Characterization of Cold and Short Day Acclimation in Grape Genotypes of Contrasting

Home , Sugar Act

Freezing Tolerance

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy

in the Graduate School of The Ohio State University

Trudi Nadia Lavinia Grant

Graduate Program in Horticulture and Crop Science

The Ohio State University

2012

Dissertation Committee:

Imed Dami, Advisor

Rebecca Lamb

Jim Metzger

Ray Miller

Eric Stockinger

Copyrighted by

Trudi Nadia Lavinia Grant

2012

Abstract

Grapes are temperate crops, frequently damaged by freezing temperatures. The economic losses that result from freezing injury are major problems for grape and wine industries in cold regions. Our central hypothesis is that soluble sugars in grape tissues provides increased freezing tolerance (FT) and the larger sugar, raffinose, accumulates earlier and to higher amounts in cold-tolerant than in cold-sensitive cultivars in response to cold temperature and short-day (SD) photoperiod. The central objective of this dissertation was to characterize the morphological, physiological and biochemical changes in response to cold acclimation (CA) and SD photoperiod. The specific objectives were to: 1) characterize the morphological, physiological and biochemical changes induced by low temperature in cold-tolerant and cold-sensitive Vitis species under controlled conditions; 2) characterize the morphological, physiological and biochemical changes induced by SD photoperiod in cold-tolerant and cold-sensitive Vitis species under controlled conditions and 3) characterize the morphological, physiological and biochemical changes induced by low temperature and SD photoperiod in cold- tolerant and cold-sensitive Vitis species under field conditions.

In response to temperature under controlled environs, grape (Vitis spp) cultivars

‘Frontenac’, ‘Couderc 3309’, ‘Concord’, ‘Cabernet Franc’, ‘Traminette’ and ‘Seyval’ were evaluated. Shoot growth slowed under cold temperature regimes in all cultivars except ‘Concord’. Under the non-acclimating temperature regime, raffinose

ii concentrations were low and similar among cultivars, whereas under CA temperature regimes raffinose accumulation was generally higher and cold-tolerant cultivars accumulated higher concentrations than did cold-sensitive cultivars. Basal leaves and buds accumulated the most raffinose.

In evaluating responses to SD, experiments were conducted in greenhouse using

‘Cabernet Franc’, ‘Couderc 3309’ and ‘Concord’ cultivars. Shoot growth slowed under

SD photoperiod in all cultivars. There was also increased periderm formation, endodormancy induction and increased FT. ‘Concord’ was the first cultivar to initiate these changes in response to SD followed by ‘Couderc-3309’ then ‘Cabernet franc’.

Under LD, raffinose concentrations were low and similar among cultivars, whereas under

SD, raffinose accumulation was higher and cold-tolerant cultivars ‘Couderc-3309’ and

‘Concord’ accumulated higher concentrations than the cold-sensitive cultivar ‘Cabernet franc’.

In the field study we evaluated ‘Couderc 3309’, ‘Concord’ and ‘Cabernet Franc’ and characterized changes in FT and soluble sugar concentrations during the dormant season for two years. Generally basal buds were more FT than middle buds, which were more tolerant than apical buds. The cold-tolerant cultivars ‘Couderc 3309’ and ‘Concord’ had the lowest LT50 compared to the cold-sensitive ‘Cabernet Franc’. There was variation in leaf sugar concentration but leaf raffinose content showed cultivar dependent response associated with early acclimation. Cold-tolerant cultivars showed early responses to SD before low temperatures. In buds, among all sugars, fructose, glucose, sucrose, raffinose and stachyose concentrations had strong correlations with LT50. Basal buds accumulated the most raffinose. Raffinose accumulation was also two to three times

iii higher in the cold-tolerant cultivars than in cold-sensitive. These results suggest that raffinose might be an early step in CA that coincides with early development of FT and can be used as a screening tool for breeding cold-tolerant genotypes and a target for improving FT via gene transfer.

Dedication

This dissertation is dedicated to my husband, Horatio Grant and

my daughter, Breanne Grant

Acknowledgments

First I must thank all my advisory committee members, Drs. Imed Dami, Rebecca

Lamb, Jim Metzger, Ray Miller and Eric Stockinger, they have provided collaboration in designing experiments, interpreting data and also valued suggestions for this dissertation and publication.

I would also like to thank my lab mates Miss Patricia Chalfant and Mr. Y. Zhang for assistance in collecting samples and data for my experiments. I am grateful to Dr. L.

Phelan for his help with the GC machine; he has provided invaluable information that has helped me in completing this dissertation. I also thank Dr Taehyun Ji, Mr. David

Scurlock, Mr. Bruce Williams and Miss Kesia Hartzler for helping with lab techniques, managing greenhouse plants and/or managing the vineyard in Wooster.

I am thankful for the support from many undergraduate interns who helped with my experiments and I also appreciate all the help that I have received from the HCS faculty, staff and students as well as other facility staff at OARDC. This dissertation was also partially funded by SEEDS, a competitive grants program from The OARDC and the

Lonz foundation.

I am very grateful for the support my husband Horatio Grant has provided, his love has encouraged me to be the best I can be. I will forever be indebted to my daughter

Breanne Grant for the joy and inspiration she gave me as I completed my PhD.

Vita

1998 ...... B.S. Biological Sciences, Northern

Caribbean University

2007...... M.S. Biological Sciences Walla Walla

College

2007 to present ...... Graduate Research Associate, Department

Horticulture and Crop Science, The Ohio

State University

Publications

Grant, T. N., Dami, I.E., Ji, T., Scurlock, D., and Streeter, J. 2009. Leaf raffinose is an early cold acclimation response in various Vitis genotypes. Can. J. Plant Sci. 89:961-968.

Fields of Study

Major Field: Horticulture and Crop Science

vii

Table of Contents

Abstract ...... ii

Dedication ...... v

Acknowledgments...... vi

Vita ...... vii

Publications ...... vii

Fields of Study ...... vii

Table of Contents ...... viii

List of Tables ...... x

List of Figures ...... xiii

Chapter 1: Introduction ...... 1

Chapter 2: Variation in leaf and bud soluble sugar concentration among Vitis genotypes grown under two temperature regimes ...... 23

Chapter3: Responses of greenhouse-grown Vitis genotypes to photoperiod regimes ...... 45

Chapter 4: Seasonal changes of freezing tolerance and soluble sugars among field-grown

Vitis genotypes ...... 88

viii Chapter 5 Cold and short-day acclimation and how they affect grape genotypes of contrasting freezing tolerance ...... 133

Bibliography ...... 140

Appendix A: GC Chromatograms ...... 148

Appendix B: LTE of grapevine Basal buds ...... 151

List of Tables

Table 2.1. Soluble sugar concentration (mg g-1 dry wt) in leaves of non-acclimated and acclimated Vitis vinifera, ‘Cabernet franc’, V. labruscana ‘Concord’, V. spp, ‘Frontenac’,

‘Traminette’ and ‘Seyval’ and V.riparia x V. rupestris ‘C-3309’ grapevines …………39

Table 2.2. Soluble sugar concentration (mg g-1 dry wt) in leaves and buds of non- acclimated and acclimated cold sensitive Vitis vinifera, ‘Cabernet Franc’ and cold hardy

V. riparia x V. rupestris, ‘C-3309’grapevines…………………………………………...40

Table 2.3. Soluble sugar concentration (mg g-1 dry wt) in leaves of acclimated and non- acclimated Vitis vinifera, ‘Cabernet Franc’ at different stages of development z………..41

Table 3.1. Shoot length, number of nodes and periderm formation for Cabernet franc

(CF), Couderc 3309 (C-3309) and Concord (CD) grapevines grown under long day (LD) and short day (SD) photoperiod regimes………………………………………………...70

Table 3.2. Number of days to 50% budburst (D50BB) and percent budburst (%BB) at 30 days for Cabernet franc (CF), Couderc 3309 (C-3309) and Concord (CD) grapevines grown under long day (LD) and short day (SD) photoperiod regimes…………………..71 x

Table 3.3. Freezing tolerance (LT50 in oC) of Couderc-3309 (C-3309), Cabernet franc

(CF) and Concord (CD) of basal buds after exposure to long day (LD and short day (SD) photoperiod at 4, 6 and 8 weeks………………………………………………………….72

Table 3.4. Freezing tolerance (LT50 in oC) of Couderc-3309 (C-3309), Cabernet franc

(CF) and Concord (CD) of basal buds after exposure to long day (LD and short day (SD) photoperiod at 4, 6 and 8 weeks………………………………………………………….73

Table 3.5. Soluble sugar concentration (mg g-1 dry wt) in leaves of Cabernet franc (CF),

Concord (CD) and Couderc 3309 (C-3309) grapevines exposed to 4, six and 8 weeks long day (LD) and short day (SD) photoperiod regimes………………………………...74

Table 3.6. Soluble sugar concentration (mg g-1 dry wt) in buds of Cabernet franc (CF),

Concord (CD) and Couderc 3309 (C-3309) grapevines exposed to 4, 6 and 8 weeks long day (LD) and short day (SD) photoperiod regimes……………………………………...77

Table 4.1.Seasonal changes of FT (LT50, oC) in relation to cultivar and bud position during two seasons……………………………………………………………………...112

Table 4.2. Seasonal changes of soluble sugar concentration (mg g-1 dry wt) in basal leaves of Cabernet franc (CF), Concord (CD) and Couderc 3309 (C-3309) grapevines during early acclimation in 2008……………………………………………………….113

Table 4.3. Seasonal changes of soluble sugar concentration (mg g-1 dry wt) in apical, middle and basal buds of Cabernet franc (CF), Concord (CD) and Couderc 3309 (C-

3309) grapevines……………………………………………………………………….114

Table 4.4. Correlation coefficients (R-values) of the relationship between LT50 and soluble sugar concentration in Cabernet franc (CF), Couderc 3309 (C-3309) and Concord

(CD) grapevine buds……………………………………………………………………121

Table 4.5. Freezing tolerance (LT50, oC) of field acclimated and artificially de- acclimated buds in Cabernet franc (CF), Couderc 3309 (C-3309) and Concord (CD) grapevines………………………………………………………………………………122

Table 4.6. Soluble sugar concentration (mg g-1 dry wt) of field acclimated and artificially de-acclimated basal buds for Cabernet franc (CF), Couderc 3309 (C-3309) and Concord

(CD) grapevines collected January 27, 2010…………………………………………...123

xii

List of Figures

Figure 1.1. Key enzymes involved in raffinose synthesis and degradation……………..22

Figure 2.1. Shoot length growth of grape cultivars, Vitis vinifera, ‘Cabernet Franc’ (cold sensitive), V. spp, ‘Frontenac’, and V. labruscana, ‘Concord’ (cold-hardy) exposed to no- acclimation (A) and acclimation (B) temperature regimes during two-week period.

Measurements are means ± SE (n = 4)…………………………………………………..42

Figure 2.2. Shoot length growth of grape cultivars, Vitis spp, ‘Traminette’ and ‘Seyval’ (cold –intermediate) and V. riparia x V. rupestris, ‘C-3309’ (cold-hardy) exposed to no- acclimation (A) and acclimation (B) temperature regimes during two-week period. Measurements are means ± SE (n = 5)…………………………………………………..43

Figure 2.3. Raffinose concentrations in leaf (A) and bud (B) tissues in relation to node position of non-acclimated and acclimated cold sensitive Vitis vinifera, ‘Cabernet Franc’ and cold hardy V. riparia x V. rupestris, ‘C-3309’ vines. Values are means ± SE (n = 5). Different letters indicate significant differences of raffinose in leaf and bud tissues by node position at p < 0.05…………………………………………………………………44

Figure 3.1. Shoot length progression of Cabernet franc (CF), Couderc 3309 (C-3309) and

Concord (CD) grapevines exposed to long day (LD) and short day (SD) photoperiod regimes during an eight-week period in 2010. Measurements are means (n = 4)………80

xiii

Figure 3.2. Number of nodes of Cabernet franc (CF), Couderc 3309 (C-3309) and

Concord (CD) grapevines exposed to long day (LD) and short day (SD) photoperiod regimes during an eight-week period in 2010. Measurements are means (n = 4)………81

Figure 3.3. Percent budburst for Cabernet franc (CF), Couderc 3309 (C-3309) and

Concord (CD) grapevines grown under long day (LD) and short day (SD) photoperiod regimes in 2010. Measurements are means (n = 4)……………………………………...82

Figure 3.4. Freezing tolerance (LT50) of Couderc-3309 (C-3309), Cabernet franc (CF) and Concord (CD) grapevines of basal buds after exposure to long day (LD) and short day (SD) photoperiod at 4, 6 and 8 weeks in 2010. Measurements are means +/- standard error (n = 4)………………………………………………………………………………83

Figure 3.5. Raffinose concentrations of Couderc-3309 (C-3309), Cabernet franc (CF) and

Concord (CD) grapevines basal leaves after exposure to long day (LD and short day (SD) photoperiod at 4, 6 and 8 weeks in 2010. Measurements are means +/- standard error (n =

4)…………………………………………………………………………………………84

Figure 3.6. Relationship of bud FT and leaf raffinose concentrations of Couderc-3309 (C-

3309), Cabernet franc (CF) and Concord (CD) grapevines basal leaves after exposure to long day (LD and short day (SD) photoperiod at (A) 4, (B) 6, and (C) 8 weeks………85

xiv Figure 3.7. Relationship of bud FT and bud raffinose concentrations of Couderc-3309 (C-

