) throughout the transition but a discontinuous change in heat capacity (second derivative of chemical potential). glass formation: A change from a fluid to a semi-solid state in the protoplasm of desiccating cells, also known as vitrification. Carbohydrates and proteins are the main compounds that can contribute to glass formation in the cytoplasm. heat-shock proteins: Proteins generally synthesized in response specifically to relatively high temperatures (e.g. 40°C): often act as chaperones (q.v.) and protein protectants. Höfler diagram: The plot of cellular water potential components against protoplast volume. homoiochlorophyllous: Retaining most or all chlorophyll through a drying–rewetting cycle. homoiohydrous: Maintaining a high water potential and active metabolism during times of low water availability. hydration force explanation: A hypothesis used to describe how compression of macromolecules can lead to their deformation. Compression of cell volume by water removal can lead to repulsive forces as molecules with similar charges come within close proximity. The repulsion between surfaces can lead to lateral tensions within structures that cause phase changes or deformations. The hydration force explanation invokes non-specific mechanisms for the protection of macromolecules by carbohydrates (as opposed to the water replacement hypothesis, which invokes specific interactions), first as osmotica that resist water loss, and then as glass formers that provide mechanical resistance to the compression. hydration levels: Ranges of water contents or water potentials that define different structures/functions of molecules or physiological activities of cells. Also, ranges of water contents or water potentials that define changes in thermodynamic or motional properties of water. hydraulic conductivity: A measure of the diffusional resistance of a water transport pathway within the tissue. hyperfine interaction: The interaction between nuclei and unpaired electrons in EPR. hyperfine splitting constant: The distance between lines of an EPR spectrum originating from hyperfine splitting. hyperfine splitting of the EPR spectrum: Separation between lines of an EPR spectrum due to the hyperfine interaction. hysteresis: The difference in the equilibrium water between desorption and adsorption isotherms. Dessication Glossary 4/4/02 2:28 pm Page 378

378 Glossary

image contrast: The differences in signal intensity in NMR imaging between different regions of the sample. imbibitional stress: Stress imposed on a dehydrated organism by imbibition of water. The injury that ensues usually encompasses the loss of plasma membrane integrity. The injury is severe when imbibition occurs at low temperature and when the organism/cell is very dry: avoidance can be achieved by prehydration in humid air and warm imbibition. in vitro: Literally ‘in glass’, referring to tissues cultured in a sterile container on an artiﬁ- cial, sterile nutrient medium. inhomogeneous broadening: The broadening of the EPR lines because of unresolved hyperﬁne structure. integrated intensity: The area beneath an absorption peak. interfacial region: The boundary between two phases. intermediate seeds: Seeds that tolerate considerable (at least to 30% RH) but not complete drying. Life spans of stored seeds progressively increase as the storage RH is decreased to about 50% and then a reversed trend is observed with storage RH < 50%. Seeds with intermediate characteristics cannot be stored using standard recommended storage protocols: though they appear to survive low water contents, they do not survive the added stress of exposure to 18°C. See orthodox, recalcitrant. IRGA (infrared gas analysis): Methods using gas-phase absorption bands in the near

infrared to measure concentration changes in CO2 (or other gases). isotropic motion: Uniform tumbling of a spin probe in all directions; the isotropic EPR spectrum originates from the averaging of the spectral anisotropy. LEA (late embryogenesis abundant) proteins: A broad family of universal plant proteins with conserved amino acid motifs which accumulate to high levels during late stages of embryo development or in response to osmotic stress in vegetative tissues. They are very hydrophilic, remain soluble at T 90°C, and are believed to protect cells during water stress through an, as yet, unknown mechanism. See also dehydrins. lichenized fungus: A fungus that has formed a lichen in association with an alga. line width: The width at half-height of absorption peak. liverworts: Bryophytes (q.v.) with leafy or (less commonly) thalloid, usually dorsiventral gametophytes and short-lived sporophytes (Hepaticae; Hepaticopsida). magic angle: The angle (54° 55) of the axis of mechanical rotation of the sample in high- resolution NMR to eliminate line broadening. magnetic susceptibility: The ratio between magnetization and magnetic field strength. magnetogyric ratio: The ratio of magnetic dipole moment to the spin angular moment of a specific nucleus. manometric methods: Techniques using change in pressure (Warburg) or volume (e.g. Gilson) in a gas space over the reaction mixture or material to follow a metabolic process (e.g. respiration or photosynthesis). minimum critical volume: See critical minimum volume. monolayer hydration: The level of hydration at which only polar sites are bounded by water. mosses: Bryophytes (q.v.) typically with (+ or ) radial leafy shoots, and sporophyte capsules borne on a long-lived seta (Musci, Bryopsida). NMR imaging: Imaging of spatial distribution of water or water properties based on proton magnetic resonance. nuclear magnetic resonance (NMR): The absorption of electromagnetic energy during the transition of a nucleus between two discrete energy levels of Zeeman splitting. oligosaccharide: A carbohydrate whose molecules are composed of a few (< 20) generally mixed (e.g. glucose, fructose, galactose) monosaccharide units: three, four and five in raffinose, stachyose, verbascose, respectively. Dessication Glossary 18/3/02 2:00 pm Page 379

Glossary 379

order parameter: Quantity calculated from the shape of the EPR spectrum, which indicates the degree of motional anisotropy. orthodox seeds: Seeds that tolerate the immediate effects of severe water loss (i.e. desiccation-tolerant). Life spans of stored seeds progressively increase as the storage RH is decreased to about 20% and then a reversed trend may be observed with storage RH < 20% (decreasing life span with decreasing RH). Seeds with orthodox characteristics can be stored using standard recommended storage protocols of drying to 0.05 ± 0.02 g water g1 dry mass and storing in a freezer at about 18°C. Storage longevity can be predicted from temperature and seed water content. See intermediate, recalcitrant. osmolytes: See compatible solutes. osmotic adjustment: The net accumulation of solutes after the plant tissue has been exposed to a predetermined rate of water deficit. osmotic excursions: Reversible shrinking and swelling of cells and protoplasts during exposure to cycles of low and high water potentials. osmotically inactive: Apoplastic water present in very small pores and strong water- binding sites of biological surfaces in plant tissues. osmotically unresponsive: Membrane vesicles that fail to swell when water stress is relieved because the lumen lacks osmotically active constituents. This most probably occurs when different membrane systems compress together during dehydration and fuse, excluding formerly contained constituents. ovule: The female (mega) spore and gametophyte of higher plants: becomes the seed after fertilization. pachychalazy: See chalaza. paramagnetic: Atom or molecule containing an unpaired electron. partitioning: Distribution of molecules, e.g. between lipid and aqueous phases. permanent wilting point: Minimum water potential tolerated by non-transpiring cells. Similar in concept to critical water potential. phase separation: A consequence of demixing. The aggregation of similar-type molecules into enriched domains leads to higher localized chemical potentials and greater likelihood of phase changes. phase transition: Change of state between solid, liquid and vapour phases. Phase changes in lipids are complex because of the diverse crystalline states of pure lipid and lipid mixtures. For polar lipids, phase changes occur when a fluid gel converts to a liquid crystalline or hexagonal phase. Phase changes in polar lipids can be induced by altering the water status (drying favours gel and hexagonal phases) or temperature (low temperatures favour gel phases). Phase changes are termed first-order transitions because of an abrupt, discontinuous change in the enthalpy, entropy and volume (first derivatives of chemical potential) and a consequent infinite heat capacity (second derivative of chemical potential). phycobiont: In lichens, the alga that is associated with the fungus, or mycobiont. phylogenetic classification: Phylogenetic classifications attempt to generate systems that reflect as closely as possible the evolutionary relationships and history of a group of organisms. They recognize only those groupings of species that are monophyletic, i.e. all the members of the group are likely to be descended from a single common ancestor. phytochrome: Chromoproteins that undergo a reversible conformational change maximally upon absorption of red or far-red light. They regulate many aspects of plant function. plasma membrane: The membrane that envelops a cell protoplast. plasmolysis: Withdrawal of the cytoplasm from the cell wall when the cell is placed in a solution of lower osmotic potential than the cell sap. poikilochlorophylly: The ability to reversibly lose chlorophyll during desiccation. Dessication Glossary 4/4/02 2:28 pm Page 380

380 Glossary

poikilohydrous: Of plants whose water content closely follows fluctuations of humidity in their environments (in contrast to homoiohydrous plants), typically suspending metabolism during periods of drought. pollen: Male gametophyte of higher plants, which functions to deliver its haploid sperm cells to the ovules in order to bring about fertilization. population (of molecules): The number of molecules occupying a particular energy level. prothallus: Gametophyte (haploid) in ferns, horsetails, Selaginella and Lycopodium that is formed from spore germination and which produces gametes. After fertilization the diploid sporophyte grows out of the prothallus. protonema: The structure (immature gametophyte) that develops from spore germination, e.g. in mosses. Pteridophyta (pteridophytes): Green plants with an alternation of haploid (gametophyte, prothallus) and diploid (sporophyte) generations, both (at least potentially) capable of living independently, the sporophyte being the dominant plant (ferns, horsetails, clubmosses, spikemosses). pulsed NMR: NMR technique based on the recording of NMR signals with time after exciting nuclei by a short intense pulse of radio-frequency radiation. PV curve: The relationship between the reciprocal of water potential and relative water content of a tissue. radical adduct: The product of the interaction of a primary free radical with a spin-trap molecule. reactive oxygen species (ROS): Molecules containing oxygen with an unpaired electron or a pair of electrons with parallel spin (singlet oxygen). These molecules, often resulting from free-radical reactions, seek an additional electron and so are highly reactive with biomolecules which are electron-rich. Reacting with ROS, biomolecules are peroxidized to become reactive themselves, initiating a cascade of degradative reactions. recalcitrant seeds: Mature seeds that do not survive if desiccated to water potentials less than about 15 MPa (about 90% RH) and hence cannot be stored in the ‘dry’ state. Hydrated storage at either cryogenic or supra-freezing temperatures appears to be the best storage option. receptivity of the isotope: The product of the sensitivity of the isotope for NMR experiments and its natural abundance. rehydrins: Proteins synthesized specifically in association with rehydration in desiccation- tolerant plants. relative humidity: Water activity multiplied by 100. relative water content (RWC): Tissue water content relative to (fraction or percentage) water content at full turgor. relaxation process: The return of magnetization to thermal equilibrium. resurrection plants: A small group of poikilohydrous higher plants which tolerate almost complete water loss in their vegetative tissues and resume normal functional activity after rehydration: occur in specific ecological niches with seasonal water availability. RH (relative humidity): Water content of air expressed as percentage (or fraction) of saturated water content at the same temperature. rotational correlation time: The time it takes for a molecule to rotate one radian around its axis. rubber: A fluid that is more viscous than a syrup but less viscous than a glass. There are fewer intermolecular interactions and larger pore sizes in rubbers compared with glasses and this allows for greater elasticity in the structure. RWC: Relative water content. second-derivative analysis: Mathematical procedure used for increasing the resolution of an FTIR spectrum. self-incompatibility: In higher plants the phenomenon of pollen being unable to establish fertilization within the same plant or some individuals of the same plant species. Dessication Glossary 4/4/02 2:29 pm Page 381

Glossary 381

sensitivity (spectral): The minimal number of spins (electrons, nuclei) which can be measured by a method (EPR, NMR, respectively). sensitivity of the isotope for NMR experiments: The quantity depending on magnetogyric ratio () and spin quantum number (I). spatial resolution: The precision in the determination of the location of a signal source (or the measure of the minimal distance between two signal sources within the sample which still allows them to be distinguished). spectral anisotropy: The dependence of EPR spectra on the orientation of the spin probes, originating from the anisotropy of the interaction of an electron with the externally applied magnetic ﬁeld. spectral resolution: Quantity that expresses how the lines in a spectrum are separated from one another. spectroscopy: The measurement of the energy differences between discrete energetic levels of atoms or molecules. spin label (spin probe): Stable free radical that contains a nitroxide fragment with an unpaired electron. spin trap: Diamagnetic molecule forming a nitroxide radical when interacting with a primary free radical. spin-echo technique: Pulse NMR based on applying a second pulse after a set of delay times to eliminate the effect of the inhomogeneity of the magnet.

spin-lattice (or longitudinal) relaxation time (T1): The time constant of magnetization decay because of the interaction of nuclear/electron magnetic moments (spin) with the environment (lattice).

spin–spin (or transverse) relaxation time (T2 ): The time constant of magnetization decay because of the interaction of nuclear/electron magnetic moments (spin) with each other. sporangia: Spore-producing structures, e.g. in pteridophytes. spore: In mosses, ferns, horsetails, Lycopodium and Selaginella, a haploid, stress-resistant cell formed by meiosis; in fungi, spores may not necessarily be the result of meiosis and also may be diploid. spore bank: Layer of accumulated spores in the soil. sporocarp: Structure in certain ferns that contains the sporangia. sporophyte: The diploid (asexual) phase of the alternation of generations in plants. structural water: Water required to maintain the configuration of macromolecules normally observed under aqueous conditions, and so is most often identified in drying systems as the minimum amount of water required to prevent a conformational change. Water, at water contents where conformational changes in macromolecules occur, has unusual thermodynamic properties or restricted mobility. Consequently, structural water is a component of bound or vicinal water in hydration models using the bound-water concept. In alternative solution-based models of hydration, structural water is a likely component of the super-viscous solutions with rubbery or glassy characteristics. sucrose: A disaccharide; molecules each contain one glucose and one fructose unit. symbiosis: A regular association between two organisms characterized by mutual benefit and interdependence.

Tg: Glass–liquid transition temperature. Tm: Gel–liquid crystalline temperature of membranes. teliospore: A thick-walled resting spore of fungi belonging to the rusts and the smuts, in which karyogamy occurs. trehalose: A disaccharide in which each molecule contains two glucose units with an 1→1 linkage: often associated with desiccation tolerance in animals and some plants. tricellular (or trinucleate): See bicellular. turgid: Swollen or ﬁrm because of the pressure of water within the cell or tissue. Dessication Glossary 4/4/02 2:30 pm Page 382

382 Glossary

turgor: Cellular pressure generated from the movement of water into a cell. Pressure exerted by the cell wall, balancing the difference between the osmotic potential of the cell contents and the water potential of the surroundings. urediniospore: A binucleate spore of Uredinales (rust fungi). vascular plants: Green land plants which possess vascular tissues for water conduction, comprising pteridophytes and seed plants (gymnosperms and angiosperms). viability: Potential ability of a cell, tissue, organ or plant part to resume full metabolism and growth under favourable conditions (e.g. of seeds). vitrification: A change from a fluid to a semi-solid state in the protoplasm of desiccating cells, also known as glass formation. volumetric elasticity module: An expression to quantify the relationship between the change in volume and the applied pressure. water activity: Proportion of water available compared with pure water. Water activity is usually measured in the vapour above a condensed phase and ranges from 0 (no water in the condensed phase) to 1 (pure water in the condensed phase). At equilibrium, the water activities of the vapour and condensed phases are the same. The natural log of water activity directly relates to the chemical potential of water. See water potential. water content: A measure of the concentration of water that is usually expressed by mass ratios on either an absolute scale of 0 to ∞ (no water to pure water) by dividing mass of water by mass of dry material, or on a relative scale of 0–1 (no water to full hydration). The denominator for the relative expression is either total mass or mass of water in fully hydrated tissues, an empirically derived value that varies with species, tissue and tissue development (a.k.a. relative water content or RWC). water potential: A measure of availability of water in terms of pressure that decreases from 0 (pure water) to ∞ as water content decreases. Units are usually expressed as MPa. The related parameter, chemical potential of water, which describes the availability of water or its potential for effecting reactions in energy terms (units are usually joules), is the difference between the molar free energy of pure water at standard temperature and pressure (STP) and the water potential times the molar volume of pure water at STP (18 cm3 mol1). Chemical potential of water inversely correlates with chemical potentials of solutes, which in turn are components of the free-energy difference that drive reactions. water replacement hypothesis: A mechanism of protecting macromolecules from structural changes during dehydration by inserting hydrophilic groups of specific solutes (usually carbohydrates) on to hydrophilic sites of macromolecules. This substitution prevents aggregation of molecules by van der Waals forces and the separation prevents deleterious interactions. water sorption isotherm: A plot that describes the relationship between water content ≤ ≥ and RH (RH 93%) or water content and water potential ( w 20 MPa) for a particular material at a particular temperature. water-clustering function: A volumetric analysis of water relation for the formation of water clusters (e.g. water–water self-association). wave number (spectroscopy): The number of waves per centimetre. xeromorphic: Having morphological characteristics particularly adapted to conserving water under dry conditions. Zeeman splitting: The splitting of the energy levels of the electron/nucleus due to the interaction of its magnetic moment with the external static magnetic field. zygospore: A resting spore that results from the fusion of two gametangia in Zygomycotina (fungi).

(Terms included do not necessarily reﬂect the collective views of the authors) Dessication Taxonomic Index 3/4/02 2:30 pm Page 383

Taxonomic Index

Authorities for names are not included and synonymy is not rationalized, but can be cross-checked via the relevant chapters.

Acanthaceae Adiantaceae vegetative desiccation tolerance 222 desiccation tolerant species 218 Acer Adiantum platanoides desiccation tolerant species cell cycle 174 incisum 218 oligosaccharides 172 gametophyte cryopreservation respiration 173 tenerum 225 seed anatomy 252 trapeziforme 225 seed development 156–157 spore desiccation tolerance seed drying time 159 capillus-veneris 192 see also Norway maple Aesculus hippocastanum pseudoplatanus axis water stress 267 cell cycle 174 critical water potential and water content 50 desiccation tolerance, and seed drying seed development 159 time 159 seed dormancy 158, 245 oligosaccharides 172 water loss curve 68 respiration 173 water and seed longevity 103 seed anatomy 252 Afrotrilepis pilosa seed development 156–157 in situ/natural habitat 12, 224 seed dormancy 158 longevity when dry 225 see also sycamore sp. specialized structures/velamen 224 free-radical scavenging 174 vegetative desiccation tolerance 220 variation in desiccation tolerance 155, Agathis 244 robusta Aceraceae seed storage classification 241 angiosperm phylogeny 242 sp. recalcitrant seeds 247 orthodox and recalcitrant seeds 244–245 seed storage classification 248 seed weight 245 Acinetobacter radioresistans Aglaonema sp. survival at 31% RH 10 desiccation sensitive pollen 187 Acorales Agrostemma githago seed storage classification 247 seed development 153 Actiniopteris Alismatales desiccation tolerant species seed storage classification 247 dimorpha 218 Allium sp. radiata 218 vegetative propagules 228

383 Dessication Taxonomic Index 3/4/02 2:30 pm Page 384

384 Taxonomic Index

Aloina aloides apple ABA-induced tolerance 216 orthodox seed 252 Alternaria TBARS assay 119 desiccation tolerant spores 195 see also Malus porri 196 Aquifoliales Amaranthaceae seed storage classification 247 angiosperm phylogeny 242 Arabidopsis seed storage classification 248 sp. Amaryllis sp. aba, abi-3 and other mutants 164–165 pollen DNA repair 356 abi-3 and other gene products 163, 332 Amborellaceae alkyl hydroperoxidase 330 angiosperm phylogeny 246 AtPer1 expression 170 Amomyrtus luma desiccation intolerant mutants 25, 253, seed storage classification 241 310, 324 Anacardiaceae ‘dormancy’ gene 29 recalcitrant seeds 247 HSP 165, 309 Anacystis sp. osmosensor 371 desiccation-tolerant cysts 17 protein denaturation 169 Anamodon viticulosus transgenic plants 326, 331–332, 334–335 chlorophyll fluorescence 212 thaliana desiccation tolerance 209, 216 1H-NMR of seeds for betaine 132 photosynthesis rate 210 EST collections 30 recovery processes 214 homologues of lea cDNAs 162 Andira inermis lea genes 164 critical water potential and water content protein–sugar glass 306 50 seed mucilage 348 Andreaea seed storage stability 304 rothii trehalose synthesis 168 CO uptake 214 2 Araceae desiccation tolerance 213 desiccation sensitive pollen 9, 187 sp. seed storage classification 248 desiccation tolerance 209 Andreaeales Araucaria desiccation tolerance 209 angustifolia Anemia recalcitrant seeds 245 phyllitidis seed respiration 173 spore desiccation tolerance 192 araucana tomentosa recalcitrant seeds 245 desiccation tolerant species 220 bidwillii Anemone coronaria recalcitrant seeds 245 tuber desiccation tolerance 228 seed size and weight 246 angiosperms brownii vegetative desiccation tolerance 220 seed size 246 Annonaceae cunninghamii seed storage classification 247–248 seed weight 245 Anomodon viticulosus heterophylla dark respiration 215 seed weight 245 in situ, desiccated and hydrated 14 hunsteinii photosynthesis and water potential 229 recalcitrant seeds 245 Anthurium seed weight 246 seed storage classification 240 water and seed longevity 103 Apiaceae mirabilis seed storage classification 248 seed size 246 see also Umbelliferae Section of Araucariaceae 245–246 Apiales sp. seed storage classification 247 orthodox and recalcitrant seeds 244 Apocynaceae sphaerocarpa seed storage classification 248 seed size 246 Dessication Taxonomic Index 3/4/02 2:30 pm Page 385

Taxonomic Index 385

Araucariaceae sp. habitat and seed storage 252 desiccation sensitive pollen 187 seed desiccation sensitivity 244–245 Avicennia marina Archidium alternifolium ABA and seed development 171 spore germination 194 axis drying curve 99 Arecaceae cell vacuolation 174 habitat and seed storage 252 desiccation sensitivity 159, 268 recalcitrant seeds 247 DNA repair 174, 354–355 seed anatomy 252 respiration 172 seed storage classification 248–249, 253 seed desiccation 153 see also Palmae seed development 157 Arecales stachyose 172 number of desiccation sensitive seeded sub-cellular de-differentiation 173 species 246–247 viviparous germination 158 Artemia Aylthonia blackii desiccation tolerant cysts 281 vegetative desiccation tolerance 222 water difussion coefficient in cysts 130 Azadirachta indica Arthropteris orientalis axis drying curve 99 desiccation tolerant species 220 EPR of chilling stress 122 Artocarpus heterophyllus imbibitional damage 102 axis drying curve 99 seed storage classification 241 Asclepiadaceae variation in desiccation tolerance 157, 266 seed storage classification 248 see also neem Asparagales Azolla seed storage classification 247 spore dormancy 192 Aspergillus japonicus sporocarp storage desiccation tolerant spores 196 filiculoides 193 Aspleniaceae desiccation tolerant species 219 Asplenium Bacillus subtilis desiccation tolerant species DNA form 350 aethiopicum 219 bourgaei 219 gene product 332–333 pringlei 219 Balsaminaceae ruta-muraria 220 pollen storage life 190 rutifolium var. bipinnatum 219 Barbacenia sandersoni 219 vegetative desiccation tolerance trichomanes 220 flava 222 vegetative desiccation tolerance longifolia 222 septentrionale 220 riedeliana 222 Asteraceae sellovii 222 seed storage classification 248 Barbaceniopsis tricellular pollen 188 vegetative desiccation tolerance see also Compositae boliviensis 222 Asterales humahuagensis 222 seed storage classification 247 Barbula sp. Athyrium filix-femina temperature and survival 213 spore bank 193 barley spore storage 193 alkyl hydroperoxidase 330 Atrichum androgynum chemical shift imaging of seeds 131 partial dehydration 216 ‘dormancy’ gene 29, 349 Austrobaileyaceae gene product 332 angiosperm phylogeny 246 lea gene 25 Avena LEA proteins 24, 334 fatua PER1 protein 170 seed DNA repair 353 pulsed (spin-echo) NMR of developing see also oat seeds 130 Dessication Taxonomic Index 3/4/02 2:30 pm Page 386