3309), Cabernet franc (CF) and Concord (CD) grapevines basal leaves after exposure to long day (LD and short day (SD) photoperiod at (A) 4, (B) 6, and (C) 8 weeks………..86

Figure 4.1. Daily minimum and maximum temperatures and LT50 for Cabernet franc

(CF), Couderc 3309 (C-3309) and Concord (CD) grapevines recorded from August to

April for (A) 2008 - 2009 and (B) 2009 - 2010. First killing frost dates are indicted by the arrow and occurred on October 23, 2008 and October 18, 2009………………………124

Figure 4.2. Monthly average day length for August to April in (A) 2008 to 2009 and (B)

2009 to 2010..…………………………………………………………………………..125

Figure 4.3. LT50 of apical, middle and basal buds of Cabernet franc (CF), Couderc 3309

(C-3309) and Concord (CD) grapevines collected (A) 2008 to 2009 and (B) 2009 to

2010……………………………………………………………………………………126

Figure 4.4. Soluble sugar concentration of Cabernet franc (CF), Couderc 3309 (C-3309) and Concord (CD) grapevines collected August 2009 to April 2010 (A) Glucose, (B)

Fructose and (C) Sucrose………………………………………………………………127

Figure 4.5. Raffinose concentration of apical, middle and basal buds of Cabernet franc

(CF), Couderc 3309 (C-3309) and Concord (CD) grapevines collected August 2009 to

April 2010………………………………………………………………………………128

Figure 4.6. Linear regression of leaf and bud raffinose concentration for Cabernet franc

(CF), Couderc 3309 (C-3309) and Concord (CD) grapevines collected August to October

2008……………………………………………………………………………………129

Figure 4.7. Correlation analysis of leaf raffinose concentration and LT50 of Cabernet franc (CF), Couderc 3309 (C-3309) and Concord (CD) grapevines collected August to

October 2008……………………………………………………………………………130

Figure A1. GC chromatogram of soluble sugar standard concentration 400 g/ul……148

Figure A2. GC chromatogram of soluble sugar in leaves of Cabernet franc grapevines exposed to long day (LD) photoperiod regime…………………………………………149

Figure A3. GC chromatogram of soluble sugar in leaves of Cabernet franc grapevines exposed to short day (SD) photoperiod regime………………………………………...150

Figure B1. LTE of Cabernet franc, Concord and Couderc 3309 basal buds collected

January 2010……………………………………………………………………………151

xvi

Chapter 1: Introduction

Grape production and its environmental limitations. Grapes are the most widely cultivated fruit crop in the world and are therefore highly economically important. In

Ohio, grape production has become one of the fastest growing sectors in agriculture and the grape and wine industries rank among the top ten in the US for grape acreage, production and number of wineries with an estimated economic impact to state revenues of more than $500 million annually (MKF Research, 2008). This growing interest is driven by the high value of wine grapes as an alternative and profitable cash crop in Ohio and its adaptability to already existing lands that are otherwise unsuitable for row crop production. Grapes, however, are temperate crops and are frequently damaged by freezing temperatures. The severe economic losses that result from freezing injury continue to be a major problem to the grape and wine industries in cold regions. In the

Northeast US, the last decade has been plagued by frequent crop losses (Guinan, 2007,

Zabadal et al., 2007) and Ohio experienced major consecutive freezing-related grape losses in the last 5 years with 2009 being the worst (Dami, unpublished data). The expansion of the industry is therefore limited by the constraints imposed by such temperature stress not only during the winter but also damage occurring in spring or fall.

1 Though all species of grapes are able to survive sub-zero temperatures, the level of freezing tolerance achieved varies between them. Generally, they are divided into groups based on the maximum cold hardiness and freezing tolerance (FT) of their dormant buds achieved in mid-winter: very tender (sensitive), tender (sensitive), moderately tender (sensitive), moderately hardy, hardy and very hardy (Zabadal et al.,

2007). The cultivars that currently dominate the market in acreage and production of premium wines are derived from the species V. vinifera and are therefore desired by grape and wine producers. These cultivars, however, are sensitive to freezing temperatures below –20oC (very tender). Cultivars from North America, though more capable of surviving freezing temperatures below –20oC, are not the preferred choices because of their lower wine quality (Zabadal et al., 2007). Currently more than 80% of grape expansion in Ohio is planted with cold sensitive cultivars of V. vinifera (Dami, personal communication). Therefore the limitation imposed by the sensitivity of the vinifera species has an effect on the sustainable growth of the industry and based on an industry survey, Ohio grape and wine producers continually ranks winter injury and freeze protection as the number one research priority (Dami et al., 2006). The understanding of the mechanisms of how grape genotypes acclimate to deal with freezing stress will improve survivability and sustainability of cold sensitive species grown in environments that are subject to frequent freezing temperatures.

Cold acclimation and its induction by environmental cues. Cold acclimation (CA) is the change associated with the adjustment to low temperature and the induction of FT.

Species unable to implement these changes are chilling sensitive and species that can are

2 chilling tolerant. Chilling tolerant species are further separated into freeze sensitive and freezing tolerant, with FT species being able to survive subzero temperatures (Levitt,

1980, Sakai and Larcher, 1987). The difference in tolerance between species, whether chilling or freezing, is dependent on their inherent genetic characteristics, but these traits are dependent on the environmental conditions that provide cues for their expression

(Guy, 1990, Levitt, 1980, Sakai and Larcher, 1987). Temperature and photoperiod are the key environmental stimuli for increasing a plant’s capacity to withstand freezing temperatures.

The first stage of CA begins in late summer or early fall when temperatures are low but above freezing and the days are shortening. This early acclimation has distinct events that occur and include physiological and morphological changes such as growth cessation, dormancy induction in buds, the partial development of FT and biochemical changes such as the synthesis of metabolites that aid in protecting cellular components from the environmental stress freezing temperatures impose (Guy, 1990, Levitt, 1980,

Sakai and Larcher, 1987). Short day (SD) photoperiod is the main factor that influences this first stage and influences the timing of growth cessation and dormancy (Levitt, 1980,

Sakai and Larcher, 1987, Weiser, 1970). Dormancy is generally defined as the temporary suspension of visible growth of any plant structure including buds, and is further categorized into paradormancy, where growth is inhibited by physiological factors outside the bud as occurs during the growing season, endodormancy, where growth is inhibited by physiological factors inside the bud as occurs during the non-growing season, and ecodormancy, where growth is inhibited by environmental factors, which occurs as the non-growing season ends and the growing season begins (Horvath et al.

3 2003, Lang et al., 1987). Physiological changes related to endo-dormancy include terminal bud set as occurs in some woody species. While they do not set terminal buds, grapevines exhibit other hallmark phenotypes of endodormancy such as periderm development and apical shoot tip abscission (Fennell and Hoover, 1991, Wake and

Fennel, 2000). There are also photoperiod sensitive ecotypes such as V. riparia and V. labrusca that will shed their leaves much earlier, even before low temperature stimulus is present.

The second phase of CA begins in late fall coincident with below freezing temperatures and this stage is photoperiod independent. During this time, CA will occur rapidly with increasing exposure to cold temperatures and an increase in FT will also coincide with the first freezing temperatures of late fall. In the plant, there are multiple changes that occur at the molecular, cellular and organ levels. Early events include an increase in intracellular calcium, a response to many abiotic and biotic stresses initiated by the destabilization of the cell membranes. This has then been shown to promote the expression of low temperature responsive genes (Knight and Knight, 2000, Orvar et al.,

2000, Plieth et al., 1999, Smallwood and Bowles, 2002). Transcriptional activators encoded by early responsive genes bind regulatory elements in the promoter region of cold responsive or regulated (COR) genes. They have been shown to increase the expression of COR genes and FT (Lui et al., 1998, Stockinger et al., 1997, Thomashow et al., 2001). The presence of these genes indicates an upstream regulatory element in CA as their expression increased upon exposure to low temperature (Lui et al., 1998,

Stockinger et al., 1997, Thomashow et al., 2001, Xiao et al., 2006). Some COR genes encode enzymes involved in the biosynthesis of osmoprotectants, enzymes responsible

4 for the synthesis of sugars or sugar derivatives, proteins associated with the dehydrative aspects of freezing stress, protective proteins that have antifreeze or cryoprotective properties and signal transducers such as transcription factors and protein kinases (Fowler and Thomashow, 2002, Thomashow 1999, Wisniewski et al. 2003).

Mechanisms of ice nucleation, freezing injury and freezing tolerance. The adaptability of varying species to develop FT involves several mechanisms for dealing with freezing stress. Plants survive ice in their tissues or avoid ice formation altogether.

The understanding of how plants deal with freezing is linked to the physical properties of liquid water as well as the physical properties of the plant cell to deal with freezing.

In pure liquid water, freezing is dependent on the presence of ice nucleators that act as catalysts for the liquid to solid phase transition. These catalysts are known as ice nuclei and can be homogeneous where the nuclei are formed without intervention of foreign bodies or heterogeneous where non-aqueous agents such as dust, or ice nucleating active bacteria such as Pseudomonas syringae are present (Levitt, 1980, Sakai and

Larcher 1987). Homogenous ice nucleation will occur when pure water is cooled to about

–38oC. In plants, ice nucleation begins internally on the surface of a cell wall in water that is being transported in xylem vessels or it begins on exterior surfaces due to frost

(Levitt, 1980, Sakai and Larcher 1987). The ice will spread throughout the extracellular regions of tissues as subzero temperatures are maintained. If the plasma membrane remains intact and the rate of cooling is slow, the ice remains confined to the external spaces and is called extracellular freezing. Cells that experience extracellular freezing have increased solute concentration because of dehydrative forces imposed by the

5 external ice crystals pulling water out of the cell. This loss of water is dependent on the vapor pressure of the ice outside the cell. The cell has a decreased freezing point but suffers from other stresses that are related to dehydration. Intracellular freezing is the crystallization of water inside the cell by internal nucleation or by the penetration of an external ice crystal through the cell membrane. This type of freezing is lethal because of the mechanical damage to the membrane (Levitt, 1980, Sakai and Larcher 1987).

Supercooling is one mechanism through which some tissues avoid freezing at subzero temperatures (Levitt, 1980, Sakai and Larcher, 1987, Wisniewski et al., 2003).

This phenomenon is maintained through small cell size, little or no intercellular spaces, low water content, absence of heterogeneous ice nucleating sites within tissues and tissues are physically separated from external nucleators in adjacent frozen tissues.

Several species have been shown to exhibit this phenomenon including the xylem tissues of apples (Hong and Sucoff, 1982), boreal hardwood species such as dogwood (Kuroda et al. 2003) and shagbark hickory (George and Burke, 1977) and bud tissues of grape (Jones at al. 1999). These tissues that supercool invariably have a limit. At this temperature limit, either the water in the tissues spontaneously freezes (around -38oC) or ice propagates from adjacent tissues because the separating mechanisms are broken. This will result in spontaneous death.

Thermal analysis (TA) has been widely used to quantify the limiting temperature of supercooling for several species (Wample et al. 1990). TA is based on detecting the latent heat of fusion that is given off when ice is formed in the tissues using attached thermocouples. The heat can be given off as a single large exotherm as is seen in species that do not supercool, or as two exotherms. The two exotherms include a high

6 temperature exotherm (HTE) which corresponds to water freezing in the extracellular spaces of tissues that don’t supercool and a low temperature exotherm (LTE), which corresponds to intracellular ice formation which leads to cell injury (Wample et al., 1990,

Wisniewski, 1995). The use of TA offers a method to explore freezing characteristics, providing a means of predicting injury due to prevailing conditions in the field, comparing genotypic differences in hardiness or evaluating environmental or cultural practices on cold hardiness (Wisniewski et al. 2003).

Typically tissues that exhibit supercooling are expected to have tightly packed cells with common cell walls (Ashworth and Abeles, 1994, Levitt, 1980, Sakai and

Larcher, 1987). Large intercellular spaces are absent and extracellular water would be in microcapillaries in the cell walls with the shape and size being irregular. The presence of the microcapillaries would not only depress the freezing point of the water in the intercellular spaces but would impede the spread of ice through out the tissues. The presence of small diameter pores would facilitate the supercooling of water in large volumes (Ashworth and Abeles, 1984).

Grape cold hardiness (Freezing tolerance). During the autumn and winter, grape tissues develop the ability to survive freezing temperature stress. The development of hardiness in grapes in response to photoperiod and/or low temperature is species specific.

They undergo the process of acclimation as any woody plant but the response to acclimating conditions is different from other woody plants in that the vines do not set terminal buds (Fennell and Hoover, 1991). Grapes survive freezing stress through various mechanisms (Fennell, 2004, Zabadal, 2007). Several tissues in the vine survive by

7 tolerating ice in the tissue and the increased solute concentrations, which lowers the cells freezing point. The woody tissues of the trunk tend to be the most freeze tolerant portion of the vine tolerating frequent freeze-thaw cycles. The roots exhibit less hardiness than the trunk but even though susceptible to freezing, these tissues are generally the least common sites of freezing injury. Cane tissues can either tolerate extracellular freezing or they supercool, the mechanism by which a liquid remains fluid below its normal freezing temperature. They will develop tolerance in early fall from about -3 to about -15oC and with the onset of subzero temperatures increase hardiness to about -32oC. The degree of temperature hardiness is species specific (Fennell, 2004, Zabadal, 2007).