386 Taxonomic Index

barley continued Brassicaceae seed development 153 seed storage classification 248 see also Hordeum species coverage 246 Bauhinia see also Cruciferae seed anatomy 252 Brassicales bean seed storage classification 247 axis water stress 267 Bromeliaceae cell contraction 270 seed storage classification 248 chilling damage 344 Bromus secalinas DSC data 306 ‘dormancy’ gene 29, 330, 349 glass formation in axes 302, Bryaceae state diagram 303, 305 induced desiccation tolerance unexplored seed imbibition damage 334 216 Beauveria Bryum desiccation tolerant spores predrying bassiana 196 caespiticium 216 brongniarti 196 capillare 216 sp. 195 pseudotriquetrum 216 bell pepper buckwheat oligosaccharides 307 fagopyritol 168 birch see also Fagopyrum esculentum DNA repair in pollen 354, 355 Bunya Blechnum spicant Section of Araucariaceae 245–246 spore storage 193 Blossfeldia liliputana vegetative desiccation tolerance 222 cabbage Boea TBARS assay 119 hygroscopica Cactaceae carbohydrates 325 seed storage classification 248 forest understorey 17 Calophyllum sp. non-xeromorphic 10 orthodox and recalcitrant seeds 244 predrying 226 Caltha palustris vegetative desiccation tolerance 222 seed storage classification 241, 244, 253 water content and survival 101 Camellia sinensis sp. seed drying curve 99 single desiccation tolerant species 217 variable seed desiccation tolerance 266 Bombacaceae see also tea seed storage classification 248 Capparaceae Boraginaceae seed storage classification 248 seed storage classification 248 Cardamine sp. Borya vegetative propagules 228 inopinata Carex physodes vegetative desiccation tolerance 221 vegetative desiccation tolerance 217, 220 nitida carrot chloroplasts 272 somatic embryos and LEA proteins 309 predrying 226 Caryophyllaceae tolerant and sensitive individuals 10 seed storage classification 248 vegetative desiccation tolerance 221 tricellular pollen 188 xeromorphic characteristics 224 Caryophyllales septentrionalis seed storage classification 247 vegetative desiccation tolerance 221 Castanea sp. sativa in situ 12 electron transport chain 173 leaf desiccation tolerance 9 HSP 166, 171, 310 Brachyachne patentiflora sp. vegetative desiccation tolerance 221 desiccation sensitive seeds 249 Brassica sp. Castaneoideae seed imbibition damage 344 subfamily phylogeny 249 Dessication Taxonomic Index 9/4/02 9:34 am Page 387

Taxonomic Index 387

Castanopsis vellea 219 orthodox and recalcitrant seeds 244, 249 wrightii 219 seed weight 249 Cheilothela chloropus Castanospermum desiccation tolerance 209 australe Chenopodiaceae axis drying curve 99 angiosperm phylogeny 242 sp. seed storage classiﬁcation 248 seed anatomy 252 tricellular pollen 188 cattail Chenopodium quinoa

pollen membrane Tm 299, 301 seed storage classification 241 Celastraceae Cibotum glaucum seed storage classification 248 gametophyte cryopreservation 225 Ceratodon purpureus Citrus predrying 216 limon spore storage 194 variable seed desiccation tolerance 266 Ceratophyllales sp. orthodox seeds 246 orthodox and recalcitrant seeds 240, 244 seed storage classification 247 see also lemon Ceratophyllum demersum Cladonia orthodox seeds 246 dark respiration Ceterach convoluta 215 desiccation tolerant species furcata 215 cordatum 220 Clusiaceae officinarum 220 seed storage classification 247–248 Chamaegigas intrepidus cocoa drying in situ 226 oil body fusion 350 leaf desiccation tolerance 9 oligosaccharide:sucrose ratio 172 morphology 10 see also Theobroma cacao natural habitat 224 Cocos nucifera vegetative desiccation tolerance 223 seed anatomy 252 Cheilanthes Coffea desiccation tolerant species arabica albomarginata 218 development and desiccation tolerance bonariensis 218 159 buchtiennii 218 effect of drying 266 capensis 218 seed water sorption 64 depauperata 218 sp. dinteri 218 compilation of desiccation sensitive seeds distans 219 240 eckloniana 218 habitat and seed storage 252 farinosa 218 seed moisture content at dispersal 252 fragillima 219 variation in desiccation tolerance 155, glauca 218 253, 266 hirta 218 water and physiological activity 52 inaequalis 218 Colletotrichum gloeosporioides integerrima 218 desiccation tolerant spores 196 lasiophylla 219 Coleochloa lendigera 219 pallidior marginata 218 vegetative desiccation tolerance 221 marlothii 218 setifera multifida 218 longevity when dry 225 myriophylla 218 vegetative desiccation tolerance 221 parviloba 218 velamen 224 paucijuga 219 Commelinales pringlei 219 seed storage classification 247 sieberi 219 Compositae sp. 10, 217 pollen germination time 188 tenuifolia 219 see also Asteraceae Dessication Taxonomic Index 3/4/02 2:30 pm Page 388

388 Taxonomic Index

Commelinaceae post-germination response 254 pollen storage life 190 tolerance of rapid desiccation 223 Convolvulaceae vegetative desiccation tolerance 60 seed storage classiﬁcation 248 Cruciferae Coprosma sp. tricellular pollen 188 orthodox and recalcitrant seeds 244 see also Brassicaceae Cordia alliodora Ctenopteris heterophylla seed storage classiﬁcation 241 desiccation tolerant species 220 Cornales cucumber

seed storage classification 247 axes CO2 production 295 Corylus avellana metabolic imbalance 169 seed storage classification 241 plasma membrane lesions 347 cotton Cucurbita lea mRNAs 171 desiccation sensitive pollen 187 LEA proteins 24 pollen proline content 190 seed imbibition damage 344 Cucurbitaceae cowpea desiccation sensitive pollen 9, 187 seed imbibition damage 344–345, 348 pollen storage life 190 Craterostigma seed storage classification 248 hirsutum Cucurbitales carbohydrates 325 seed storage classification 247 lanceolatum Cupressus macrocarpa carbohydrates 325 seed storage classification 241 monroi Cyathea vegetative desiccation tolerance 223 spore storage nanum delgadii 193 tolerance of rapid desiccation 224 spinulosa 193 vegetative desiccation tolerance 223 Cyperaceae plantagineum in situ 188 ABA 25 seed storage classification 248 carbohydrates 28, 325 vegetative desiccation tolerance 220, 243 desiccation tolerance 217 Cyperus bellis drying in situ 226 vegetative desiccation tolerance 221 gene expression 30–31, 326, 330 glass formation 27, 302 homologues to TIPs and PIPs 166 Dactylis glomerata HSP 26, 165, 307, 309 pollen storage 192 2-octulose 26 Davallia fejeensis LEA proteins 25, 162 gametophyte cryopreservation 225 lipoxygenase inhibitor 296 Davalliaceae molecular studies 321 desiccation tolerant species 220 water uptake 227–228 Dendrographa minor rehydrins 349 photosynthesis and water potential 229 RNA 323 dicotyledons tolerance of rapid desiccation 224 vegetative desiccation tolerance 222 transgenic calli 335 Dicranales vegetative desiccation tolerance 223 desiccation tolerance 209 sp. Dicranoweisia control of water loss 348 cirrata gene product 332 desiccation tolerance 209 molecular studies 322 crispula transgenic plants 332 spore storage 194 vegetative desiccation tolerance 321, 327 Dicranum elongatum wilmsii temperature and photosynthesis 213 anthocyanin levels 224, 296 Diffenbachia ascorbate peroxidase activity 296 desiccation sensitive pollen 187 desiccated and hydrated state 11 Dioscoreales folded cell walls 25 seed storage classification 247 Dessication Taxonomic Index 3/4/02 2:30 pm Page 389

Taxonomic Index 389

Diospyros sp. brachyphylla 221 orthodox and recalcitrant seeds 244 nardioides 221 Diphyscium foliosum Eragrostis ABA-induced tolerance 216 vegetative desiccation tolerance Dipsacales hispida 221 seed storage classification 247 invalida 221 Dipterocarpaceae nindensis 221 habitat and seed storage 252 paradoxa 221 recalcitrant seeds 247 post-germination response Dipterocarpus nindensis 254 seed anatomy Ericaceae alatus 252 seed storage classification 248 tuberculatus 252 Ericales variation in seed desiccation tolerance number of desiccation sensitive seeded sp. 155 species 246–247 Doryopteris Erythrina caffra desiccation tolerant species respiratory enzymes 173 concolor 219 Escherichia coli kitchingii 219 gene product 332 pedata 219 LEA-like proteins 162 triphylla 219 Euphorbiaceae Dovyalis hebecarpa seed storage classification 248 seed storage classification 241 Eurhynchium pulchellum Drymaria quercifolia vegetative desiccation tolerance 209 gametophyte cryopreservation 225 Eutacta Dryopteris Section of Araucariaceae 245–246 filix-mas Exormotheca holstii spore desiccation tolerance 192 ABA-induced vegetative desiccation paleacea tolerance 216 chlorophyll fluorescence and germination 192 spore desiccation tolerance 192 faba bean Dumortiera hirsuta seed imbibition damage 348 negative turgor pressure 57 Fabaceae dwarf French bean seed anatomy 252 seed imbibition damage 348 seed storage classification 248 see also Leguminosae Ekebergia capensis Fabales seed drying curve 99 seed storage classification 247 Elaeis guineensis Fagaceae seed storage classification 241 seed storage classification 246, 248–249, see also oilpalm 253 Encalypta species coverage 246 sp. Fagales desiccation tolerance 209 seed storage classification 247 streptocarpa (contorta) Fagoideae predrying 216 subfamily phylogeny 249 Encalyptales Fagopyrum esculentum desiccation tolerance 209 seed storage classification 241 Equisetum see also buckwheat arvense Fagus sp. spore germination 194 desiccation tolerant seeds 249 hyemale seed weight 249 spore longevity 194 Fimbristylis Eragrostiella vegetative desiccation tolerance vegetative desiccation tolerance dichotoma 221 bifaria 221 sp. 221 Dessication Taxonomic Index 3/4/02 2:30 pm Page 390

390 Taxonomic Index

Fissidens adiantoides Gymnocarpium dryopteris predrying 216 spore bank 193 Flacourtia indica Gymnosperm seed storage classification 241 desiccation tolerance 220 Fragraea fragrans pollen germination time 188 seed storage classification 241 Funaria hygrometrica ABA-induced tolerance 216 Haberlea rhodopensis carbohydrates 325 Garcinia sp. vegetative desiccation tolerance 222 orthodox and recalcitrant seeds 244 sp. Garryales desiccation tolerant species 10 seed storage classification 247 Hamamelidales Gentianaceae vegetative desiccation tolerance 243 seed storage classification 248 hazelnut Gentianales seed storage classification 241 seed storage classification 247 Geraniales Hedera helix seed storage classification 247 seed storage classification 241 Gesneriaceae Hedwigia carbohydrates 325 ciliata (albicans) desiccation tolerant species 10 predrying 216 vegetative desiccation tolerance 220, 243, sp. 321 desiccation tolerance 209 Gramineae Hedwigiales cryogenic storage of pollen 191 desiccation tolerance 209 desiccation sensitive pollen 187–188 Helminthosporium desiccation tolerance and seed oryzae 196 development 155 sp. 195 pollen germination time 188 Hippocastanaceae pollen shape 188 seed storage classification 248 tricellular pollen 188 Homalothecium lutescens vegetative desiccation tolerance 192 photosynthesis and water potential 229 see also Poaceae Hookeria lucens Grammitidaceae chlorophyll fluorescence 211 desiccation tolerant species 220 Hookeriales Grimmia desiccation sensitivity 209 apocarpa Hopea sp. in situ 13 variation in seed desiccation tolerance 155 desiccation tolerance 209 Hordeum sp. laevigata desiccation sensitive pollen 187 desiccation tolerance 213 see also barley in situ 13 Hymenophyllaceae longevity 209 desiccation tolerant species 220 survival after storage 7 Hymenophyllum pulvinata chlorophyll fluorescence 212 desiccation tolerant species tunbridgense 220 CO2 uptake 214 predrying 216 wilsonii 220 recovery of carbon fixation 230 vegetative desiccation tolerance temperature and survival 213 sanguinolentum 220 Grimmiales Hypnales desiccation tolerance 209 desiccation tolerance 209 groundnut Hypnobryales seed coat and rehydration 348 desiccation studies needed 216 Guifoylia monostylis Hypnum sp. respiratory enzymes 173 desiccation tolerance 209 Dessication Taxonomic Index 3/4/02 2:30 pm Page 391