The tissues of dormant buds also use supercooling to avoid freezing stress

(Fennell, 2004, Zabadal, 2007). Grape buds contain primary, secondary and tertiary buds, with the primary and secondary buds being mixed buds having both leaf and flower primordia and the tertiary bud being mostly vegetative. Within the bud, the hardiest are the tertiary buds with the secondary bud being less hardy and the primary buds exhibiting the least hardiness (Zabadal, 2007). Hardiness also develops from the basal buds upward

(Fennell and Hoover, 1991, Zabadal, 2007).

Differential analysis using nuclear magnetic resonance (NMR) imaging of buds that display endodormancy induced by short days gives an indication of changes in the bud tissue that may effect supercooling (Fennel and Line, 2001). Measurements of T1 and T2 times are indicators of water that is in association with macromolecules (bound state) with shorter times indicating more bound water. T1 and T2 are indicators for dormancy by highlighting the change in the state of bound water, and changes in the regions around the bud tissue. Decreased T2 times are observed in the gap region (region

8 at the base of a bud that separates the bud from the stem tissue) two weeks into short days compared to bud and stem tissue, which did not show changes until after four weeks of short days (Fennell and Line, 2001). The T2 times also decreased more quickly in the gap than in the bud tissue, suggesting that the SD signal impacted the gap tissue, then the bud, then the conducting tissue (Fennell and Line, 2001). The nature of the gap region is a physical barrier (Jones et al. 1999). Permeability test indicate that the pores within the gap region decline as acclimation increases possibly because a secondary wall forms at the base of the base of the bud axis and pectins fill the intercellular space. This barrier helps to maintain the separation of the bud from regions with ice nucleators allowing the bud to supercool (Jones et al. 1999).

Molecular regulation of freezing tolerance. Genetic experiments have shown that the induction of FT is a quantitative trait that is controlled by additive genes that lead to multiple changes from the molecular level to the organ level (Guy 1990). A large group of the genes that have been strongly induced by cold acclimation had been characterized initially in the model herbaceous plant, Arabidopsis thaliana as the cold responsive or regulated (COR) genes (also termed LTI for low temperature induced, CAS for cold acclimation specific or RD for responsive to desiccation). Cis-acting elements in the promoter region of COR genes have been identified as a DNA regulatory element, the C- repeat (CRT) dehydration responsive element (DRE), which has a conserved core sequence of CCGAC (Lui et al., 1998, Stockinger et al., 1997, Thomashow, 2001).

Transcriptional activators that bind the CRT/DRE include CBF 1, 2 and 3 (DREB1b, 1c and 1a) found in Arabidopsis (chromosome 4) have been shown to increase the

9 expression of COR genes and the freezing tolerance of overexpressing transgenic

Arabidopsis plants (Thomashow et al., 2001).

In the freezing-tolerant grape V. riparia and the freezing-sensitive V. vinifera the transcriptional activators CBF/DREB1-like genes have also been characterized

(Thomashow et al., 2001, Xiao et al., 2006, 2008). There are five CBF/DREB1-like genes in grapes; CBF 1-4 (Xiao et al., 2006, 2008) and a CBF-like transcription factor, CBFL reported by Takuhara et al. (2011). The proteins encoded by three of these genes (CBF 1-

3) from the two species are almost identical. Their expression increases upon exposure to low temperature and is not significantly different between the two species of grapes.

They also increased in response to drought and exogenous abscisic acid treatment (Xiao et al., 2006, 2008). The presence of these genes indicates an upstream regulation in grape cold acclimation. CBF4 is a unique member of the transcription family in V. riparia and

V. vinifera and has some similarity to Arabidopsis CBF1. It was reported as being unique in that it’s expression is maintained for several days, it was expressed in old and young tissue and it may represent a second type of CBF that might be more important for overwintering of grape plants (Takuhara et al. 2011, Xiao et al., 2008).

COR genes have been shown to include genes that encode enzymes responsible for the synthesis of sugars or sugar derivatives that have antifreeze or cryoprotective properties (Castonguay et al. 1998, Wisniewski et al., 2003). In the xylem parenchyma cells of larch (Larix kaempferi), differential screening and differential analysis of the genes associated with supercooling identified some genes expressed most abundantly during winter and included metabolic enzymes such as galactinol synthase (GS), the enzyme that catalyzes the first committed step in the synthesis of raffinose (Takata et al.

10 2007). Transcript profiling of Arabidopsis also confirmed this finding with transcripts for three different genes encoding putative galactinol synthases accumulating in response to low temperature and in one case, induction of 160-fold within 24 hours (Fowler and

Thomashow 2002). The changes observed may be linked to the adjustment in metabolic activity to survive the constraints imposed by low temperature such as structural protein changes and the increase or decrease in activity of various enzymes and enzymes identified included those for sugar metabolism.

Proteins. The changes of cold acclimation involve both the induction of FT and the adjustment in metabolic activity to survive the constraints imposed by low temperature

(Guy, 1990). Annual changes in the pattern of total soluble proteins were characterized in olive trees and were related to cold hardiness (Eris et al. 2007). This may include structural protein changes as well as the increase or decrease in activity of various enzymes but aside from the obvious catalytic and structural function, their activity may directly be linked to cryoprotective roles. The expression of an acidic dehydrin gene was found to increase during cold acclimation of wheat and the protein accumulated in the vicinity of the plasma membrane (Danyluk et al. 1998). Dehydrins are proteins that accumulate in vegetative tissues that have been subject to environmental stresses. These stresses include cold, drought and salinity. They are largely hydrophilic proteins with specific segments of amino acids such as a lysine rich repeat. Dehydrin like proteins have also been isolated from the floral buds of blueberry that had accumulated in response to chilling (Muthalif and Rowland, 1994).

11 The expression of two highly similar dehydrin genes (DHN1-a and DHN1-b) was characterized in V. riparia and V. vinifera (Xiao and Nussuth, 2006). The expression of each dehydrin gene was varied in both species. When cold treated, DHN1-a and DHN1-b are expressed as spliced and un-spliced transcripts in V. vinifera but only DHN1-a was expressed as un-spliced transcripts in V. riparia. The activity of spliced transcripts of both genes was observed in response to photoperiod, cold, drought and ABA treatments

(Xiao and Nussuth, 2006).

Carbohydrates. Organic compounds including sugars have been implicated in the hardening process of plants (Levitt, 1980, Sakai and Larcher 1987). In both herbaceous and woody plants it has been indicated that soluble sugars increase in the fall to winter when plants acclimate and decrease in the spring when de-acclimation occurs (Levitt,

1980, Sakai and Larcher 1987). Changes associated with the accumulation of sugars have been suggested. These include the decrease in the crystallization of water, which reduces freeze-induced dehydration, production of metabolites that produce other protective substances or metabolic energy, glass formation which may stop all biochemical and most physical activity and the protection of cellular constituents (Sakai and Larcher, 1987). The correlation has not only been quantitative but also qualitative.

Plants may accumulate glucose, sucrose, fructose, raffinose and stachyose. Raffinose and stachyose was shown to accumulate in Alfalfa (Cunningham et al., 2003), all sugars but especially sucrose in red raspberry (Palonen and Junttila, 2002) and fructans in the bluegrass P. annua L. (Dionne et al., 2001).

12 Among sugars, the raffinose family of oligosaccharides (RFO) appears to be the most important to cold hardiness as it has been frequently observed to change exclusively with cold acclimation, and has been reported to provide cryoprotection to cell membranes and stabilization of proteins (e.g. enzymes) during freezing stress. It has generally been observed that RFO concentrations increase in the fall as a response to low temperature, reach a maximum during the coldest months in mid-winter, and decrease in the spring

(Stushnoff et al. 1993). Several of the woody plant species investigated by Stushnoff et al. (1993) had only raffinose and stachyose concentrations correlating strongly with cold hardiness. Other researchers have also found that RFO correlates better than total soluble sugars with cold hardiness in both herbaceous and woody plants (Bachmann et al. 1994,

Castonguay and Nadeau 1998, Hamman et al. 1996, Stushnoff et al. 1998, Taji et al.

2002).

Raffinose is a minor carbohydrate in grape tissues (Badulescu and Ernst 2006,

Barka and Audran, 1996, Hamman et al. 1996, Jones et al. 1999, Wample and Bary

1992). Nevertheless, it is important in cold acclimation and FT of Vitis species.

Hamman et al. (1996) established a relationship in Vitis vinifera ‘Chardonnay’ and

‘Reisling’ between freezing tolerance and endogenous levels of sugars such as raffinose, but no relationship with sucrose. Stuchnoff et al. (1993) found RFO, sucrose, glucose, but not fructose, accumulated in the cold hardy ‘Valiant’ but only RFO was correlated with cold hardiness. There is also differential accumulation of raffinose in sensitive and hardy grape cultivars in side-by-side controlled environment experiments observing early responses. Cold hardy cultivars such as ‘Frontenac’, ‘Couderc 3309’ (C-3309), and

‘Concord’ (CD) accumulated the highest amount of raffinose in both bud and leaf tissues

13 and cold sensitive cultivars such as ‘Cabernet franc’ (CF) accumulated the least (Grant et al. 2009).

Several roles have been proposed in the literature for raffinose and how it protects plant tissues during cold stress. At low temperatures, raffinose delays the crystallization of sucrose (Caffrey et al. 1988, Koster and Leopold, 1988). It does not change its configuration with decreasing temperatures (Jeffrey and Huang, 1990), allowing it to have structure-preserving effect upon binding to proteins and membranes (Lineberger and

Steponkus, 1980, Santarius, 1973). The sugar molecules may function by forming hydrogen bonds with macromolecules and thus may substitute for water during desiccation stress, allowing them to maintain their hydrated confirmation (Crowe et al.,

1988). Accumulation may also decrease the osmotic potential, which depresses the freezing point of water. This is possibly through a colligative effect where the sugars change the bulk properties of the solution (Burke et al. 1976). Soluble sugars may also protect cells by forming intracellular glass, an undercooled liquid with the viscosity of a solid and its formation would ensure stability during periods of dormancy by preventing further desiccation and stabilizing cell structures (Burke, 1986). Glass forms at the glass transition temperature - Tg. Raffinose is a trisaccharide, has a higher molecular weight than monosaccharides and disaccharides and therefore has a higher Tg and will form glass more readily (Franks, 1985). In other words, raffinose is more protective than the disaccharide sucrose or the monosaccharides glucose and fructose. It is possible that the characteristics of raffinose allow it to function by any or all of the above mechanisms.

14 Enzymatic activity. The accumulation of soluble sugars is linked to the regulation of sugar concentration through enzyme activity. Enzyme activities linked to the synthesis and biodegradation of key sugars has been investigated in relation to cold acclimation.

Two enzymes catalyze the synthesis of raffinose: raffinose synthase (RS) and galactinol synthase (GS). Synthesized from sucrose through the sequential addition of molecules of galactosyl residues to sucrose, raffinose is the first compound formed in the RFO pathway and RS catalyses this first step (Figure 1.1). It is specific for sucrose as the galactosyl acceptor. Raffinose is then used for the synthesis of stachyose by the enzyme stachyose synthase. The galactosyl residues introduced in the RFO metabolic pathway come from the galactosyl transfer from UDP-D-galactose to myo-inositol, a reaction catalyzed by GS. GS is temperature dependent and its activity has been found to increase during exposure to cold temperatures (Castonguay et al. 1998). Arabidopsis plants under drought and cold stress showed an increase in the activity of GS isoforms and the expression of stress responsive genes such as GolS, galactinol and raffinose have roles in the response to these stresses (Fowler and Thomashow, 2002, Taji et al. 2002). An increase in GS activity in response to cold stress was also shown in kidney beans species

(Liu et al. 1998).

Enzymes that catalyze the degradation of raffinose have also been implicated in cold acclimation. The degradation of raffinose is catalyzed by the enzyme α- galactosidase (AG), and its activity has been shown to increase during de-acclimation in spring when RFO levels decreased (Castonguay and Nadeau, 1998). Two subtypes of this enzyme exist: acid and alkaline, and their activity differ in that the alkaline forms showed high affinity to stachyose and the acid form showed high affinity for raffinose

15 (Gaudreault and Web, 1996). Expression of AG has been correlated to low temperature artificial de-acclimation at 25oC in Petunia and the down-regulation of the gene enhances freezing tolerance (Pennycooke et al. 2003, 2004).

While the role of sugars in the development of cold acclimation has been well documented in grapes, there is a critical need to identify relationships between RFO synthesis profiles and FT to develop new strategies for protecting grapevines from freezing stress and to develop hardier grape cultivars. Previous research has indicated the accumulation of RFO in the sensitive grape species V. vinifera (Hamman et al. 1996,

Jones et al. 1999); however, differences in RFO accumulation between cold hardy and cold sensitive cultivars have not been documented, and no studies have included a comparison of soluble sugars between cold-hardy and cold-sensitive grape species determined in the same experiment. Furthermore, all published reports on grape describe sugar concentration changes in dormant tissues including canes and buds but not in tissues prior to leaf fall. There are also challenges to separate the effects of photoperiod and temperature on cold acclimation and dormancy as a plant may need both external signals to fully implement biophysical changes needed for survival (Garris et al., 2009, Li et al., 2002). There is a need to understand what interplay there may be between these environmental cues that will help to determine possible downstream targets of the environmental signals and the effects of the timing of the growth cessation and the depth of dormancy induced and cold acclimation. It may be that the beginning of the development of dormancy, growth cessation and initial changes in cold acclimation and the biochemical changes that occur related to these changes may be why some grapevines are more tolerant than others.