Taxonomic Index 391

Illicales lemon angiosperm phylogeny 246 variable seed desiccation tolerance 266 Ilysanthes see also Citrus vegetative desiccation tolerance lettuce purpurascens 223 EPR imaging of seeds 126 wilmsii 223 Leucondon sciuroides Impatiens sp. desiccation tolerance 209 desiccation tolerant pollen 346 Liliaceae oligosaccharides 307 vegetative desiccation tolerance 10, 221, 243 pollen membrane phase transition 349 Liliales thermal events in seeds 136 seed storage classification 247 Indian wild rice Limosella seed storage classification 241 gradiflora see also Zizania desiccation tolerant corms 9 Inga sp. sp. seed anatomy 252 vegetative desiccation tolerance 223 Intermedia Lindernia Section of Araucariaceae 245–246 carbohydrates Iridaceae acecularis 325 seed storage classification 248 brevidens 325 Isoetaceae vegetative desiccation tolerance desiccation tolerant species 218 sp. 223, 321 Isoetes australis Litchi chinensis desiccation tolerant species 218 desiccation tolerance and seed development 159 Lobaria pulmonaria Juglans sp. high-light damage to tissue 229 compilation of desiccation sensitive seeds lucerne 240 transgenic plants 332, 334 Juncaceae Lunularia cruciata tricellular pollen 188 lunularic acid 216 Lycopsida (clubmosses) desiccation tolerant species 218 Kyllinga alba Lygodium japonicum vegetative desiccation tolerance 221 spore desiccation tolerance 192

Labiatae Magnolia sp. vegetative desiccation tolerance 223, 243 orthodox and recalcitrant seeds 244 Lamiaceae Magnoliales seed storage classification 248 orthodox and recalcitrant seeds 246–247 Lamiales maize seed storage classification 247 ABA-deficient mutants 163–164 Landolphia kirkii antioxidants and desiccation tolerance 296 development and desiccation tolerance 160 chemical shift imaging of kernels 131 seed drying curve 99 chromatin 278, 299 Lauraceae dehydrin 328 recalcitrant seeds 246 DNA repair 357 seed storage classification 248 LEA proteins 24, 308, 334 Laurales pollen DNA repair 355 number of desiccation sensitive seeded pollen proline content 190 species 246–247 pollen shape 189 Leguminosae pollen storage 192 seed anatomy 252 protein secondary structure 280 see also Fabaceae seed imbibition damage 344 Lemnaceae sugars and vitreous state 305 seed storage classification 248 see also Zea mays Dessication Taxonomic Index 3/4/02 2:30 pm Page 392

392 Taxonomic Index

Malpighiales Mnium seed storage classification 247 hornum Malus sp. recovery time 230 compilation of desiccation sensitive seeds marginatum 240 predrying 216 see also apple Mohria caffrorum Malvaceae desiccation tolerant species 220 seed storage classification 248 monocotyledons Malvales vegetative desiccation tolerance 220 number of desiccation sensitive seeded Moraceae species 246–247 seed storage classification 247–248 Mariscus capensis moth bean vegetative desiccation tolerance 221 gene product 332 Mauritia sp. mung bean compilation of desiccation sensitive seeds chemical shift imaging of seeds 131 240 1H-NMR spectra of water 77 Melastomataceae Muntingia calabura seed storage classification 248 seed storage classification 241 species coverage 246 Myrothamnaceae Meliaceae carbohydrates 325 habitat and seed storage 252 vegetative desiccation tolerance 10, 223, multiple criteria 250 321 recalcitrant seeds 247 Myrothamnus Mesembryanthemum crystallinum flabellifolius (flabellifolia) gene product 332 anthocyanin levels 227 Metarhizium carbohydrates (flabellifolia) 325 desiccation tolerant spores desiccated and hydrated state 11 anisopliae 196 desiccation tolerance 217 sp. 195 natural habitat 224 flavoviride negative turgor pressure 57 desiccation tolerant spores 195–196, 242 rehydration 227 drying rate and survival 195 trehalose (flabellifolia) 168, 324 spore imbibitional injury 197 vegetative desiccation tolerance Michelia champaca (flabellifolia) 220, 223 seed storage classification 241 moschata Micraira vegetative desiccation tolerance 223 vegetative desiccation tolerance Myrtaceae adamsii 221 seed storage classification 247–248 spinifera 221 Myrtales subulifolia 221 seed storage classification 247 tenuis 221 Microchloa vegetative desiccation tolerance caffra 221 Najas flexilis indica 221 seed desiccation tolerance 253 kunthii 221 Nanuza plicata Microdracoides squamosa vegetative desiccation tolerance 222 vegetative desiccation tolerance 221 neem Mielichhoferia elongata EPR of axis chilling stress 122 predrying 216 imbibitional stress 121, 344 Millettia sp. seed storage classification 241 seed anatomy 252 thermal events in seeds 136 Mimordica variable seed desiccation tolerance 266 desiccation sensitive pollen 187 see also Azadirachta indica Mniaceae nematodes desiccation tolerance studies needed 216 trehalose 168 Dessication Taxonomic Index 3/4/02 2:30 pm Page 393

Taxonomic Index 393

Neosartorya fischeri Orthotrichales desiccation tolerant spores 196 desiccation tolerance 209 heat resistant spores 197 Orthotrichum Nicotiana plumbaginifolia anomalum gene product 332 in situ, desiccated and hydrated 14 Norway maple desiccation tolerance 209 seed development 156 Osmunda japonica see also Acer platanoides spore proline and arginine content 193 Nostoc sp. Oxalidales desiccation-tolerant cysts 17 seed storage classification 247 water and physiological activity 52 Oxalis sp. Nothochlaena bulbils/vegetative propagules 228 seed storage classification 240 marantae vegetative desiccation tolerance 217, 219 parryi Paecilomyces temperature and photosynthesis 224 desiccation tolerant spores vegetative desiccation tolerance 219 farinosus 195–196 Nothofagus sp. fumosoroseus 196 desiccation tolerant seeds 249 Palmae seed weight 249 recalcitrant seeds 247 Nymphaea seed anatomy 252 gigantea see also Arecareae orthodox seeds 249 Pandanales sp. seed storage classification 247 angiosperm phylogeny 246 Papaveraceae Nymphaeaceae pollen shape 188 recalcitrant seeds 247 Papaver Nymphaeales dubium angiosperm phylogeny 246 pollen development 189 Nyssa aquatica rhoeas seed storage classification 241 pollen shape 189 papaya intermediate seed 268 oak Paraceterach muelleri state diagram for cotyledon 305 desiccation tolerant species 219 see also Fagaceae and Quercus pea oat axis water stress 267 gene product 332 chemical shift imaging of seeds 131 plasmalemma fusion in leaves 274 carbon dioxide production in axes 294 see also Avena fatua desiccation tolerance 280 glass formation in axes 303 oilpalm lipid peroxidation in axes 118 seed storage classification 241 metabolic imbalance 169 see also Elaeis guineensis mitochondrial activity on seed Oligotrichum hercynicum rehydration 359 spore storage 194 seed imbibition damage 344, 348 Onoclea sensibilis sHSP 309 spore desiccation tolerance 192 TBARS assay 119 Orchidaceae see also Pisum seed storage classification 248 Pellaea Oropetium desiccation tolerant species carbohydrates atropurpurea 219 thomaeum 325 boivinii 219 vegetative desiccation tolerance calomelanos 219 capense 221 falcata 219 roxburghianum 221 glabella 219 thomaeum 221 hastata 219 Dessication Taxonomic Index 3/4/02 2:30 pm Page 394

394 Taxonomic Index

Pellaea continued Plagiomnium rostratum desiccation tolerant species continued predrying 216 longimucronata 219 Plagiothecium undulatum ovata 219 predrying 216 quadripinnata 219 water potential and survival 211 rotundifolia 219 Platycerium stemaria sagittata var. cordata 219 vegetative desiccation tolerance 217, 220 ternifolia 219 Platyhypnidium rusciforme viridis 219 predrying 216 Penicillium bilaji Pleurochaete squarrosa desiccation sensitive conidia 195 chlorophyll fluorescence 212 spore storage 195 Pleurosorus rutifolius Pennisetum desiccation tolerant species 220 desiccation sensitive pollen Pleurostima sp. purpureum 188 vegetative desiccation tolerance 222 sp. 187 Pleurozium schreiberi desiccation tolerant pollen predrying 216 americanum 188 Poa bulbosa typhoides 188 vegetative desiccation tolerance 221 Pentaclethra sp. Poaceae seed anatomy 252 carbohydrates 325 Pentagramma triangularis desiccation sensitive pollen 9 forest understorey 17 desiccation tolerant species richness 10 in situ, desiccated and hydrated 13 seed storage classification 248 Petunia sp. vegetative desiccation tolerance 221, 243 pollen DNA repair 354, 356 see also Gramineae Phacelia tanacetifolia Poales 31P NMR and seed metabolism 132 seed storage classification 247 Phaseolus vulgaris Podocarpaceae ABA and seed development 171 seed desiccation sensitivity 244 NMR imaging of seeds 131 Podocarpus premature drying 161 henkelii seed development 152–153, 159 radical tip and axis drying curves 99 Phegopteris connectilis usambarensis spore bank 193 orthodox seed 244 Philonotis seriata Pohlia elongata predrying 216 predrying 216 Phycomyces blakesleeanus Polygonaceae desiccation tolerant spores 196 seed storage classification 248 Phyllisis scolopendrium Polypodiaceae spore storage 193 desiccation tolerant species 220 Pilotrichella ampullacea Polypodium recovery time on remoistening 214 desiccation tolerant species Piper hispidum cambricum 220 seed storage classification 241 vulgare 220 Piperaceae polypodioides seed storage classification 248 illuminated drying 226 Piperales specialized structures 224 orthodox seeds 246–247 vegetative desiccation tolerance 217, 220 Pisum sp. virginianum seed anatomy 252 desiccation tolerant species 220 see also pea repair processes 28 Pithecellobium sp. Polystichum setiferum seed anatomy 252 spore storage 193 Pittosporum sp. Polytrichales orthodox and recalcitrant seeds 244 desiccation tolerance 209 Dessication Taxonomic Index 9/4/02 9:35 am Page 395

Taxonomic Index 395

Polytrichum desiccation tolerance 213, 216 formosum recovery processes 215 recovery time 230 temperature and photosynthesis 213 piliferum water potential and survival 211 desiccation tolerance 209 sp. Porphyra sp. desiccation tolerance 209 large scale drying 97 survival pattern 213 Pottiales Ramalina maciformis desiccation tolerance 209 water storage and gas difussion 20 proso millet wetting and drying 18 testa colour and imbibition 348 Rammondia sp. Proteaceae desiccation tolerant species 10 seed storage classification 248 Ramonda Proteales carbohydrates seed storage classification 247 myconi 325 Pseudopezicula vegetative desiccation tolerance desiccation tolerant spores 195 myconi 223 Pteridaceae nathaliae 223 desiccation tolerant species richness 10 pyrenaica 223 Pteropsida (ferns) serbica 223 desiccation tolerant species 218 Ranunculaceae Puccinia seed storage classification 248 desiccation tolerant spores Ranunculales graminis 196 seed storage classification 247 recondita 196 Ranunculus seed priming arvensis 354 sceleratus 354 Quercus tuber desiccation tolerance robur asiaticus 228 ABA and seed development 171 vegetative propagules critical water content 66 ficaria 228 critical water potential 50 red rice cytoskeleton 174, 273 13C labelling and seed metabolism 132 matrix-bound water 53 Rhizocarpon geographicum protectant against oxidative stress 174 adaptation to climate 230 seed development and desiccation Rhizophoraceae tolerance 158–9 recalcitrant seeds 247 soluble sugars 172 Rhynchostegium riparioides volatiles and unregulated respiration 173 predrying 216 rubra Rhytidiadelphus critical water potential or water content loreus 50 chlorophyll fluorescence 211 pressure–volume curve 58 dark respiration 215 sorption isotherm of cotyledon tissue 67 recovery time 230 soluble sugars 172 sp. water hydration sites 66 predrying 216 sp. Riccia desiccation sensitive seeds 249 fluitans variation in desiccation tolerance 155 effect of ABA 216 see also oak macrocarpa gametophyte longevity 209 survival after storage 7 Racomitrium rice aciculare lea gene 25 spore storage 194 non-detection of Tg by DSC 136 lanuginosum seed chilling injury 344 chlorophyll fluorescence 212 transgenic plants 331–334 Dessication Taxonomic Index 3/4/02 2:30 pm Page 396

396 Taxonomic Index

Ricinus communis Schizophyllum commune desiccation intolerance 155 survival after storage 8 seed development 152–153 Scleropodium tourretii Rosaceae desiccation tolerance 209 seed storage classification 248 Sclerotinia sclerotinum Rosales desiccation tolerant spores 196 seed storage classification 247 Scrophulariaceae Roystonea carbohydrates 325 seed storage classification 240 seed storage classification 248 Rubiaceae vegetative desiccation tolerance 223, 243, seed storage classification 248, 253 321 Rutaceae Secale seed storage classification 247–248 cereale rye DNA repair 358 DNA repair in embryos 357–358 see also rye pollen cryogenic storage 192 sp. transcription in embryos 352 desiccation sensitive pollen 187 see also Secale cereale Selaginella desiccation tolerant species caffrorum 218 Sabal sp. convoluta 218 seed storage classification 240 digitata 218 Saccharomyces imbricata 218 cerevisiae njam-njamensis 217–218 desiccation tolerant cells 196 peruviana 218 HSP 310 pilifera 218 LEA-like proteins 162, 308 sartorii 218 mutants 196 lepidophylla see also yeast desiccation tolerant species 218 uvarum drying rate 6 desiccation tolerant cells 196 folded cell walls 60 Saccharum sp. illuminated drying 226 desiccation sensitive pollen 187 membrane organization 22 Salix sp. predrying 226 seed storage classification 240 resurrection 217 Santalales sellowii seed storage classification 247 desiccation tolerant species 218 Santalum album in situ 12 seed storage classification 241 sp. Sapindaceae desiccation tolerant species richness 10, angiosperm phylogeny 242 217 seed storage classification 248 evolution of desiccation tolerance 243 Sapindales heat tolerance 8 seed storage classification 247 photosynthesis 17, 227 Sapotaceae trehalose 324 seed storage classification 247–248 Selaginellaceae Satureja gilliesii desiccation tolerant species 218 desiccation tolerant organ/tissue 9, 223 Septoria nodorum Saxifraga sp. hydrated storage of spores 197 vegetative propagules 228 Shorea Saxifragales robusta seed storage classification 247 free-radical scavenging 174 Schistidium rivulare sp. spore storage 194 variation in seed desiccation tolerance Schizaea sp. 155 desiccation tolerant species 220 Solanaceae Schizaeaceae pollen shape 188 desiccation tolerant species 220 seed storage classification 248 Dessication Taxonomic Index 3/4/02 2:30 pm Page 397

Taxonomic Index 397

Solanales sugarbeet seed storage classification 247 transgenic plants 332–333 Sordaria sunflower desiccation tolerant spores ABA-deficient mutants 164 macrospora 196 sHSP 309 survival of cavitation 57 Swietenia sorghum seed storage classification 240 desiccation sensitive pollen 187 sycamore seed imbibition damage 344 seed development 156 soybean see also Acer pseudoplatanus antioxidant in seed membranes 296 Syntrichia glass composition in axes 305 desiccation tolerance 209 glycoproteins in seed coat 348 Syzigium guiniense LEAs in axes 308 axis drying curve 99 lipid-soluble antioxidants 169 13C NMR and seed metabolism 132 seed development and desiccation Talaromyces flavus tolerance 153 desiccation tolerant spores 196 seed imbibition 155 heat resistant spores 197 seed imbibition damage 344 Talbotia elegans water clustering in axes 66 vegetative desiccation tolerance 222 Sphagnum sp. Taxus brevifolia net photosynthesis 17 orthodox seed 252 Spondias sp. tea orthodox and recalcitrant seeds 244 axis viability loss 102 Sporobolus metabolic imbalances in seeds 280 vegetative desiccation tolerance variable seed desiccation tolerance 266 see also Camellia sinensis atrovirens 221 Telphairia occidentalis elongatus 221 viviparous germination 158 festivus 221 Theobroma cacao fimbriatus 222 ABA and seed development 171 lampranthus 222 drying curves of axes 69–71, 99 pellucidus 222 free-radical scavenging 174 sp. see also cocoa desiccation tolerance 327 Thrinax sp. gene expression 330 seed storage classification 240 molecular studies 322 Thuidium delicatulum stapfianus protein analysis 328 carbohydrates 325 Tiliaceae control of water loss 348 seed storage classification 248 desiccated and hydrated 15 Timmia austriaca EST collections 30 predrying 216 LEA proteins 307 tobacco molecular studies 321 non-detection of Tg by DSC 136 protein synthesis 329 transcription factors 326 rehydrins 349 transgenic plants 326, 331–334 repair processes 28 Todea barbara trehalose 168, 324 spore storage 193 vegetative desiccation tolerance 220 tomato xeromorphic characteristics 224 ABA-deficient mutants 163 Stagonospora convolvuli fruit shedding 252 desiccation tolerant spores 196 Tortella spore longevity 197 desiccation tolerance 209 Sterculiaceae Tortula seed storage classification 248 latifolia Streptocarpus sp. in situ, desiccated and hydrated 14 desiccation tolerant vegetative tissue 223 (ruralis subspecies) ruraliformis Dessication Taxonomic Index 9/4/02 9:35 am Page 398

398 Taxonomic Index

Tortula continued Typha (ruralis subspecies) ruraliformis continued latifolia sucrose 26 membrane permeability 346 31 CO2 uptake 214 P NMR and phospholipids 133 (syn Syntrichia) ruralis pollen imbibitional leakage and EPR ABA 216 spectra 123 cellular integrity 327 pollen storage life 191 cellular protection and dehydrins 25 sp. chlorophyll fluorescence 211–212 pollen monolayer hydration 65 chloroplast 328 dark respiration 215 desiccation and hydration in situ 15 Ulota desiccation tolerance 216 crispa drying rate 216 chlorophyll fluorescence 212 EST collections 30–31 sp. growth in dark 229 desiccation tolerance 209 leaf longevity 209 Umbelliferae metabolism and protein synthesis tricellular pollen 188 328–329 see also Apiaceae metabolism on de- and rehydration 29 Uromyces appendiculatus molecular studies 321, 327, 329–330 desiccation tolerant spores 195–196 phosphorus and potassium content and Urticaceae nitrate reductase activity 19 seed storage classification 248 predrying 216 Ustilago scitaminea rapid adaptation 230 desiccation tolerant spores 195–196 recovery time on remoistening 214–215 spore longevity 197 rehydrins 349 sucrose 27 TEM of leaf cells 16 temperature and survival 213 Vellozia sp. water potential and longevity 211 vegetative desiccation tolerance 222 sp. Velloziaceae desiccation tolerance 209 carbohydrates 325 Trichilia dregeana desiccation tolerant species 222 axis drying curve 99 desiccation tolerant species richness 10 axis viability loss 102 in situ 12 cytoskeleton 273 vegetative desiccation tolerance 243 water and seed longevity 103 Venturia inaequalis Trichoderma harzianum desiccation tolerant spores 195–196 desiccation tolerant spores 196 Vicia narbonensis Trilepis sp. ADP-glucose pyrophosphorylase activity vegetative desiccation tolerance 221 112 Trimeniaceae Vitellaria paradoxa angiosperm phylogeny 246 habitat and seed storage 252 Tripogon Vitex sp. vegetative desiccation tolerance orthodox and recalcitrant seeds 244 capillaris 222 Vochysia honurensis curvatus 222 seed storage classification 241 filiformis 222 jacquemontii 222 lolioformis 222 walnut lisboae 222 serotonin accumulation 170 minimus 222 Washingtonia sp. polyanthus 222 seed anatomy 252 spicatus 222 Welwitschia mirabilis Triticum sp. foliage desiccation tolerance 217, 220, 225 desiccation sensitive pollen 187 Welwitschiaceae see also wheat desiccation tolerant species 220 Dessication Taxonomic Index 3/4/02 2:30 pm Page 399

Taxonomic Index 399

wheat villosa amphiphile partitioning 370 carbohydrates 325 dehydrins in embryos 309 molecular studies 321 DNA repair 357 viscosa EM protein 25 anthocyanin levels 227 EPR imaging of kernels 126 ascorbate peroxidase activity 296 EPR of seed tissues 124 control of water loss 348 LEA transgene 332, 334 desiccated and hydrated state 11 NMR imaging of kernels 131 proembryo desiccation tolerance and EPR 121 yeast seed development 152 trehalose 168, 324

T2 water relaxation 129 see also Saccharomyces cerevisiae see also Triticum Wollemia nobilis desiccation tolerant seeds 245 Zea mays seed size 245 desiccation sensitive pollen 187–188 Woodsia ilvensi LEA proteins 307 desiccation tolerant species 220 see also maize Zingiber sp. desiccation sensitive pollen 187 Xerophyta Zingiberaceae humilis desiccation sensitive pollen 187–188 chloroplast 272 seed storage classification 248 pinnifolia Zingiberales velamen 224 seed storage classification 247 retinervis Zizania desiccated and hydrated state 11 aquatica scabrida seed storage classification 241

CO2 and photosynthesis 19 palustris rehydration and respiration 18 drying temperature 98 sp. imbibitional damage 102, 344 adaptation to habitat 230 post-germination response 266 desiccation tolerant species richness 217 tetrazolium test 104 vegetative desiccation tolerance 222 see also Indian wild rice squarrosa Zygodon longevity when dry 225 desiccation tolerance 209 Dessication Taxonomic Index 3/4/02 2:30 pm Page 400 Dessication Subject Index 3/4/02 2:30 pm Page 401