16 Our central hypothesis is that the accumulation of soluble sugars in grape tissues provides increased cold hardiness and the larger soluble sugars such as RFO’s accumulate earlier and to higher amounts in cold tolerant than in cold sensitive cultivars in response to cold temperature and SD photoperiod. The central objective of this dissertation was to characterize the morphological, physiological and biochemical changes in response to cold acclimation and SD photoperiod. The specific objectives were to: 1) characterize the morphological, physiological and biochemical changes in induced by low temperature in various cold tolerant and cold sensitive Vitis species grown under controlled conditions; 2) characterize the morphological, physiological and biochemical changes in induced by SD photoperiod in various cold tolerant and cold sensitive Vitis species grown under controlled conditions; and 3) characterize the morphological, physiological and biochemical changes induced by low temperature and

SD photoperiod in various cold tolerant and cold sensitive Vitis species grown under field conditions.

17 References

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Knight, H. and Knight, M. R. 2000. Imaging Spatial and Cellular Characteristics of Low Temperature Calcium Signature after Cold Acclimation in Arabidopsis. J. Exp. Bot. 51(351):1679-1686.

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Muthalif, M. M. and Rowland, L. J. 1994. Identification of Dehydrin-Like Proteins Responsive to Chilling in Floral Buds of Blueberry (Vaccinium, section Cyanococcus). Plant Physiol. 104:1439-1447.

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Pennycooke, J. C., Vepachedu, R., Stushnoff, C. and Jones, M. L. 2004. Expression of an α-Galactisidase Gene is Upregulated during Low Temperature Deacclimation. J. Amer. Soc. Hort. Sci. 129(3)

Plieth, C., Hansen, U., Knight, H. and Knight, M. R. 1999. Temperature Sensing by Plants: the Primary Characteristics of Signal Perception and Calcium Response. The Plant Journal. 18(5):491-497.

19 Sakai, A. and Larcher, W. Frost Survival of Plants. 1987. Responses and Adaptation to Freezing Stress. In Billings, W. D., Golley, F., Lange, O.L., Olson, J. S. and Remmert, H. (eds). Ecological Studies, Vol 62. Springer-Verlag. Berlin, Germany.

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Streeter, J.G. and Strimbu, C. E. 1998. Simultaneous extraction and Derivitization of Carbohydrates from Green Plant Tissues for Analysis by Gas-liquid Chromotography. Anal. Biochem. 259:253-257.

Stockinger, K. J., Gilmour, S. J. and Thomashow, M. F. 1997. Arabidopsis thaliana CBF1 Encodes and AP2 Domain-Containing Transcriptional Activator that binds to the C-Repeat/DRE, a Cis-Acting DNA Regulatory Element that Stimulates Transcription in Response to Low Temperature and Water Deficit. PNAS. 94:1035-1040.

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20 Wisniewski, M., Bassette, C. and Gusta, L. V. 2003. An Overview of Cold Hardiness in Woody Plants: Seeing the Forest Through the Trees. Hortscience. 38(5):952-959

Wisniewski, M. Deep Supercooling in Woody Plants and the Role of Cell Wall Structure. In Lee Jr, R. E., Warren, G. J. and Gusta, L.V. (eds). 1995. Biological Ice Nucleation and its Applications. APS Press, St. Paul Minnesota. 163-181.

Xiao, H. and Nassuth, A. 2006. Stress- and Development-Induced Expression of Spliced and Unspliced Transcripts from Two Highly Similar Dehydrin 1 Genes in V. riparia and V. vinifera. Plant Cell Rep. 25:968-977.

Xiao, H., Siddiqua, M., Braybrook, S. and Nassuth, A. 2006. Three grape CBF/DREB1 Genes Respond to Low Temperature, Drought and Abscisic Acid. Plant, Cell and Environment. 29:1410-1421.

Xiao, H., Tattersall, E. A. R., Siddiqua, M., Cramer, G. R. and Nassuth, A. 2008. CBF is a Unique Member of the CBF Transcription Factor Family of V. vinifera and V. riparia. Plant, Cell and Environment. 31:1-10.

Zabadal, T. J., Dami, I. E., Goffinet, M. C., Martinson, T. E. and Chien, M. L. 2007. Winter Injury to Grapevines and Methods of Protection. Michigan State University. MI, USA.

UDP-Galactose + Myo-Inositol Galactinol Synthase Galactinol + UDP

Galactinol + Sucrose Raffinose Synthase Raffinose + Myo-Inositol

Raffinose + Sucrose Stachyose Synthase Stachiose + Myo-Inositol

α- Galactosidase Raffinose Sucrose + Galactinol

Figure 1.1. Key enzymes involved in raffinose synthesis and degradation

Chapter 2: Variation in leaf and bud soluble sugar concentration among Vitis genotypes grown under two temperature regimes

Trudi N. Grant, Imed E. Dami, Taehyun Ji, David Scurlock, and John Streeter

Soluble sugar accumulation was determined in the grape (Vitis spp) cultivars ‘Frontenac’,

‘Couderc 3309’, ‘Concord’, ‘Cabernet Franc’, ‘Traminette’ and ‘Seyval’ grown under two temperature regimes. Shoot growth slowed under cold temperature regimes in all cultivars except Concord, which was the least responsive. Among all sugars, raffinose showed distinctive responses associated with the two temperature regimes. Under a non- acclimating temperature regime, raffinose concentrations were low and similar among cultivars, whereas under cold acclimating temperature regimes raffinose accumulation was generally higher and cold hardy cultivars accumulated higher concentrations than did cold sensitive cultivars. Basal leaves and buds accumulated the most raffinose. ‘Cabernet

Franc’ vines exhibited no differences in sugar accumulation at different stages of development. The results suggest that raffinose accumulation might be an early step in the process of cold acclimation that coincides with slowed shoot growth, and may precede the onset of dormancy and freezing tolerance. Leaf raffinose concentration might be useful as a detection tool to distinguish various Vitis genotypes with contrasting FT.

Key words: buds, cold acclimation, leaf, raffinose, Vitis

23 The severe economic losses that often result from freezing injury to grapevines, especially in the Midwest and Northeast U.S., are a major problem to the grape and wine industry in these regions (Zabadal et al. 2007). In fact, crop losses due to freezing injury have been unusually frequent in this decade with consecutive occurrences since 2003 resulting in losses of hundreds of millions of dollars (Guinan 2007, Zabadal et al. 2007).

In spite of the widely appreciated magnitude of the problem of freeze injury to grapes, little progress has been made in understanding the mechanisms of freeze resistance or in developing economic and sustainable new approaches for freeze protection of commercially important cultivars. Simply stated, why a cold-hardy grape cultivar is several degrees hardier than a cold-sensitive cultivar is still not well known and is under- studied.

Grapevines adapt to cold climates by undergoing seasonal changes that result in a transition from a cold-sensitive to a cold-hardy state, a process known as cold acclimation. This process leads to an increase in freezing tolerance or cold hardiness, which is the ability of dormant grapevine tissues to survive freezing temperature stress

(Zabadal et al. 2007). Cold acclimation and the subsequent increase in cold hardiness are genetically regulated and involve the action of multiple mechanisms which result in the production of several metabolites including polypeptides, amino acids, and sugars (Levitt

1980, Li 1984, Sakai and Larcher 1987). In general, total soluble sugars in plants increases during the onset of cold acclimation, reaches a maximum during full cold hardiness, and decreases during de-acclimation (Sakai and Larcher 1987).

Among sugars, the raffinose family of oligosaccharides (RFO) appears to be the most important to cold hardiness as it has been frequently observed to change exclusively

24 with cold acclimation, and has been reported to provide cryoprotection to cell membranes and stabilization of proteins (e.g. enzymes) during freezing stress. It has generally been observed that RFO concentrations increase in the fall as a response to low temperature, reach a maximum during the coldest months in mid-winter, and decrease in the spring

(Stushnoff et al. 1993). Several of the woody plant species investigated by Stushnoff et al. (1993) had only raffinose and stachyose correlating strongly with cold hardiness.

Other researchers have also found that RFO correlates better than total soluble sugars with cold hardiness in herbaceous and woody plants (Bachmann et al. 1994, Hamman et al. 1996, Dami 1997, Castonguay and Nadeau 1998, Stushnoff et al. 1998, Taji et al.

2002).

Raffinose is a minor carbohydrate in grape tissues (Wample and Bary 1992, Barka and Audran, 1996, Hamman et al. 1996, Jones et al. 1999, Badulescu and Ernst 2006).

Nevertheless, there is reason to believe that it is important in cold acclimation and freezing tolerance of Vitis species. Hamman et al. (1996) established a relationship in

Vitis vinifera ‘Chardonnay’ and ‘Reisling’ between freezing tolerance and endogenous levels of sugars such as raffinose but no relationship with sucrose. Stuchnoff et al. (1993) found RFO, sucrose, glucose but not fructose, accumulated in the cold hardy ‘Valiant’ but only RFO was correlated with cold hardiness. Although RFO is found in grape species, the relative concentrations in cultivars varying in cold hardiness have not been established. There is, therefore, a critical need to identify relationships between RFO synthesis profiles and cold hardiness to develop new strategies for protecting grapevines from freezing stress and to develop hardier grape cultivars. Although recent reports indicated the accumulation of RFO in the sensitive grape species Vitis vinifera (Hamman

25 et al. 1996, Jones et al. 1999), differences in RFO accumulation between cold hardy and cold sensitive cultivars have not been documented, and no studies have included a comparison of soluble sugars between cold-hardy and cold-sensitive grape species determined in the same experiment. Furthermore, all published reports on grape describe sugar concentration changes in dormant tissues including canes and buds but not in tissues prior to leaf fall. Therefore, the objectives of this study were to: 1) determine levels of specific soluble sugars, especially raffinose, in leaves of various Vitis genotypes with contrasting cold hardiness in response to cold acclimation; 2) characterize sugar accumulation in bud and leaf tissues relative to their position on the shoot; and 3) investigate the influence of cold acclimation on sugar concentrations in leaves of vines at different stages of development. Our hypotheses are that grapevines, like many other temperate plants, accumulate raffinose in their leaves in response to cold exposure well before sub-freezing temperatures in preparation for winter and that raffinose accumulation varies among grape genotypes according to their freezing tolerance.

Materials and Methods

Plant Materials: Grape cultivars of contrasting cold hardiness, as rated previously (Dami et al. 2005, Zabadal et al. 2007) were used in this study: Vitis spp, ‘Frontenac’ (very cold hardy), V. riparia x V. rupestris ‘Couderc 3309’(cold hardy), V. labruscana ‘Concord’

(cold hardy), V. vinifera, ‘Cabernet Franc’ (cold sensitive or moderately cold tender); and

V. spp, ‘Traminette’ and ‘Seyval’ (moderately hardy). Two-year-old plants were dormant pruned to retain three to four buds. After bud break, the strongest shoot was retained and

26 trained vertically using a stake. All flower clusters were removed. The plants were grown in climate-controlled glass greenhouses under the following conditions: 25oC/20oC and

40/70% relative humidity (day/night). Supplemental light was provided automatically when photosynthetic photon flux (PPF) density was below 600 μmol m-2s-1 using high pressure sodium lamps and to maintain a 16-hr photoperiod. All plants were watered daily and fertilized every other day with 100 ppm 20-20-20 fertilizer. Five vines per cultivar having uniform growth and 12-15 leaves were selected for each of the acclimating experiments, which were conducted in growth chambers. The experiments described below were conducted between 2005 and 2007 and were repeated at least once.

Cultivar Differences in Growth and Sugar Accumulation under two Temperature

Regimes: Experiments to examine cultivar differences in sugar accumulation were conducted in growth chambers (Conviron, Winnipeg, Manitoba, Canada) to provide two temperature regimes. The no-acclimation regime consisted of a constant temperature at

23oC, light for 16 hr/dy at 600 μmol m-2s-1 PPF and 50% relative humidity. The cold- acclimation regime consisted of 23oC/15oC (day/night) for one week followed by

15oC/7oC (day/night) for the second week, 50% relative humidity, and light for 8 hr/dy at

600 μmolm-2s-1 PPF. Due to the large number of plants, each study was split into two experiments. One included three cultivars, V. vinifera ‘Cabernet Franc’, V. labruscana

‘Concord’, and V. spp ‘Frontenac’. The other included V. spp ‘Traminette’ and ‘Seyval’, and V. riparia x V. rupestris ‘C-3309’. Five pots per cultivar were used under each regime.

27 Leaf and node numbers and shoot length were measured on each vine prior to exposure to the temperature regimes and every two days thereafter for two weeks. The same measurements were taken at the end of the experiment and periderm development was visually rated. Other data collected included new leaves grown during the acclimation period, numbers of abscised shoot tips and dormancy assayed by cutting back shoots to two basal buds and recording the number of days until 50% budbreak.

Qualitative and quantitative analyses of soluble sugars were conducted on leaves at the end of the two-week temperature regimes. Basal leaves (nodes two to eight) were collected in the morning at approximately the same time and were immediately placed at

-80oC. The frozen leaves were lyophilized and ground to pass through a 20-mesh screen.

Approximately 5 mg of ground leaf tissue was extracted and derivitized simultaneously according to Streeter and Strimbu (1998). This involved mixing the ground tissue with pyridine containing hydroxylamine and an internal standard, phenyl -D glucopyranoside. After incubation at 70oC, the oxime derivatives were reacted with hexamethyldisilazane (HMDS) to form the tri-methylsilyl (TMS) derivatives of the oximes. The derivatives were injected into a gas chromatograph (Hewlett Packard 5890

Series II, Hewlett Packard, Boulder, CO) with a 30 m capillary column (HP5-MS, 250

m inner diameter and 0.25 m thickness). Helium, the carrier gas, was at a constant flow rate of 1.0 ml.min-1. Soluble sugars were identified and peaks quantified (Agilent

Technologies, Wilmington, DE).

Sugar Accumulation in Bud and Leaf Tissues Relative to Node Position: An experiment was conducted with two cultivars, V. riparia x V. rupestris ‘C-3309’ and V. 28 vinifera ‘Cabernet Franc’ to compare soluble sugars in leaves and buds at different shoot node positions in cultivars with contrasting cold hardiness. Vines were exposed to the two temperature regimes as described above, then all buds and leaves were collected and divided into three sub-samples: basal (nodes two to eight), middle (nodes nine to 14), and apical (nodes 15 to 20). Leaf preparation was similar to the procedure described above.

Buds were excised, plunged into liquid nitrogen and pulverized with mortar and pestle, then lyophilized. The dry bud tissue was mixed with 75% ethanol in a tube. The mixture was vortexed occasionally while at room temperature for two to four hours then centrifuged for five minutes at 10 000 rpm. The supernatant was transferred to a vial then dried under air at 45oC. The extraction was repeated twice. The extracted bud metabolites and the leaf tissue were derivatized using the method by Streeter and Strimbu (1998) as described above.

Sugar Content in Leaf Tissues of a Cultivar at Different Developmental Stages: V. vinifera ‘Cabernet Franc’ plants at different stages of development were selected based on node number and grouped into four subsets: stage 1, 12-14 nodes; stage 2, 15-17 nodes; stage 3, 18-23 nodes; and stage 4, 23-27 nodes. Developmental stages were related to plant age with stage one being the youngest plants and stage four the oldest plants. Potted vines at various stages of development were simultaneously placed under the two temperature regimes for two weeks as described above. Leaves from basal nodes two to eight were collected for sugar analysis. Morphological changes in the vines such as shoot growth, node number and periderm development were also monitored. Only sugar concentrations in the basal leaves are reported.

Statistical Analysis: Statistical analysis of sugar concentrations was conducted using the

PROC ANOVA/GLM procedure of SAS (SAS Institute, Cary, NC). LSD was used to compare means at α = 0.05.

Results

Morphological Changes: Exposure of vines to the low-temperature regime slowed shoot elongation. Shoots exposed to the acclimation and no-acclimation regimes continued growing throughout the experiments (Fig. 2.1, 2.2). All the cultivars behaved similarly under the cold acclimation regime and showed no growth increase after day seven. It was noted that the shoots of cultivars V. riparia x V. rupestris ‘C-3309’ and V. sp. ‘Frontenac’ grew the most and those of V. vinifera ‘Cabernet Franc’, V. sp. ‘Seyval’, and

‘Traminette’ grew the least under the cold acclimation regime (Fig. 2.1, 2.2). Shoots of V. labruscana ‘Concord’ plants, however, grew slowly under both temperature regimes, indicating that shoot growth rate can be controlled by factors other than temperature (Fig.

2.1). The ‘Concord’ plants had abscised shoot tips and did not resume growth when forced (data not shown), indicating the onset of dormancy. The other cultivars did not exhibit shoot tip abscission and all had actively growing shoot tips under both temperature regimes and resumed growth when forced (data not shown).

Sugar Concentration in Leaf Tissues of Various Grape Cultivars: The most abundant sugars detected in all cultivars were fructose, glucose, myo-inositol, sucrose, raffinose

30 and galactinol, with sucrose being the predominant sugar on a dry weight basis. In the no- acclimation regime, there were no consistent differences in leaf sugar concentration among most cultivars (Table 2.1). For example, sucrose concentration was the highest in

V. labruscana ‘Concord’ (53 mg.g-1 dry weight), but the same in V. spp ‘Frontenac’ and

V. vinifera ‘Cabernet Franc’. ‘Traminette’, ‘Seyval’ and ‘C-3309’ had similar concentrations of sucrose in the range of 40–50 mg.g-1 dry weight. Variations among cultivars under both temperature regimes were seen in the concentrations of fructose, glucose, and myo-inositol. Raffinose was consistently low and similar among cultivars under the no-acclimation regime, but differed among cultivars under the acclimation.

‘Cabernet Franc’ and ‘Traminette’ had low concentrations of raffinose, whereas

‘Concord’, ‘Frontenac’, ‘C-3309’, and ‘Seyval’, all cold hardy cultivars, had high concentrations under the acclimation regime.

Sugar Accumulation in Bud and Leaf Tissues Relative to Node Position: Exposure to the acclimation regime resulted in similar sugar concentrations between the two cultivars except for raffinose concentrations, which were higher (2.5-fold) in leaves of the cold hardy cultivar, ‘C-3309’ (Table 2.2). The sugars in bud tissues did not differ between the cultivars except for raffinose, which was higher in ‘C-3309’ under the acclimation regime. Under both temperature regimes, fructose and glucose concentrations were higher in bud than in leaf tissues in both cultivars, and sucrose, galactinol, and raffinose were higher in leaves than in buds.

Sugar concentrations differed in the bud and leaf tissues collected from among apical, middle and basal positions. Under the cold acclimation regime, higher

31 concentrations of raffinose were generally found in leaf and bud tissues at basal and middle node positions than at apical node positions (Fig 2.3).

Sugar Content in Leaf Tissues of a Cultivar at Different Developmental Stages:

Under the no-acclimation regime, there were small differences among developmental stages in concentrations of glucose, myo-inositol and raffinose, but not a clear decline or accumulation of these sugars over development. Under the acclimation regime no differences in sugar concentrations among the stages were detected (Table 2.3). These results indicate that the plant developmental stage has little if any impact on the accumulation of sugars.

Discussion

The accumulation of RFO during cold acclimation has been documented in a wide variety of plants, including leaves of Puma rye (Secale cereale L.) (Koster and Lynch

1992), cabbage Brassica sp. (Santarius and Milde 1977), Ajuga reptans L. (Bachman et al. 1994), and alfalfa (Medicago sativa L.) (Castonguay et al. 1995). Accumulation of

RFO has also been reported in dormant tissues of woody plants including crabapple

(Malus sp.), apple (Malus sylvestris), grape (Vitis sp.), dogwood (Cornus sp.) (Stushnoff et al. 1993), red raspberry (Rubus idaeus L.) (Palonen and Juntilla 2002), aspen (Populus tremuloides Michx.) (Cox and Stushnoff 2001), conifer species (Hinesley et al. 1992) and

Norway spruce (Picea abies) (Lipavska et al. 2000). Some of the recent studies have indicated that sucrose is not the only sugar influencing cold hardiness and that RFOs such

32 as raffinose are more important. In grape, several researchers have examined seasonal patterns of sugar accumulation in dormant tissues, including buds and canes, and have found strong correlations between the accumulation of soluble sugars, specifically RFO, and freezing tolerance (Wample and Bary 1992, Barka and Audran 1996, Hamman et al.

1996, Jones et al. 1999). We are the first to report differences in the accumulation of raffinose in leaves of different grapevine cultivars that is associated with the cultivars’ established hardiness.

The first stages of acclimation relate firstly to photoperiod and later to low non- freezing temperatures, which might function in preparing plants for freezing temperatures

(Weiser, 1970, Zabadal et al. 2007). This early acclimation is later followed by full freezing tolerance initiated by the subsequent exposure to sub-freezing temperatures

(Zabadal et al. 2007). The distinct events that occur during the early phases include growth cessation and the synthesis of metabolites. The leaves then become important as sites of photoperiod signal reception and later of low temperature stimuli. Previous research has demonstrated that signal transduction pathways starting from the perception of either short-day or low-temperature stimuli and leading to cold acclimation are operational in leaves of birch (Betula pendula) and this early development is a possible protective mechanism during autumn, enabling prolonged periods for photosynthesis (Li et al. 2002). It has also been shown that in Vitis labruscana and Vitis riparia, short days initiated growth cessation and dormancy development as well as an increase in freezing tolerance (Fennell and Hoover 1991).

It has generally been shown that in grapes RFO correlates with cold hardiness

(Dami, 1997, Hamman et al. 1996, Stushnoff, et al. 1993), however, a side-by-side 33 comparison of RFO accumulation in cold hardy and cold sensitive cultivars has not been documented until the present study. We found that the cold hardy cultivars we tested, V. spp ‘Frontenac’, V. riparia x V. rupestris ‘C-3309’, and V. labruscana ‘Concord’, accumulated higher concentrations of raffinose than did the cold sensitive cultivar, V. vinifera ‘Cabernet Franc’ when exposed to low non-freezing temperatures. These results corroborate with previous findings of differences between RFO accumulations in

Medicaga (alfalfa) cultivars associated with cultivar hardiness (Castonguay et al. 1995,

Castonguay and Nadeau 1998). Our findings confirm our hypothesis that grape genotypes adapted to very low freezing temperatures accumulate relatively higher amounts of RFO levels under low but non-freezing temperatures than do sensitive plants. We also demonstrated that the pattern of sugar accumulation in leaves coincides with sugar accumulation in buds. Raffinose concentrations in leaf and bud tissues are, however, not uniform throughout the plant. Basal tissues often had higher concentrations of raffinose compared to middle or apical tissues, which may indicate differences in hardiness progression. Our observations corroborate those of Badulescu and Ernst (2006) who reported that basal bud tissue had the highest sugar concentration. It is not known, however, whether the RFO differences in apical, middle and basal tissues are associated with the varying tissue responses to the acclimating stimuli or to development progression, but it is likely the latter as cold acclimation of bud and stem tissues is known to proceed from the base to the shoot tip with the basal buds being the most developed and cold hardy. Dehydration of basal stem tissues leads to periderm formation (Zabadal et al. 2007). The pattern of raffinose accumulation follows that of periderm formation and tissue dehydration in grape shoots. Therefore, it is suggested that the high amount of

34 raffinose accumulated in basal tissues plays a role on protecting plant tissues during dehydration, which takes place prior to exposure to subfreezing temperatures. This needs further investigation but we have observed that cold hardy and cold sensitive cultivars exposed to the low non-freezing temperatures accumulated different levels of raffinose, but their bud freezing tolerance changed little or not at all (data not shown). This is another indication that raffinose accumulation may be part of a suite of changes that occur during the first stages of the acclimation process coinciding with slowed shoot growth and in some cases with shoot tip abscission (i.e. in Concord), but preceding periderm formation and subsequent acquisition of freezing tolerance.

Raffinose concentration in the leaf tissues before completion of dormancy and hardiness is developed could be used as an indication of hardiness among Vitis genotypes. Studying the preparation for cold acclimation by using a non-dormant plant part (i.e. leaves) may help elucidate the processes taking place during the early phases of cold acclimation and provide new clues for understanding the mechanism of freezing tolerance of grape cultivars.

References

Bachmann, M., Matile, P. and Keller, F. 1994. Metabolism of the raffinose family of oligosaccharides in leaves of Ajuga reptans: cold acclimation, translocation and sink to source transition: discovery of chain elongation enzyme. Plant Physiol. 105:1335-1345.

Badulescu, R. and Ernst, M. 2006. Changes of temperature exotherms and soluble sugars in grapevine (Vitis vinefera L.) buds during winter. J. Appl. Bot. Food Qual. 80: 165-170

Barka, E.A. and Audran, J.C. 1996. Response des vignes champenoises aux temperatures negatives: effet d’un refroidissement controle sur les reserves glucidiques du complexe gemmaire avant et au cours du debourrement. Can. J. Bot. 74: 492-505.

Castonguay, Y., Nadeau, P., Lechasseur, P. and Chouinard, L. 1995. Raffinose synthase and galactinol synthase in developing seeds and leaves of legumes. J. Agric. Food. Chem. 38: 351-355.

Castonguay, Y. and Nadeau, P. 1998. Crop Physiology and Metabolism: Enzymatic control of soluble carbohydrate accumulation in cold-acclimated crowns of Alfalfa. Crop Sci. 38: 1183-1189.

Cox, S. E. and Stushnoff, C. 2001. Temperature related shifts in soluble carbohydrate content during dormancy and cold acclimation in Populus tremuloides. Can. J. For. Res. 31: 730-737

Dami, I. E. 1997. Physiological responses of grapevines to environmental stresses. Ph.D. Dissertation, Colorado State University, Fort Collins, Colorado.

Dami, I. E., Bordelon, B., Ferree, D. C., Brown, M., Ellis, M. A., Williams, R. N. and Doohan, D. 2005. Midwest Grape Production Guide. Ohio State University Extension. OH, USA.

Fennel, A. and Hoover, E. 1991. Morphological, physiological and dormancy responses of three Vitis genotypes to short photoperiod. Physiol. Planta. 109: 203-210

36 Guinan, P. 2007. The Easter Freeze of 2007-What Happened? Pages 7-12 in Workshop Proceedings: Understanding and preventing freeze damage in vineyards. University of Missouri, Columba, MO.

Hamman, R.A., Dami, I.E., Walsh, T.M., and Stushnoff, C. 1996. Seasonal carbohydrate changes and cold hardiness of Chardonnay and Riesling grapevines. Am. J. Enol. Vitic. 47: 31-36.

Hinesley, L.E., Pharr, D.M., Snelling, L.K. and Funderburk, S.R. 1992. Foliar raffinose and sucrose in four conifer species: relationship to seasonal temperature. J. Am. Soc. Hort. 117: 852-855

Jones, K.M., Paroschy, J., McKersie, B.M., and Bowley, S.R. 1999. Carbohydrate composition and freezing tolerance of canes and buds in Vitis vinifera. J. Plant Physiol. 155:101-106.