Subject Index

ABA see Abscisic acid Anoxia 8 ABRE see Abscisic acid Antarctica 17, 18 Abscisic acid (ABA) 24 et seq, 154 et seq, 190, Anthesis 189 194, 216, 226, 308, 309, 323 et seq, 334, Anthocyanins 18, 118, 165, 227, 265, 296, 348 356, 367 et seq, Antioxidants 167, 169, 228, 264, 294 et seq, analog 164 311, 371 mutants 25, 163 et seq, 310, 326 et seq, 335 Antisense 112 response element (ABRE) 164 Aquaporins 166 Abscission 172 Aquatic species 241, 249, 250, 253 Accelerated ageing 113 Arthropods 7 Acetaldehyde 114, 115, 169, 173, 295 Ascopore 57, 196, 197 Activity, water 50 et seq Ascorbate 296 ADP 294 Ascorbate peroxidase 296 ADP-glucose pyrophosphorylase 112 Ascorbic acid 174, 280 Arginine 190, 195, 196 Adsorption 53 Arginine decarboxylase 333 Ageing 229, 304, 311, 345, 351, 357 Aspartyl protein methyl transferase 28, 170, 351 Aldehydes 114 Aspartyl residues 28, 170, 351 Algae 4 et seq, 320 ATP 351, 358, 359 Aleurone layer 135, 163, 170, 334 Axes see Embryo Alkanes 114 Alkenes 114 Alkones 114 Alkyl hydroperoxidase 33 Basal meristems, desiccation tolerance 9 Amino acids 190, 198, 229, 277 Betaine 132 Ammonia 170 Bilayers 133 Amphipaths 280, 296, 308, 347 et seq compression 281 Amphiphiles 22 et seq, 113 et seq, 294, 296 et Boreal zone 17 seq, 311, 347, 370 Bovine serum albumin 308 antioxidant 296 et seq Broad leaved forests 249 endogenous 297, 371 Broadening agents 120 et seq -Amylase 308 Browning 113, 303 Angiosperms 7 et seq, 150 et seq, 220 et seq Brunauer-Emmet-Teller (BET) model 60 et seq Anhydrobiotes (anhydrobiosis) 116, 186, 198, Bryophytes 6 et seq, 207 et seq, 320 et seq, 368 293 et seq, 349 et seq Bulbils 228 Annuals, desiccation tolerant 10 Bulbs 228

401 Dessication Subject Index 3/4/02 2:30 pm Page 402

402 Subject Index

C3 plants 117, 213 Compaction, of molecules 273 C4 plants, desiccation tolerant 12 Compartmentation 274, 344 Calcium 168, 278, 371 Compatible solutes 190, 198, 264, 276, 294, 298, Callus 25, 26, 165, 335 301, 308, 311, 331 Calorimetry 74, 112 et seq, 305 Compensation point 17 Calvin cycle 117 Competition 20 Capillary action 227 Conidia 195, 196, 243 Carbohydrates 296, 321 et seq Conservation 240 et seq Carbon 18 Corms 9 balance 18, 20, 208, 215, 327 Cotyledons 150, 152, 166, 170, 245, 294, 347, gain 19 354, 367 loss 211, 217 Crassulacean acid metabolism 10 Carbon dioxide 169, 210, 294 Critical water activity 65 exchange 114 Critical water content 157, 303 Carnitine 298 Critical water potential 241, 268 Carotenoids 18, 23 Crustacea 207 Catalase 295 Cryopreservation 160, 225 Cavitation 12, 20 Cryoprotection 196, 197 Cell Crystallization 54, 347, 303, 311 compartmentation 129 Cuticle 217 compartments 132 Cyanobacteria 4, 115 contraction 270 Cyclitols 369 cycle 215, 352, 359 Cytokinesis 351, 353 damage 328 et seq Cytosine 351 division 122, 150, 215, 279, 352, 367 Cytoskeleton 273 enlargement 122 Cytosol 307 expansion 150, 264, 347, 352 integrity 303, 350 pH 126, 132 Damage 21 et seq, 28, 65, 151, 159 et seq, 263 et recovery 328 et seq seq, 328 et seq, 344 et seq shrinkage 271 desiccation induced 113, 263 et seq, 294 size 269 free radical 114 et seq, 321 ultrastructure 16, 22 et seq, 136 and metabolism 295 volume 112, 226, 265 et seq D’Arcy–Watt model 61 Cell walls 21 De-esterification 294 convolution 227, 270 Dehydrins see LEAs elasticity 59 Dehydroascorbic acid 296 folding 270 Dephosphorylation 322 Chalaza 252 Deserts 17 Chaparral 17 Desiccation-sensitive plants 116 Chaperonin 26, 167, 310 Desiccation tolerance 150 et seq Chemical potential 51 et seq animals 7, 207 Chlorophyll 18, 23, 165, 208 et seq, 223, 265, and bacterial infections 10 320, 333 constitutive 208 et seq, 226, 327 fluorescence 104, 116, 192, 209 et seq, 334, continuum 151, 242, 246 368 definition 4, 320 Chloroplast 16, 23 et seq, 223, 227, 271 et seq, developmental programme 9 307, 328, 368 vs. drought tolerance 5, 207, 230, 320 Chromatin 166, 308, 344, 357 and drying rates 100 Chromium oxalate 120 ecology 9, 13 et seq, 224, 320, 327 cis-Acting elements 326 environmental induction 9 Cladistics 242 evolution 10, 12, 20, 171, 208, 240 et seq, Cladogram 253 321 Classification 244 et seq genes 31, 161 et seq, 243, 321 et seq molecular data 246 geographic range 8, 10, 320 Climbers 250 and germination 9, 37 Cold tolerance 8 glasses and 303 Dessication Subject Index 9/4/02 9:32 am Page 403

Subject Index 403

habitats 8, 17, 208, 209, 217 et seq phosphate 350 heat-shock proteins 306, 309 et seq polymerase 352, 358 higher plant vs. bryophyte 321 repair 28, 174, 215, 350 et seq induced 216 et seq, 226 replication 352 injury 150 et seq, 263 et seq sequence data 242 LEAs and 24 et seq, 161 et seq, 307 et seq stability 278 level 242 synthesis 351 and longevity 368 et seq and water 350 metabolism and 5, 7 Dormancy 9, 29, 150, 173, 186 et seq, 224, 253, molecular responses 320 et seq 330, 349 morphological types 10 Drought 298 mutants 163 et seq, 310, 324 avoiders 230 and nutrient availability 10, 18 deﬁnition 5 oligosaccharides and 27, 168, 306, 368 evaders 230 physiological types 10 hardening 209, 215 and productivity 9 stress 30, 298, 335, 369 provenance and 157 Drought tolerance 4, 186, 207, 264 quantitative 268 vs. desiccation tolerance 5, 207, 230, 320 seeds 150 et seq, 239 et seq Dry matter sensitivity 239 et seq accumulation 113 taxonomic range 8 et seq, 207 et seq, 321 and cell shrinkage 271 and temperature 213, 224, 280, 327 Drying 113 et seq, 263 et seq, 294 et seq, 368 et timing 100 seq water potential 157, 368 in air 4, 94 Devonian–Mississippian 250 air movement 94 et seq Dew 13, 18, 20 boundary layer 94 et seq Dew-point depression 53 curves 68 et seq Diaspores 193 cycles 6, 9, 14, 17 et seq, 224 Dicotyledons equilibration to low humidities 7 desiccation sensitivity 249 excised axes 95 et seq desiccation tolerance 10, 321 fast 68, 208 et seq, 216 LEAs 323 ﬂash 95 et seq Dictysomes 272 free radicals 116 Dielectric relaxation 74 gene expression 369 Differential respirometer 114 in light 6 Differential scanning calorimetry 54, 74 et seq methods 96 et seq Diffusion 113 rapid 6, 19, 195, 225, 328 Diffusional correlation time 72 rate 6, 29, 68 et seq, 79, 94 et seq, 321, 327, Disaccharides 294 et seq, 300 et seq 328 Dispersal fruit 254 seed shape 98 pollen 186 seed size 98 seed 254 seeds 152 et seq DNA 350 et seq in shade 94 et seq amounts 249 silica gel 95, 152 binding proteins 299 slow 29, 195, 208 et seq, 216, 328 breaks 357, 358 and sucrose 27 conformation 299, 344, 356 in sun 94 et seq damage 52, 174 surface/volume ratio 94 et seq dehydration 350 temperature 94, 98, 157 forms 350 time 71, 96, 112, 226 free radical damage 117, 357, 359 tissues 94 et seq hypersensitive sites 359 levels 352 ligase 352 Editosome 351 mitochondrial 117, 353, 357 Electron microscopy 22 nuclear 117, 350 Electron paramagnetic resonance (EPR) 112 et nuclease 351, 352, 357 seq, 297, 304 346 Dessication Subject Index 3/4/02 2:30 pm Page 404

404 Subject Index

Electron spin resonance (ESR) 112 et seq, 297, Free energy 51, 61 304 and drying 94 Electron transport 116, 173, 294 Gibbs 50 Em Free radicals 5, 65, 114 et seq, 116 et seq, 173, gene 163 227, 265, 277, 279, 293 et seq, 351 protein 25 et seq attack 169 Embryo 150, 245, 252, 270 et seq, 299, 334, 344 desiccation tolerance 116 et seq, 293 et et seq seq, 321 axes 78, 95 et seq, 150 et seq, 166, 169, effects on cells 117 294, 295, 303, 305, 354 et seq generators 119 drying 95 et seq, 150 et seq processing 296 somatic 169, 295, 306 scavenging 174, 265, 279, 295 et seq, 311 Embryogenesis 164, 266, 278 Freezing 53, 225, 265, 270, 295, 298, 335 Endoplasmic reticulum 16, 271, 272, 278 Freezing point depression 53 et seq Endosperm 121, 135, 252 Freezing stress 48 Enthalpy 61, 74 Freezing tolerance 7, 8, 59 Entropy 61 Fructan synthase 333 Enzymes 5 Fructans 298 activities 113 Fructose 322 lability 277 phosphate 322 repair 263 Fruit structure 243, 244, 252 stabilization 169, 303 Fungal spores 115 Eocene 245 Fungi 4 Ethanol 114, 115, 125, 173, 195, 196, 295 infection 103 Ethylene 115, 131 Funiculus 152 Eudicots 247 Evolution, desiccation tolerance 10, 12, 20, 171, 208, 240 et seq, 264 Galactinol 369 Exotherm 54 Galactinol synthase 369 Expressed sequence tags (ESTs) 30 Galactopinitol 168 Extraction of metabolites 113 Galactosyl cyclitols 306 Extracts 116 Gametophytes 7 et seq, 29, 186, 209, 245, 253 Extrusion Gas analysis 209 protein 347 Gas diffusion 20 starch 347 Gases 20, 114 GCMS 114 Genes Fagopyritol 168, 369 ABA responsive 25, 163 Fatty acids 167, 294 ABI-3 326 diunsaturated 271 desiccation sensitivity 253 and free radicals 117 desiccation tolerance 31, 175, 254, 321 et polyunsaturated 188 seq, 356 spin labelled 123 Em 163 Fermentation 114, 169 enzymatic antioxidants 296 Ferns 7 et seq, 217 et seq, 320, 344 expression 157, 320 et seq, 329 et seq, 369, Ferricyanide 120 370 Fixatives 270, 347 fus3 163 Flavonoids 296, 297, 347 LEA 24, 161 et seq, 309, 326, 335, 336 Fluidizing compounds 310, 347 et seq lec1 165 Fluorescein 196 osem 163 Fluorescence spectroscopy 114 promoters 326 Fog 13 Rab2 28, 163 Forbs 10, 250 replacement 336 Forest tree species 250 Vp 163 et seq Fourier transformation 128, 161 Genetic engineering 210 Fourier transformation infra-red spectroscopy Genome ﬁdelity 356 (FTIR) 112 et seq, 134, 161, 297, 299, 301 Genomics 372 Dessication Subject Index 3/4/02 2:30 pm Page 405