Koster, K. L. and Lynch, D. V. 1992. Solute accumulation and compartmentation during the cold acclimation of Puma rye. Plant Physiol. 98:108-113

Levitt, J. 1980. Responses of plants to environmental stresses: Vol. 1. Chilling, freezing and high temperature stresses. 2nd ed. Academic Press, New York.

Li, C., Puhakainen, T., Welling, A., Viherä-Arrnio, A., Ernsten, A., Junttila, O., Heino, P and Palva, E.T. 2002. Cold acclimation in Silver birch (Betula pendula). Development of freezing tolerance in different tissues and climatic ecotypes. Physiol. Plant. 116: 478-488.

Li, P.H. 1984. Subzero temperature stress physiology of herbaceous plants. Hort. Rev. 6: 373-416.

Lipavska, H. Sbodova, H and Albrechtova, J. 2000. Annual dynamics of the content of non-structural saccharides in the context of structural development of vegetable buds of Norway spruce. J. Plant Physiol. 157: 365-373.

Palonen, P. and Juntilla, O. 2002. Carbohydrate and winter hardiness in red raspberry. Acta Horticulturae 585: 573-577.

37 Sakai, A., and Larcher,W. (eds) 1987. Frost survival of plants, responses and adaptations to freezing stress. Ecological Studies 62. Springer-Verlag, Berlin, Germany.

Santarius, K. A. and Milde, H. 1977. Sugar compartmentation in frost hardy and partially dehardened cabbage leaf cells. Planta 136: 163-166.

Streeter, J.G. and Strimbu, C.E. 1998. Simultaneous extraction and derivatization of carbohydrates from green plant tissues for analysis by gas-liquid chromatography. Anal. Biochem. 259: 253-257.

Stushnoff, C., Seufferheld, M.J. and Creegan, T. 1998. Oligosaccharides as endogenous cryoprotectants in woody plants. Pages 301-309 in P.H. Li and T.H.H. Chen, eds. Plant cold hardiness: Molecular biology, biochemistry and physiology. Plenum Press, New York,

Taji, T., Ohsumi, C., Iuchi, S., Seki, M., Kasuga, M., Kobayashi, M., Yamaguchi- Shinozaki, K. and Shinozaki, K. 2002. Important roles of drought- and cold-inducible genes for galactinol synthase in stress tolerance in Arabidopsis thaliana. The Plant Journal. 29: 417-426.

Wample, and Bary, A. 1992. Harvest date as a factor in carbohydrate storage and freezing tolerance of Cabernet Sauvignon grapevines. J. Am. Soc. Hort. Sci. 117: 32-36.

Weiser, C. J. 1970. Cold Resistance and Injury in Woody Plants. Science 169: 1269- 1278.

Zabadal, T. J., Dami, I. E., Goffinet, M. C., Martinson, T. E. and Chien, M. L. 2007. Winter injury to grapevines and methods of protection. Michigan State University. MI, USA.

38 Table 2.1. Soluble sugar concentration (mg g-1 dry wt) in leaves of non-acclimated and acclimated Vitis vinifera, ‘Cabernet franc’, V. labruscana ‘Concord’, V. spp, ‘Frontenac’, ‘Traminette’ and ‘Seyval’ and V. riparia x V. rupestris ‘C-3309’ grapevines

No acclimation temperature regime Genotype Fructose Glucose Myo-inositol Sucrose Galactinol Raffinose Experiment 1 Cab Franc 10.0 ab 11.4 ab 13.7 a 40.4 b 3.3 a 2.3 Concord 12.6 a 14.5 a 10.5 b 53.0 a 3.0 b 2.4 Frontenac 8.9 b 9.9 b 12.8 a 37.2 b 3.3 a 2.3 Experiment 2 Traminette 9.1 9.1 13.5 a 47.0 3.2 2.3 Seyval 8.9 8.4 13.1 a 43.8 3.2 2.4 C-3309 9.2 8.6 11.2 b 50.7 3.2 2.4

Acclimation temperature regime Experiment 1 Cab Franc 10.2 12.0 12.4 39.3 b 3.6 2.6 b Concord 9.5 9.8 12.8 58.3 a 3.6 4.1 a Frontenac 9.7 10.8 11.7 44.0 b 3.5 3.7 a Experiment 2 Traminette 9.2 ab 8.8 13.8a 59.1 a 4.0 b 3.0 b Seyval 9.3 a 8.6 13.9 a 50.6 b 4.9 a 5.1 a 8.8 b 8.3 9.7 b 60.4 a 3.8 b 3.9 b C-3309 a, b Means with different letters in columns of each experiment are significantly different at p < 0.05

39 Table 2.2. Soluble sugar concentration (mg g-1 dry wt) in leaves and buds of non-acclimated and acclimated cold sensitive Vitis vinifera, ‘Cabernet Franc’ and cold hardy V. riparia x V. rupestris, ‘C-3309’grapevines

No acclimation temperature regime Fructose Glucose Myo-inositol Sucrose Galactinol Raffinose Leaf Bud Leaf Bud Leaf Bud Leaf Bud Leaf Bud Leaf Bud Cab Franc 3.7 15.7 5.9 15.1 26.6a 9.07 48.8 16.8 0.6b 0.0 0.7b 0.0 C-3309 4.9 12.5 4.8 17.7 9.5b 10.7 26.0 19.1 1.5a 0.0 1.3a 0.2 Acclimation temperature regime Cab Franc 5.8 13.3 10.4 11.5 21.6 12.3 63.2 21.6 4.0 0.9 1.8b 0.8b C-3309 1.9 7.0 4.2 8.3 14.8 11.2 31.3 16.1 4.9 1.1 4.6a 5.5a

40 a, b Means with different letters in columns of each experiment are significantly different at p < 0.05

40 Table 2.3. Soluble sugar concentration (mg g-1 dry wt) in leaves of acclimated and non- acclimated Vitis vinifera, ‘Cabernet Franc’ at different stages of development z

No acclimation temperature regime Stages of

Development Glucose Fructose Myo-Inositol Sucrose Galactinol Raffinose Stage 1 9.8b 3.0 10.0a 44.2 0.4 0.1b

Stage 2 10.9ab 3.5 9.4a 51.0 0.5 0.3ab

Stage 3 15.2a 3.6 9.5a 50.6 0.4 0.1b

Stage 4 12.3ab 2.8 9.2b 52.4 0.5 0.5a

Acclimation temperature regime

Stage 1 11.1 3.8 12.4 75.7 2.6 1.1 Stage 2 13.7 5.2 12.5 73.6 2.2 1.6 Stage 3 11.3 4.4 11.6 95.2 1.9 1.1 Stage 4 14.1 4.3 11.0 72.7 3.2 2.0 z Stages of development as related to plant age, with stage 1 corresponding to vines with 12-14 nodes; stage 2, 15-17 nodes; stage 3, 18-23 nodes; and stage 4, 23-27 nodes a, b Means with different letters in columns of each experiment are significantly different at p < 0.05.

41 Cabernet A. No Acclimation Temperature Regime Franc Concord

Frontenac

120

100

Shoot length (cm) length Shoot 20

0 3 5 7 10 12 14

Days

B. Acclimation Temperature Regime

120

Cabernet Franc

100 Concord

80 Frontenac

40 Shoot length (cm) length Shoot 20

0 3 5 7 10 12 14

Days

42 A. No Acclimation Temperature Regime

Traminette Seyval

120 C-3309

100

40 Shoot length (cm) length Shoot

0 3 5 7 10 12 14

Days

B. Acclimation Temperature Regime

120 Traminette Seyval 100 C-3309 80 60 40

Shoot length (cm) length Shoot 20 0 3 5 7 10 12 14

Days

a ab

B. Bud Cabernet Franc C-3309

25 a

dry dry

1 - 20 b

15 c

10 wt) 5

0 Basal Middle Apical Basal Middle Apical Non-acclimated Acclimated

Raffinose concentration (mg g (mg concentration Raffinose

Chapter 3: Responses of greenhouse-grown Vitis genotypes to photoperiod regimes

Trudi N. Grant and Imed E. Dami

The purpose of this study was to characterize the morphological, physiological and biochemical changes that occur in grape genotypes in response to short day (SD) photoperiod. Experiments were conducted under greenhouse conditions using cold sensitive Vitis vinifera ‘Cabernet Franc’ (CF) and cold tolerant V. riparia x V. rupestris

‘Couderc 3309’ (C-3309) and V. labruscana ‘Concord’ (CD) cultivars. Potted vines were exposed to SD (8-hour) or long day (LD) (16 hour) photoperiod, for four, six and eight weeks. Shoot growth, periderm formation, dormancy, freezing tolerance (FT) (LT50) and soluble sugar accumulation were examined for each photoperiod duration. Shoot growth slowed under each SD photoperiod duration and in all cultivars. There was also increased periderm formation and endodormancy induction which resulted in increased FT. CD was the first cultivar to initiate these changes in response to SD followed by C-3309 then CF.

FT of all grape genotypes increased by 0.7, 2.0, and 2.7oC after four, six, and eight weeks under the SD regime, respectively. CD reached an LT50 of -10.5oC in week six, whereas

C-3309 and CF reached -12.5oC and -8.5oC respectively in week eight. The three cultivars did not gain FT under the LD regime and LT50 remained the same and ranged

45 between -6.1 and -8.1oC during the duration of the experiment. Among all sugars, raffinose showed distinctive responses associated with the two photoperiod regimes.

Under the LD regime, raffinose concentrations were low and similar among cultivars, whereas under SD regimes raffinose accumulation was generally higher than under LD regimes and cold hardy cultivars C-3309 and CD accumulated higher concentrations than did the cold sensitive cultivar CF. Basal leaves and buds accumulated the most raffinose.

The results suggest that raffinose accumulation might be an early step in the cold acclimation process that coincides with slowed shoot growth, and induction of endodormancy, which leads to the acquisition of FT.

Key Words: buds, cold acclimation, freezing tolerance, dormancy, leaf, raffinose, Vitis

46 Woody plants including Vitis sp. go through an annual cycle of growth and dormancy related to seasonal changes in the critical environmental components: photoperiod and temperature. The first stages of acclimation relate to photoperiod and the later stages to low non-freezing temperatures, both of which function in preparing plants for freezing temperatures (Sakai and Larcher, 1987, Weiser, 1970). This early acclimation has distinct events that occur and include physiological changes such as growth cessation, dormancy induction in buds, partial development of FT and the synthesis of metabolites that aid in promoting these changes (Guy, 1990, Sakai and Larcher, 1987). These environmental stimuli then become important factors in winter survival. SD photoperiod influences the timing of growth cessation and dormancy at the end of the growing season (Sakai and

Larcher, 1987, Weiser, 1970). Plant leaves receive the photoperiod stimulus and a factor, possibly phytochrome, moves to the bark tissues, stems and buds and initiates the changes in the plant. The changes in photoperiodic conditions perceived probably induce the synthesis of hormones such as ABA and/or facilitate the release of ABA, which is then translocated to the various plant tissues inducing temporary suspension of metabolic activities and totally inhibiting mitotic activity (Chao et al., 2007).

Dormancy, generally defined as the temporary suspension of visible growth of any plant structure including buds, is further categorized into paradormancy, where growth is inhibited by physiological factors outside the bud, endodormancy, where growth is inhibited by physiological factors inside the bud, and ecodormancy, where growth is inhibited by environmental factors (Lang et al. 1987). Some species of grapes begin to acclimate in response to SD prior to the low temperature stimulus, allowing the grapevines to initiate the changes involved in cold acclimation and a state of

47 endodormancy (Fennell and Hoover, 1991, Garris et al. 2009, Wake and Fennel, 2000).

While grapevines do not set terminal buds, they exhibit other hallmark phenotypes such as periderm development, growth cessation, shoot tip abscission and the induction of endodormancy in auxillary buds (Fennell and Hoover, 1991, Wake and Fennel, 2000).

There are also photoperiod sensitive ecotypes such as V. riparia and V. labrusca that will shed their leaves much earlier, even before low temperature stimulus is present. The understanding of why these grapevines respond differentially to photoperiod is not known but genetic marker studies by Garris et al. (2009) have revealed some candidate genes for further study such as the phytochrome genes PHYA and PHYB and the

Flowering Locus T gene family FT/TFL1.

External signals such as light and internal signals such as hormones and sugar act through specific overlapping signal transduction pathways to regulate endo-, eco- and paradormancy. The physiological changes that occur in response to the external signal -

SD photoperiod and the physiological effects of such signal have been documented for some Vitis sp. However, internal responses of sugars to SD have not been documented.

The role of sugars in the development of cold acclimation has been well documented in grapes. Total soluble sugars in grapevines generally increase during the initial stages of cold acclimation and among sugars, the raffinose family of oligosaccharides (RFO) is the most important since it has been frequently observed to change exclusively with cold acclimation (Badulescu and Ernst, 2006, Hamman et al. 1996, Stushnoff et al. 1993,

Wample and Bary, 1992). There is also differential accumulation of raffinose in cold sensitive and cold hardy grape cultivars in side-by-side controlled environment experiments. Cold hardy cultivars such as ‘Frontenac’, C-3309, and CD accumulated the

48 highest amount of raffinose in both bud and leaf tissues and cold sensitive cultivar, CF accumulated the least (Grant et al., 2009).

There are challenges to separate the effects of photoperiod and temperature on cold acclimation and dormancy as the plant may need both external signals to fully implement biophysical changes needed for survival (Garris et al., 2009, Li et al., 2002). There is however, a need to understand what interplay there may be between these environmental cues that will help to determine possible downstream targets of the environmental signals and the effects of the timing of the growth cessation and the depth of dormancy induced and cold acclimation. It may be that the beginning of the development of dormancy, growth cessation and initial changes in cold acclimation and the biochemical changes that occur related to these changes may be why some grapevines are more tolerant than others. The purpose of this study was, therefore, to characterize the morphological, physiological and biochemical changes that occur in cold sensitive and cold tolerant grape species in response to SD and how it may influence their dormancy and FT.

Materials and Methods

Plant Materials and Treatments: Two-year old grape cultivars of contrasting cold hardiness, as rated previously (Dami et al., 2005, Zabadal et al., 2007) were used in this study: Vitis riparia x V. rupestris ‘Couderc 3309’ (cold hardy), V. labruscana ‘Concord’

(cold hardy), and V. vinifera, ‘Cabernet Franc’ (cold sensitive). Plants were dormant pruned to retain three to four buds. After bud break, two of the strongest shoots were retained and trained vertically on stakes. All flower clusters were removed. The plants

49 were grown under climate-controlled un-shaded glass greenhouses with the following conditions: 25oC/20oC and 40/70% relative humidity (day/night). Supplemental light was provided automatically when photosynthetic photon flux (PPF) density was below 600

μmol m-2s-1 using high pressure sodium lamps (Sunlight Supply, Woodland, WA) and to maintain a 16-hr photoperiod. All plants were watered daily and fertilized every other day with 100 ppm 20-20-20 fertilizer (Peter’s Professional, Marysville, OH). Vines having uniform growth and 12-15 leaves were selected and randomly assigned to each treatment. The experiment was set up using a split plot design with three blocks: photoperiod as the main plot and cultivar as the sub plot. Four pots were used per cultivar, each with two vines.

Photoperiod experiments were conducted for four, six and eight weeks. Plants were grown under long day (LD): 16/8 hr day/night or short day (SD): 8/16 hr day/night in a climate controlled un-shaded greenhouse. To maintain eight hour darkness, black plastic sheeting (Sunbelt plastic, Minneapolis, MN) was draped over the SD plants and opened and closed at time points to maintain the specified length of time. The experiments were conducted twice using the same vines in 2009 and 2010.

Physiological and Morphological Assessment: Leaf and node numbers, and shoot length were measured on one vine in each pot prior to the exposure to the photoperiod treatment and every week thereafter. Data was recorded as a change in growth at four, six and eight weeks of treatment. Periderm development was also assessed at the end of each time period, by counting shoot internodes that changed color from green to tan or brown.

50 Periderm formation was expressed as the ratio of number of brown to total number of internodes per shoot.

Dormancy induction was determined at the end of each time period: four, six and eight weeks. Shoots were cut back to two basal buds and plants placed under LD conditions (16/8 hr day/night) to determine the depth of dormancy induced. Budburst was recorded as EL stage five and monitored every two days for 32 days. Dormancy was estimated as the number of days until 50% budburst (D50BB) and also the percent of budburst (%BB) at 32 days. The higher D50BB, the more buds are dormant. An increased D50BB indicated that buds were endodormant.

FT was determined using thermal analysis, which measures the low temperature exotherm (LTE) detected at the ice nucleation temperature for the buds. Canes were collected and placed in plastic bags to prevent moisture loss. To determine changes in freezing tolerance five buds from each replication, node positions three to eight, were excised from collected canes in the laboratory and loaded onto thermoelectric modules

(MELCOR, Trenton, NJ). The loaded modules were placed in a Tenny programmable freezer (Tenny Inc., New Columbia, PA) and subjected to a controlled freezing rate of

4oC/hr. by lowering the temperature from 2oC to -45oC. The FT of each treatment- replication corresponds to the mean LTE and is expressed as LT50 in oC.

Qualitative and quantitative analyses of soluble sugars: Analyses of soluble sugars were conducted on leaves and buds at the end of four six and eight weeks of the photoperiod treatment. Leaves and buds (nodes positions two to eight) were collected and were immediately placed at -80oC. The frozen leaves were lyophilized (Vitis

51 Freezemobile, New York, NY) and ground to pass through a 20-mesh screen. Buds were excised, plunged in liquid nitrogen and pulverized with mortar and pestle, and then lyophilized. The dry bud tissue was mixed with 75% ethanol in a tube, vortexed and allowed to stand at room temperature for two to four hours vortexing occasionally. The tubes were centrifuged for five minutes at 10 000 rpm. The supernatant was transferred to a vial then dried under air at 45oC. The extraction was repeated twice. The extracted bud metabolites and approximately five mg of leaf tissue were derivitized using the method by Streeter and Strimbu (1998). This involved mixing the ground leaf tissue or extracted bud metabolites with pyridine containing hydroxylamine and an internal standard, phenyl

-D glucopyranoside (Sigma-Aldrich, St. Louis, MO). After incubation at 70oC, the oxime derivatives were reacted with hexamethyldisilazane (HMDS) and trifluoroacetic acid (TFA) (Sigma-Aldrich, St. Louis, MO) to form the tri-methylsilyl (TMS) derivatives of the oximes. The derivatives were injected into a gas chromatograph (Hewlett Packard

5890 Series II, Hewlett Packard, Boulder, CO) with a 30 m capillary column (HP5-MS,

250 m inner diameter and 0.25 m thickness). Injection temperature was 280oC and oven ramp was: 180oC hold for two minutes, 6oC/minute ramp to 215oC, hold one minute, 40oC/minute ramp to 320oC, hold for 22 minutes. Helium, the carrier gas, was at a constant flow rate of 1.0 ml.min-1. Soluble sugars were identified and peaks quantified

(Chemstation Quantitation Process Program, Agilent Technologies, Wilmington, DE) by comparison to standard sugars and the internal standard, phenyl -D glucopyranoside.

Statistical Analysis: Statistical analysis of morphological, physiological characteristics and soluble sugar concentrations was conducted using SAS (SAS Institute, Cary, NC) 52 and LSD was used to compare means at p  0.05. Correlation analyses were also conducted to determine the association between LT50 and raffinose concentrations for four, six and eight weeks for both leaves and buds.

Results

Shoot length: Grapevines grown under SD photoperiod had reduced shoot length compared to that in grapevines grown under LD for all time periods and in both years. In

2009, there was a 23% reduction in shoot length at week six and 37% at week eight

(Table 3.1). In 2010, there was a 20% reduction in shoot length at week four and a 33% reduction at both week six and week eight (Table 3.1). Comparisons between cultivars in

2009 revealed significant differences of shoot length for all time periods, but no differences were seen between cultivars in 2010 (Tables 3.1). When comparing LD to SD among cultivars, all three cultivars showed a decrease in shoot length beginning after two weeks exposure to the SD treatment and no shoot elongation after week four (Figure 3.1).

These differences were seen in both years. There were no interactions between cultivar and photoperiod in either year.

Node number: Similar to shoot length, the change in the number of nodes also decreased significantly in SD grapevines compared to LD for both 2009 and 2010 (Table 3.1). The number of nodes per vine increased under LD conditions from 23 nodes at week four to

29 nodes at week eight in 2009, but under SD conditions, the increase over time was very minimal, with the grapevines gaining only one node between four and eight weeks. A

53 similar trend was also seen in 2010 with vines having 14-41% differences from week four to week eight. The reduced node number is also comparable to that seen for reduced shoot length. Both morphological traits showed approximately a 23% reduction at week six and 37-38% at week eight for 2009. Reductions in node number were, however, higher in 2010. There were also differences that were cultivar related at four, six and eight weeks for both 2009 and 2010. CD showed no increase in the number of nodes after week three under SD, while C-3309 and CF did not stop growing until week five (Figure

3.2). There were, however, no interactions between cultivar and photoperiod except at four weeks in 2009.

Periderm development: There was an increase of periderm formation on SD grapevines four to six weeks into the photoperiod treatment compared to LD (Table 3.1). No periderm was seen at four weeks in 2009 but there was some development in the 2010 experiment showing 94% difference between treatments. At six weeks and in both years, marked increases in the periderm development with SD plants having more than 50% of their nodes turning brown, a 92% difference compared to LD grapevines. The trend continued at eight weeks under SD with grapevines showing periderm on more than 52-

58% of the vine compared to only between zero to 16% in LD, a 68 to 100% difference between LD and SD in 2009 and 2010 (Table 3.1). Cultivar differences were only apparent at six weeks in 2009, with CF having the highest percentage of periderm compared to C-3309 and CD. No cultivar differences were observed in 2010. There was some interaction between cultivars and photoperiod at six weeks in 2009 (p = 0.014) and

54 eight weeks in 2010 (p = 0.003). This interaction is from CF which consistently had higher percentage of periderm compared to the other two cultivars.

Dormancy: The shoots from grapevines that were exposed to LD had 100% budburst within two to three weeks after 32 days under forcing conditions in both 2009 and 2010

(Figure 3). However, exposure to SD photoperiod decreased budburst with 40 to 80% budburst when exposed for four weeks, 16 to 60% for six weeks and 0 to 4% for eight weeks after 32 days under forcing conditions (Figure 3.3). The delay of budburst indicated that the SD grapevines entered endodormancy, which was initiated after four weeks exposure to SD and reached full endo-dormancy after eight weeks. There were also differences among cultivars in response to SD. CD showed increased response to the

SD photoperiod having the least percentage of budburst after 30 days compared to CF and C-3309. After four weeks in SD, CD showed significant difference with only 63 to

74% budburst compared to approximately 78 to 99% for CF and C-3309 in 2009 and

2010, respectively (Table 3.2). This trend continued for grapevines exposed to six weeks

SD, with CD having the least budburst followed by C-3309 then CF in 2009, but there were no differences among cultivars in 2010. After eight weeks exposure to SD in 2009 there was a 48 to 52% difference with CD having only 24% budburst compared to 50% for C-3309 and 47% for CF. In 2010, however, there were no differences among cultivars all having only approximately 50% budburst after 30 days under forcing conditions.

For vines exposed to SD for four weeks, LD plants took only 16 D50BB whereas

SD plants needed 19-28 D50BB in 2009 and 2010 (Table 3.2). For vines exposed to six weeks SD plants needed more than 32 days for 2009 and 24 days in 2010. For eight

55 weeks SD, even after 32 days under forcing conditions, there was no increase in budburst frequency with percent budburst remaining well below 50%. CD showed the highest number of days for D50BB under SD, 25-30 days in 2009 and 23 to 25 days in 2010. CF and C-3309 had lower D50BB and showed no differences between each other until after eight weeks SD conditions.

The delay in budburst for CD plants indicated that CD was the first grapevine to initiate endo-dormancy starting after four weeks exposure to SD compared to the other two cultivars, which did not initiate endodormancy until after six weeks treatment. C-

3309 having 0% budburst was more endodormant than CF, which had 10% budburst after

8 weeks (Figure 3.3). SD treatment affected all cultivars, which had almost no budburst indicating they were all endo-dormant (Figure 3.3).

Freezing tolerance: The LT50s were the same among all cultivars under the LD regime for four, six and eight weeks (Table 3.3). However, under SD regime, FT increased

(LT50 decreased) (Figure 3.4). LT50 in buds was different between LD (-7.7oC) and SD

(-8.4oC) even after 4 weeks of treatment (Table 3). After six weeks treatment, the LT50 continued to decrease showing a 2oC difference between LD and SD. After eight weeks of SD treatment the LT50 dropped another degree to approximately -10.5oC for SD grapevines. While there were no differences among cultivars until week eight (Table 3.3), there were significant interactions that were photoperiod dependent (Table 3.4). There were no differences among cultivars after four weeks of treatment except for CD, which had the lowest killing temperature of -9.5oC (Table 3.4). After six weeks, the LT50 of C-

3309 and CD continued to decrease and were 2 to 3oC lower than that for CF, which

56 remained the same between photoperiod treatments. After eight weeks CF began to show significant differences between treatments with a decrease of LT50 by -2.4oC. CD and C-

3309 continued to show decreased LT50s reaching -10.5oC and -12.5oC, respectively

(Table 3.4).

Qualitative and quantitative analyses of soluble sugars: The sugars detected in both leaves and buds for all cultivars were fructose, glucose, myo-inositol, sucrose, raffinose and galactinol. Sucrose was the predominant sugar in leaf tissues with 34 to 44% of total soluble sugar concentration on a dry weight basis. In buds tissues, sugar concentrations were generally much lower (43% less) than in leaves and fructose, glucose and sucrose were the predominant sugars, each sugar ranging between 25 to 35% of total soluble sugar concentration (Tables 3.5 and 3.6). Concentrations for sucrose ranged between 25 and 52 mg.g-1 for LD and between 33 and 60 mg.g-1 for SD treated leaves and 10 to 14 mg.g-1 and 12 to 27 mg.g-1 for LD and SD treated buds, respectively (Table 3.5 and 3.6).

All of the sugars detected also showed increases in concentration in response to the photoperiod treatment but the changes were not always significant or consistent with the photoperiod treatment or the length of the photoperiod treatment. While myo-inositol and galactinol showed little to no variation in concentration, there was variation in the concentration of fructose, glucose and sucrose and this was seen for both years at photoperiod durations (Table 3.5). In 2009, significant differences were observed between LD and SD for fructose but no differences were observed for glucose or sucrose after four weeks of treatment (Table 3.5). After six weeks of treatment, there were significant differences for glucose and sucrose but no differences for glucose (Table 3.5).