Subject Index 405

Germinability 152 et seq in desiccation tolerance 165 et seq, 309 et Germination 122, 162, 253, 266, 306, 309, 353 seq and desiccation tolerance 9, 174, 367 in seeds 26, 165 et seq and drying 152 et seq transcription factor 326 precocious 154, 164 in vegetative tissues 26 and repair 28 Heat tolerance 8 test 103, 242 Helices, amphipathic 330 Germplasm preservation 30 -Helix 135, 161, 166, 277, 323 Gibberellins 164, 193 Herbs desiccation tolerant 10 biosynthesis 165, 193 Hexagonal phase 274, 346 Glass 72, 78, 135, 169, 191, 269, 281 298, 301 et Histidine kinase 371 seq, 311, 324, 351, 345 Histodifferentiation 150, 268 composition 302 Homoiochlorophylly 227, 230, 368 deﬁnition 302 Homoiohydry 217, 228, 243, 252 in desiccation tolerance 303 Hornworts 10 formation 113, 298, 301 et seq HPLC 114 and longevity 303 Human cells, desiccation tolerance 12 maltodextrin in 306 Humic substances 126 and membranes 303 Hydration 50, 52, 66, 72, 114, 155, 267 proteins in 306 levels 173, 268, 294 Hydraulic conductivity 59 and sucrose 26, 27, 303 et seq Hydraulic ﬂow 345 and sugars 27, 306 Hydrins 29, 215, 329 et seq temperature 302, 303 Hydrogen bonds 72, 135, 169, 277, 278, 298, transitions 27, 75, 136, 301 et seq 300, 301, 307, 311, 324, 349 water content 302 Hydrometer 53 Globulin 163, 166 Hydroperoxidase 29 Glucose 303 Hydroperoxides 296 Glucose-glycerol 325 Hydrophilins 162 -Glucuronidase 165 Hydrophilly 162 Glutamate 190, 196, 298, 331 Hydrostatic pressure 51 et seq, 60, 68 Glutathione 296 Hydroxyl groups 26, 167, 168, 300 Glutathione reductase 174, 295 Hygrometry 51 Glyceraldehyde-3-phosphate 322 Hysteresis 60, 105 dehydrogenase 322 Glycerol solutions 55, 195, 196 Glycine-betaine 298 Imbibition 344 et seq, 351 Glycolysis 117 Imbibitional injury (stress) 136, 191, 194, 197, Graminoids 10, 250 344 et seq Grana 16, 227, 272 phase change 346 Grasses 20, 336 temperature 344 Gravitational potential 51 et seq Iminonitroxides 126 Growth, effects of desiccation 14 Infra-red spectroscopy 74 cell 52 Inositol D-ononitol 333 Growth, g.rate 14, 19, 20 Insect larvae 7 GTP – binding protein 28 Insertional mutagenesis 335 Guanidine-HCl 298 Intermediate seeds 150, 172, 198, 241, 266 Guggenheim-Anderson-de Boer (GAB) model 60 International Plant Genetic Resources Institute et seq 241 Gymnosperms 8, 9, 321, 323 International Seed Testing Association 48 Intracellular gas 57 Invasive techniques 112 et seq Hairs 19 Ions Hardening 216 distribution 131 Headspace analysis 114 leakage 215 Heat-shock proteins 26, 167, 265, 294, 309 et NMR 131 seq, 371 sequestration 26, 308 Dessication Subject Index 3/4/02 2:30 pm Page 406

406 Subject Index

Isopleth 64 surfaces 320 Isotherm water content 49 et seq curves 51 water potential 53 hysteresis 60, 105 waxes 348 sorption 60 et seq, 79, 105, 277 Leeuwenhoek, Anthony von 6 Leucine zipper 165, 322, 326 Lichens 6 et seq, 114, 136, 320 K-segment 25, 323 Light 192, 193, 211, 224, 272 Kernel development 121 damage by 18, 226, 272 Kinetics limiting 20 non-equilibrium 70 Lipid bilayer 22 water loss 114 oxidation 65 peroxidation 117 et seq Lipid bodies 307 Lactate dehydrogenase 277, 308 Lipids 188, 193 Late embryogenesis abundant proteins see LEAs Liposomes 26 et seq, 274 et seq, 310, 346 Late Palaeozoic 245 membranes 169, 297 et seq Late Precambrian 263 Lipoxygenase 296 Leakage 18, 21, 153, 160, 191, 214, 216, 223, Liquid helium 213 297, 300 et seq, 308, 334, 344 et seq, 348 Liquid nitrogen 126, 192 damage 104, 328 Longevity 27, 297 et seq, 303 et seq, 368 et seq viability 104 oligosaccharides and 305 et seq, 368 et seq vigour 104 pollen 27 vital dyes 104 seeds 27, 241 et seq, 303 et seq Leathers 268, 280 Lyophilization 192 LEAs 24 et seq, 254, 264, 294, 322 et seq, 367 et Lysine 196, 323 seq Lysozyme 52, 72 actions 308 binding properties 308 Macromolecules 228, 273 dehydrins 24 et seq, 161 et seq, 309, 370 hydration 298 and desiccation tolerance 307 et seq integrity 321 genes 24 et seq, 161 et seq, 309, 326, 335, stabilization 294, 298, 303, 307 336 Maillard reaction 172 in glasses 306, 309 Malonyldialdehyde 117, 118 groups 25, 161 et seq, 307, 323 Maltose 301 HVA1 25, 31, 334 Mannitol 298 hydration 308 Marker molecules 371 nuclear 309 Marsh species 253 phosphorylation 309 Matric potential 53 et seq pollen 190 forces 59 proteins 24 et seq Maturation drying 150 et seq, 296, 370 RAB17 308 Megagametophyte 150 structure 25, 323 Meiosis 192 synthesis 153 et seq Membranes TAS14 308 and amphiphiles 296 et seq transcripts 24 et seq, 161 et seq conformation 346 Leaves convolution 227 carbohydrates in 325 damage to 5, 21 et seq, 104, 155 et seq, 173, curling 226 328 cuticle 348 disruption 59, 155 et seq desiccation tolerance 9 et seq, 18, 320 et dynamics 133 seq effects of water loss 271 et seq drying 101, 223 fatty acid domains 273 growth 320 ﬂuidity 124, 134, 191, 279 hairs 348 folding 271 et seq LEAs in 307 fusion 168, 303, 311 rolling 265 hydration 73 Dessication Subject Index 3/4/02 2:30 pm Page 407

Subject Index 407

integrity 121, 160, 169, 214, 228, 276, 293, Mucilage 348 310, 346 et seq Multigenic traits 336 isolated 124 Mutants 25, 112, 163 et seq, 196, 253, 310, 324 liposomal 26 et seq nuclear 227 Myo-inositol O-methyltransferase 333 packing 273 partitioning into 113, 296 et seq phase 265, 274 et seq, 299 et seq Nematodes 7, 168, 207 phospholipid 133, 167 Neoteny 9 physical properties 123, 347 Nitrate 192 plastid 227 Nitrate reductase 19 preservation 298 Nitroxide 120 et seq protection 26 et seq NMR spectroscopy 74 et seq rehydration 274 Non-invasive techniques 112 et seq repair 155, 167 Nuclear magnetic resonance (NMR) 112 et seq rigidity 274, 297 Nucleases 351, 352, 357 sarcoplasmic reticulum 168 Nucleic acids stability 160, 168 dehydration 278 stucture 303 hydration 52 and sugars 168 integrity 321 tearing 166 synthesis 150 tonoplast 227 and water 350 transition temperature 169, 195, 299 Nucleolus 170, 309 vesicles 28, 168 Nucleus 16, 170, 307, 309 Metabolic activities Nutrient availability 10, 18 and drying 112 Nutrient capture 208 and water 114 Metabolism, regulation 294 et seq Metabolites 2-Octulose 26, 28, 324, 325 ﬂux 115 Oil NMR 131 bodies 124, 130, 349 seeds 115 NMR signals 128 Microsomes 296 in seeds 115 et seq, 242 Microtubules 215 Oleosins 349 Minimum critical volume 270 Oligosaccharides 27, 118, 167, 170, 189, 294 et Mitochondria 16, 116, 161, 189, 227, 271, 273, seq, 303, 368 et seq 279, 296, 297, 344 and desiccation tolerance 306, 368 et seq dehydrogenases 359 and longevity 305 et seq, 368 DNA 117, 353, 357 Ordovician 243 genome 357 Ornithine 333 Mitotic division 150, 264 Orthodox seeds see Seeds Modulus of elasticity 55, 59 Osmole 54 Moist forests 17 Osmolytes 298, 331 Moist tropics 250 Osmometer 54 Moisture content (MC) 158, 197, 241 et seq Osmosensor 371 Molecular marker analysis 331 Osmotic potential 52 et seq, 68 et seq, 264 Molecular movement (mobility) 27, 72, 191, Ovule 186, 252 229, 269, 280, 302, 306 351 Oxidation 5 and ageing 304 Oxidative stress 30, 114, 116, 192, 296 Molecular spin probes 78 damage 293, 297, 311 Monocotyledons Oxygen desiccation sensitivity 246, 249 availability 169 desiccation tolerance 10, 18, 321 exchange 114 LEAs 323 protection against 198, 333 Monosaccharides 172, 303 reactive species (ROS) 116, 170, 265, 279, Mosses 29 et seq, 207 et seq, 319 et seq, 344 et 294 seq scavenging 333 Dessication Subject Index 3/4/02 2:30 pm Page 408

408 Subject Index

Oxygen continued phase transitions 346 solubility 113 rehydration stress 346 tolerance of low 8 Plasmadesmata 21, 270 et seq uptake 5, 211, 294 Plasmalemma 129, 270 et seq, 281 Plasmolysis 56, 209, 270 Plastids 271, 279, 344 Paramagnetism 120 Plastoglobuli 23, 271 Partitioning Pleiotropic effects 112 of amphiphiles, amphipaths 22 et seq, 294 Poikilochlorophylly 18, 23, 104, 208, 227, 230, et seq, 311, 347 368 within cells 120 Poikilohydry 320 into membranes 113, 280, 371 Polar lipids, effects of drying 273 Pathogens 229, 344 Pollen 7 et seq, 20, 22, 24, 27, 112 et seq, 133, Pectic substances 152 150, 186 et seq, 297 et seq, 344 et seq, Perennating structures 9 354 et seq, 368 Perennials, desiccation tolerant 10 ABA 190 Permafrost 194 ageing 113, 187 Permanent wilting point 270 amino acids 190 Permo-Carboniferous 245 bicellular 188 Peroxidases 295 compatible solutes 190 Peroxidation 189, 198, 279, 294 dispersal 186 Peroxide 295 dormancy 186 Peroxiredoxin 170, 296 germination 186 et seq pH 126, 132 glasses 191 Phase transition 22, 134, 167, 274 et seq, 280, hydration 186 281, 297, 299 et seq, 346 et seq LEAs 190 and sugars 300 longevity 186 Phenolics 296, 297 mitochondria 272 Phenols 113 molecular mobility 72 Phloem 323 recalcitrant 187 Phosphatase 322, 326 shape 188 Phosphatidylcholine 299 sperm cells 186 et seq, 355 Phosphofructokinase 168, 301 storage 191, 198 Phosphoglycerate 322 sucrose 189 Phospholipase D 322, 326 tricellular 188 Phospholipid 123, 167, 188, 271 et seq, 294 tube 186 et seq bilayers 133, 293, 298, 300, 346 viability 186 et seq, 347 composition 349 vigour 347 hexagonal phase 133 water in 48, 65 and sugars 281, 300 et seq, 307 Pollination 186 vesicles 169, 188 Pollinia 191 see also Polar lipids Polyamines 333 Phospholipid:sterol ratio 270 Polyethylene glycol solutions 55, 104, 295, 344 Phosphorus 19 Poly-L-lysine 305 Phosphorylation 309, 322 Polyols 296, 298 Photo-oxidation 5, 264 Polypeptides 277 Photoprotection 227 Polyphosphates 132 Photosynthesis 5, 17 et seq, 29, 52, 114, 209 et Poly(ribo)somes 161, 272, 278, 327 seq, 268, 327, 333 Polyubiquitin 29, 330, 349 Photosystem II 29, 104, 116, 194, 211 et seq, Potassium 19 226, 272, 322 Preferential exclusion 298 Phytochrome 192 Pressure chamber 53 Pioneers 224 Pressure–volume analysis 49 et seq, 106 Plasma membrane 21 et seq, 153, 189, 196, 226, Priming 353 et seq 270 et seq, 327, 345 et seq Productivity folding 347 crop 333 permeability 122, 346 and desiccation tolerance 9, 20 Dessication Subject Index 3/4/02 2:30 pm Page 409

Subject Index 409

Proembryonic cells 121 Putrescine 333 Proline 190, 195, 331, 333 Pyrolline carboxylate 331 Promoter analysis 326 Pyrolline carboxylate synthetase 331 Promoters 326 Proteases 167, 351 Protectants 165, 228, 264 et seq, 282, 294, 303 Quantitative trait locus (QTL) 336 Protection 268, 321, 322, 327 et seq, 347 et seq, Quasi elastic neutron scattering 75 368 et seq Quiescence 150, 245, 369 Protein 24 et seq Quinones 296 ABI 165, 322 bodies 161, 307 channel 166 Rab2 protein 28 conformation 113, 135, 277, 301, 303, 322 RAB17 163 denatured 167, 169, 311, 320 Rachis 152 desiccation related 161 Rafﬁnose 27, 167, 168, 172, 300 et seq, 369 early light inducible 322 Rain 13, 20 elF1 322 Raman spectroscopy 74 Em 25 et seq Random coil 135 extraction 113 Recalcitrant seeds see Seeds extrusion 347 Recovery 228, 328 et seq and free radicals 117 Regeneration niche 250 folding 293, 298 Rehydration 17 et seq, 22 et seq, 28, 113, 153, FUS3 165 166, 169, 209 et seq, 227, 242, 269 et heat shock 26, 167, 265, 294, 309 et seq, seq, 294, 321 et seq, 344 et seq, 368 et 371 seq heat stable 190, 194, 198 damage 344 et seq hydration 52, 72, 73, 276, 349 Rehydrins 29, 215, 329 et seq, 349 hydrins 29, 329 et seq Relative humidity 53 et seq, 188, 216, 266 et integrity 321 seq, 320 et seq interaction with sugars 135, 301, 311 air 4, 5 kinase 322, 326 and drying 97 et seq labile 293 equilibrium 51 et seq, 190, 225, 252, 265 LEC1 165 tolerance of 5 L-isoaspartyl residues 28 Relative water content (RWC) 49 et seq, 106, LEA see LEAs 207 et seq, 225 major intrinsic (MIPs) 166, 322 Relaxation times 127 myb 322, 326 Repair 5, 21, 28 et seq, 151, 173, 215, 229, 263 phosphatase 326 et seq, 294, 321, 327, 350 et seq preservation 298 Reserve deposition 150, 166, 172 protective 151 Reserve mobilization 369 Rab2, 17, 28, 29, 163 Respiration 19, 52, 114, 150, 173, 209 et seq, rehydrins 24, 29, 215, 329 et seq, 349 268, 279, 294, 298, 351 repair 28, 167 Resurrection plants 48, 114, 162, 166, 170, 217, secondary structure 135, 277, 301 225, 226, 244, 268, 281 stabilization 298, 333 drying 101, 296 et seq, 320 et seq, 344 et storage 164, 165, 193 seq, 367 et seq synthesis 20, 150, 154, 161, 189, 213, 215, Retrotransposons 335 272, 278, 327 et seq, 351 Ribonucleoprotein Vp1 322 messenger (mRNP) 29, 320 et seq, 330 Proteomics 175, 372 RNA (mRNA) 329, 330, 351 Proteosome 349 Rock pools 226 Prothalli 192 Root pressure 227 Proton exchanges 277 Roots 163, 264, 331 Protonema(ata) 193, 194, 209, 216 Rosette plants, desiccation tolerant 10 Protoplasts 271 Rotifers 7, 207 Psychrometer 53 Rubbers 269, 280 Pteridophytes 9 et seq, 217 et seq Rutin 297 Dessication Subject Index 3/4/02 2:30 pm Page 410

410 Subject Index

Salinity stress (see salt stress) 25, 48 water in 48 et seq, 151 et seq Salinity tolerance 8, 334 water loss 151 Salt solutions 53, 55, 97 viability 27, 28, 66, 102, 241, 278 Salt stress 25, 333, 335 weight 249 Savannah 252 Selection pressure 159, 253 Scales 19, 224 Self incompatibility 186 Scutellum 367 Semi-arid grasslands 18 Seed ferns 250 Senescence 152 Seedling 254, 367 Serine residues 25, 308, 323 Seeds 7 et seq, 112 et seq, 149 et seq, 239 et seq, Serotonin 170 320, 344 et seq, 367 et seq Shade plants 17 ageing 113 Shedding 155, 157, 172 banks 241, 253 -Sheet 135 chromosomal aberrations 350 Signalling pathways 326, 334, 369 et seq coat (see testa) 22, 347 et seq Silica gel 95, 152, 225 colour 348 Silurian 243, 263 desiccation sensitive 239 et seq Solutes 53 desiccation tolerance 150 et seq, 239 et seq leakage 160, 223, 297 development 24, 31, 122, 123, 150 et seq, Sorbitol 298 334, 350 Sorption 269 dormant 150, 253, 330, 349 Sorption properties 242 dry weight 151 Sorption sites 129, 167 drying 152 et seq Spectroscopy 119 et seq expansion 166 Sperm cells 186, 355 ﬁlling 112 Spin labels 120 et seq free radical damage 117 Spin probes 22, 120 et seq fresh weight 151 Spin trapping 126 gene expression 369, 370 Spores 4, 7 et seq, 150, 186 et seq germinating 115, 132 dispersal 186 germination 152, 253 fungal 115 imbibition 166, 344 et seq, 351 Sporocarp 193 imbibitional injury 136, 191, 194, 197, 344 Sporophytes 7 et seq, 253 et seq Stachyose 27, 167, 172, 300 et seq intermediate 150, 172, 241, 266 synthase 369 longevity 27, 241 et seq Starch extrusion 347 maturation 24, 115, 150 et seq, 253, 266, Steroids 123 294, 295, 307, 350, 369 Stigma 186 maturity 155, 254 Stomata 208, 217, 320 metabolites 115 Storage 155, 242, 297 et seq moisture content 158, 241 et seq, 250, 252 Stress 264 et seq, 294 non-endospermic 252 chilling 348 oils 115 et seq, 135 drought 30, 298, 335 orthodox 9, 24, 52, 65, 95 et seq, 150 et seq, duration 102, 103 172, 240 et seq, 266 et seq, 344 et seq, freezing 298 368 et seq imbibition 344 et seq production 30 intensity 102, 103 recalcitrant 9, 50, 53, 66, 95 et seq, 105, mechanical 70, 346 et seq 151 et seq, 172, 240 et seq, 266 et seq, multiple 321 294 et seq, 344 et seq, 367 et seq osmotic 229, 298, 333 reserves 157 oxidative 30, 114, 116, 192, 296 size 98, 244, 250 physico-chemical 70 shape 98, 250 salt 25, 333, 335 storage behaviour 155 et seq, 241, 250 water 25, 154, 162, 167, 172, 226, 266 et structure 243 seq sugars 115 Stress strain 69, 266, 347 tree 9 Stress tolerance 30 volume 345 Stroma 227 Dessication Subject Index 3/4/02 2:30 pm Page 411

Subject Index 411

Succulents, desiccation tolerant 10 water uptake 348 Sucrose 298, 324 et seq, 368 et seq waxy 348 alcohols 229 Tetrazolium test distribution 131 fungi 104 glass formation 26 et seq, 302 et seq viability 104 and membranes 299 Thiobarbituric acid 117, 118 in mosses 26 et seq, 328 Thylakoids 23, 116, 227 phosphate 322 Tocopherol 167, 174, 280, 296 phosphate synthase 322 Tonoplast 129, 132, 272 in pollen 189 Toxin 321 protection 164, 167, 227, 328 Transcription 351 in seeds 26 et seq activator 163, 322, 335 synthase 322, 323 factors 165, 326, 334, 336 Sugars 20, 24, 26 et seq, 115, 118, 168 et seq, regulators 322 294, 335, 368 et seq Transduction see Signalling pathways alcohols 229 Transgenic plants 112, 330 et seq hydrophilic 27 Transgenic studies 25, 31, 298, 330 et seq hydroxyl groups 27 Translation 351 interaction with protein 135, 301, 311 Translation factor 322 and membranes 168, 276 Transpiration 320 phosphates 322 Transposon tagging 31, 335 and phospholipids 281, 300 et seq Tree seeds 9 protective 151, 276, 277, 297 Trehalase 168 Sulphuric acid 225 Trehalose 13, 26, 115, 133, 168, 195, 197, 298 et Superoxide 295 seq, 310, 324 et seq Superoxide dismutase 174, 295, 334 stabilization by 333 Syrups 269 Trehalose phosphate phosphatase 168, 331 Trehalose phosphate synthetase 31 Triacylglycerol 118 Tannins 348 Tropics 17, 252 Tardigrades 7, 207 Tubers 228 t-DNA 335 Tundra 17 Teliospores 196 Turgor 49, 55, 106, 226, 267 et seq, 321, 371 Telomeres, telomerase 357, 359 pressure 55 et seq, 264 et seq Temperature and desiccation tolerance 213, 224, 225, 327 Ubiquitin 25, 167, 334, 349 evaporation rate 62 see also Polyubiquitin extremes 265 UDP-glucose 322 and free energy difference 94 Ultrastructure 153 and glasses 113 Umbelliferose 169 and injury 344 Urea 298 and longevity 241 Urediniospores 196 monolayer hydration 65 Uridine 351 and survival time 213 UV radiation 119, 354 et seq transition 169, 195, 297, 300, 302 et seq, UV-B tolerance 8 346 et seq TEMPO 123 TEMPONE 121 et seq Vacuoles 21, 227 Testa 252, 347 et seq and cell shrinkage 271 amphiphiles 349 Van der Waals interactions 135, 273 glycoproteins 348 Van’t Hoff relationship 63 et seq and imbibitional injury 348 Vascular bundle 129, 323 leakage 348 Vascular factors 153 lignin polymers 348 Vascular separation 171, 268 phenolics 348 Vascular system 227 pigmentation 348 Vegetative tissues 207 et seq, 272, 320 et seq Dessication Subject Index 3/4/02 2:30 pm Page 412

412 Subject Index

Vesicles 168 matrix bound 160 Vesicular trafficking 28 molecular interactions 76, 79 Viability 161, 186 et seq, 241, 278, 303, 353 monolayer 61, 65 Vicilin 166 multimolecular clusters 62 Viscosity 124, 280, 281, 301 et seq non-freezable 73, 151, 160 cytoplasmic 78, 113, 159, 165 et seq osmotic potential 52 et seq, 68, 264 Vitrification see Glass osmotically inactive 73 Vivipary 154, 158, 164, 241, 245, 253 partial molar volume 51 Volatiles 114, 173 potential 49 et seq, 97, 157, 208 et seq, 241 et seq, 267 et seq, 344, 368 et seq rate of loss 28 Water status 48 et seq,79 activity 50 et seq, 65 storage 20 apoplastic 55 et seq, 68, 73, 106 stress 25, 154, 162, 226, 266 et seq binding 25, 59 et seq, 65, 67 strongly bound 128 bulk 73, 126 et seq, 130, 186, 190,298 symplastic 55 et seq, 68, 73, 106 chemical potential 51 et seq transfer routes 131 clustering 65 et seq vapour 18 compartments 130 vapour pressure 51 et seq, 94 et seq concentration 105 wet weight basis 48 et seq, 105 et seq conservation 4 Water channel proteins 166, 323 content 48 et seq, 68, 99, 105 et seq, 112 et Water relations 55 et seq seq, 128 et seq, 151 et seq, 213, 266 et Water replacement hypothesis 12, 276, 280, 299 seq , 345 et seq diffusion 94 Woody plants 250 dissociation 63 distribution 130 dry weight basis 48 et seq, 105 et seq Xanthophyll 18, 265 equilibrium water content 48 Xeromorphs, desiccation tolerant 10, 224, 226 exchange 73 X-ray diffraction 75 fractions 130 Xylem cavitation 12, 20 freezable 73, 151, 160 Xylem potential 53 gradient 113, Xylem refilling 227 gravitational potential 51 hydration levels 74 immobilized 73 intercellular 48, 57 et seq Zeaxanthin 18, 227 and life 4, 48 et seq Zeeman splitting 120, 127 loss 114, 208, 264 et seq Zygospores 196 matric potential 53 et seq Zygote 156