57 After eight weeks of treatment, differences were observed for fructose and glucose, but not for sucrose (Table 3.5). This variation in concentration was also observed in buds.

The only sugar that consistently changed in response to the photoperiod treatment was raffinose. In both years and for both leaves and buds, raffinose concentrations were consistently higher in SD compared to that in LD treated grapevines (Figure 3.5). After four weeks of treatment, raffinose concentrations were more than 14 times higher than

LD in 2009 and about five times higher in 2010 for leaves and two to 11 times higher in buds (Table 3.5 and 3.6). After six weeks of treatment the concentrations increased to

3.9 and 1.3 mg.g-1 for leaves and buds respectively in 2009 and 2.75 and 1.4 mg.g-1 in

2010, also five times higher than LD tissues (Table 3.5 and 3.6). After eight weeks treatment, the increase was two to three times higher for both tissues and years (Table 3.5 and 3.6). Differences of raffinose among cultivars were also observed. CF had the lowest concentrations of raffinose ranging from 0 to 1.4 mg.g-1 for leaves and buds, whereas CD and C-3309 had much higher concentrations of up to 3.7 and 2.3 mg.g-1, respectively.

After four weeks treatment concentrations were one to two times higher for CD and C-

3309, respectively and this was consistent in both years. After eight weeks treatment the concentrations were the highest, more than three times the concentration of CF. Cultivar variation in the raffinose concentrations were also photoperiod dependent as is indicated by significant interaction. All cultivars had higher concentrations under the SD regime compared to LD for 2009 and 2010 for both leaves and buds (Figures 3.5 and Tables 3.5 and 3.6). Concentrations generally increased over time, with six and eight weeks having higher concentrations for all cultivars.

58 Correlation analyses conducted between leaf and bud raffinose concentration with

LT50 showed an inverse relationship between leaf raffinose and LT50 for all 3 photoperiod durations (Figure 3.6). There was, however, no correlation between bud raffinose concentration and LT50 (Figure 3.7). This showed that in response to photoperiod, the relationship between raffinose and FT was different between the two tissue types.

Discussion

SD consistently inhibited the shoot growth of the grapevines starting two weeks into the experiment for all three cultivars tested and this also resulted in short internode length in these grapevines (Figure 3.1). The responses to SD photoperiod were cultivar and time dependent with CD being the first to slow in shoot growth and complete growth cessation by week three. C-3309 and CF also had reduced shoot growth but plants did not cease growth until after four weeks of SD treatment. This reduced growth response is well documented in grapevines. Fennell and Hoover (1991) demonstrated this response in

V. labruscana (Bailey) and V. riparia that responded to SD and initiated growth cessation. While several woody plants cease growth in response to both temperature and photoperiod, it has been demonstrated that grapevines are able to initiate growth cessation in response to SD only (Fennel and Hoover, 1991, Salzman et al., 1996,

Schnabel and Wample, 1987, Wake and Fennel, 2000). Growth cessation is considered an early step in the process of dormancy development and has been widely documented as an early response for FT development (Sakai and Larcher, 1987). It involves the

59 termination of cell division in apical and auxillary meristematic tissues that causes growth inhibition and also the suppression of internode elongation, occurring before leaf fall and allowing the plant to begin the process of FT development through the redirection of resources into overwintering plant tissues (Kalcsits et al., 2009, Sakai and

Larcher, 1987). There are other woody plants that also show growth cessation in response to SD including poplar (Horvath et al., 2003), cottonwood (Populus trichocarpa) (Howe et al., 1995), and birch (Betula pubescens) (Juntilla et al., 2003).

The mechanisms of these woody species include setting terminal apical buds, a feature not expressed by grapevines, which have apical tip abscission.

Wood maturation or the progression of periderm from the base of the shoots in grapevines is one of the indicators of acclimation and dormancy development (Fennel and Hoover 1991, Salzman et al., 1996). As the green stem matures, the cortex senesces and many compounds are translocated to interior tissues that are acclimating (Zabadal et al., 2007). In this study, periderm development on grapevines occurred in both photoperiod treatments, but with SD-treated grapevines having significantly higher numbers of lignified nodes, forming at a faster rate than those in LD-treated vines. The development of periderm was observed in all three cultivars with its progression increasing throughout the experiment. This is consistent with previous experiments and confirms that the plants are initiating early responses to the SD photoperiod stimulus.

Periderm formation from the base of the shoot of grapevines to the tip has been reported to be related to a development program that divides the vine into distinct zones (Salzman et al., 1996). Tissues that develop periderm are programmed to begin endodormancy and will begin developing FT, but those tissues that are without periderm are not programmed 60 to do so (Fennel and Hoover, 1991, Salzman et al., 1996). The results of this study agree with previous reports. SD-treated grapevines displayed significant increases in periderm formation, entered endo-dormancy resulting in the acquisition of some FT. Even though

LD plants developed some periderm, it was at a significantly reduced rate and these grapevines did not enter endo-dormancy and did not increase in FT. Wake and Fennel

(2000) had similar observations and proposed that periderm may be more related to grape tissue development and is enhanced by SD, but it cannot only be used as an indicator of dormancy.

The main environmental factor that induces dormancy development is SD photoperiod (Lang et al., 1987). CD grapevines responded to the SD treatment and had the least percentage of budburst after four weeks SD treatment as compared to the other two cultivars indicating it had achieved endodormancy earlier and it was deeper than the other cultivars. C-3309 and CF also responded to the SD treatment by entering endodormancy but both cultivars needed at least six weeks of SD treatment. This study confirms the observation that variation in response to SD is cultivar dependent in grapevines. Fennell and Hoover (1991) showed that SD induced dormancy in both V. labruscana ‘Bailey’ and V. riparia. Wake and Fennell (2000), however, demonstrated that V. spp. ‘Seyval Blanc’ did not go dormant when treated with SD and they suggested that this cultivar might need low temperature treatment for dormancy induction. Schnabel and Wample (1987) demonstrated this synergistic effect for V. vinifera L. ‘Reisling’. SD and low temperatures individually were able to promote some dormancy and FT, but when in combination both produce an additive effect. In this study, all three cultivars initiated dormancy development and reached a state of deep endo-dormancy after eight 61 weeks of SD without cold temperature treatment. The differences seen here indicate that cultivar responses to photoperiod have an effect on dormancy induction, which may relate to the differences in FT observed for these cultivars. Overall, bud tissues were able to withstand freezing stress at significantly lower temperatures for the SD buds compared to LD buds. CD, however, was first initiating this change at four weeks then C-3309 at six weeks followed by CF at eight weeks. Fennel and Hoover (1991) also demonstrated that there was a change in the LT50 of grapevine buds after SD treatment. They also concluded that the change in FT corresponds with the timing of dormancy induction. In this study, CD plants initiated dormancy at four weeks of SD treatment and also had an increase in FT at the same time. The timing of the development of dormancy and then FT development are therefore linked to the responses of plants to the environmental cue - photoperiod.

SD’s initiate reduced growth, dormancy and FT and the changes in the plant related with FT may first be associated with endo-dormancy induction (Kalcsits et al.,

2009). In this study, grapevines had growth cessation from as early as three weeks into the photoperiod treatment, long before the beginning of deep endo-dormancy. The changes related to increased FT, however, coincided with dormancy development. CD grapevines that had ceased growing three weeks into the SD treatment, began to see changes in FT at week four and were the only vines to also have some dormancy initiation at four weeks indicating that the process has begun. Previous research has demonstrated that signal transduction pathways starting from the perception of short-days and leading to initial changes in dormancy and FT are operational in leaves of birch

62 (Betula pendula) and this early development is a possible protective mechanism during autumn, enabling prolonged periods for photosynthesis (Li et al., 2002).

Changes in soluble carbohydrates are correlated with the subsequent increased FT in grapevines (Hamman et al., 1996, Wample and Bary, 1992). In this study, myo-inositol and galactinol showed little to no variation in concentration, but there was variation in the concentration of fructose, glucose, sucrose and raffinose. The variation among fructose, glucose and sucrose was, however, not consistent indicating that photoperiod treatment may have little to no control over the changes observed in these grapevines. Photoperiod might probably not be involved in the accumulation of these sugars in the tissues tested and the changes observed may be related to other physiological changes occurring in the plant. Raffinose concentration did, however, change consistently in response to the photoperiod treatment and also in this study, FT was closely associated with leaf raffinose concentrations but not with bud raffinose. This could indicate that early the change in LT50 is more related to early response in the leaves and later in the buds.

The accumulation of RFO during cold acclimation has been documented in a wide variety of plants, including leaves of Ajuga reptans L. (Bachman et al., 1994), and alfalfa

(Medicago sativa L.) (Castonguay et al., 1995). Accumulation of RFO has also been reported in dormant tissues of woody plants including grape (Vitis sp.) (Grant et al.,

2009), crabapple (Malus sp.), apple (Malus sylvestris), dogwood (Cornus sp.) (Stushnoff et al., 1993), red raspberry (Rubus idaeus L.) (Palonen and Juntilla, 2002), and aspen

(Populus tremuloides Michx.) (Cox and Stushnoff, 2001). The response in grapes has also been cultivar related with differential accumulation of RFO’s in sensitive and hardy

63 grape cultivars. It was found that cold hardy cultivars such as Frontenac, C-3309, and CD accumulated the highest amount of raffinose in both bud and leaf tissues and cold sensitive cultivars such as CF accumulated the least (Grant et al., 2009). This pattern of accumulation is consistent with this study with CD and C-3309 also accumulating higher concentrations of raffinose compared to CF. To our knowledge, this is the first report demonstrating the accumulation of raffinose in grapevines in response to SD.

The beginning of these changes in response to SD without any temperature stimulus may indicate a role outside of cryo-protection. Water content decreases with induction of both dormancy and FT and therefore results in desiccation stress (Fennell et al., 1996, Salzman et al., 1996, Wolpert and Howell, 1986). It is suggested that raffinose is important in dormancy and FT because it aids in protecting the tissues from desiccation stress. Raffinose accumulation was seen to occur concurrently with the reduction in water content as occurs in seed maturation and desiccation (Hannah et al., 2006,

Peterbuer and Ricter, 2001). Many overwintering plant parts have to survive desiccation stress so it may be that the factors that are activated by changes in photoperiod are the same factors that up-regulate changes in raffinose concentration.

Raffinose has special properties that go beyond reserve and several roles have been proposed in the literature. At low temperatures, raffinose delays the crystallization of sucrose (Caffrey et al., 1988, Koster and Leopold, 1988) and it does not change its configuration with decreasing temperatures (Jeffrey and Huang, 1990) allowing it to have structure-preserving effect upon binding to proteins and membranes (Lineberger and

Steponkus, 1980, Santarius, 1973). The sugar molecules may function by forming hydrogen bonds with macromolecules and thus may substitute for water during

64 desiccation stress, thus allowing them to maintain their hydrated orientation (Crowe et al., 1988). Accumulation may also decrease the osmotic potential, which depresses the freezing point of cell water. This is possibly through a colligative effect where the sugars change the bulk properties of the solution (Burke et al., 1976). Soluble sugars may also protect cells by forming intracellular glass, an undercooled liquid with the viscosity of a solid and its formation would ensure stability during periods of dormancy by preventing further desiccation and stabilizing cell structures (Burke, 1986). Glass forms at the glass transition temperature - Tg. Raffinose is a trisaccharide, therefore has a higher molecular weight than monosaccharides and disaccharides and therefore it is more effective because it has a higher Tg and will form glass more readily (Franks, 1985). In other words, raffinose is more protective than the disaccharide sucrose or the monosaccharides glucose and fructose. It is possible that the characteristics of raffinose allow it to function by any or all of the above mechanisms.

In an earlier study, exposure to low temperature increased raffinose concentration that led to a suite of changes that occurred during the first stages of the acclimation process coinciding with slowed shoot growth, but preceding periderm formation and subsequent acquisition of FT (Grant et al. 2009). In this study we demonstrated there are many contributing factors, including morphological, physiological, and biochemical factors that take place in a wide range of grape genotypes. These responses are genotype dependent, which may explain changes that make a grape cultivar more FT than another.

It is concluded that grapevines responded similarly when environmental cues (SD and low temperature) were administered separately. Although responses such as slowed shoot growth, periderm formation, dormancy induction, and the acquisition of FT have been

65 previously reported, to our knowledge, this is the first report on the accumulation of raffinose in leaves and buds of grapevines in response to either SD or low temperature prior to any exposure to sub-freezing temperatures.

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70 Table 3.1. Shoot length, number of nodes and periderm formation for Cabernet franc (CF), Couderc 3309 (C-3309) and Concord (CD) grapevines grown under long day (LD) and short day (SD) photoperiod regimes. Shoot length (cm) Nodes/vine Periderm (%) z 4wks 6wks 8wks 4wks 6wks 8wks 4wks 6wks 8wks 2009 Photoperiod (P): LD 148 151a 162a 23a 26a 29a 0 0b 0b SD 130 115b 102b 20b 21b 21b 0 24a 58a p-value 0.14 0.05 0.03 0.04 0.05 0.02 0.04 0.002

Cultivar (C): CF 160a 164a 159a 26a 29a 30a 0 17a 30 C-3309 154b 146a 139a 23b 25b 25b 0 11b 29 CD 105c 89b 97b 15c 15c 17c 0 8b 28 p-value 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.014 0.76

71 (P x C) 0.56 0.49 0.27 0.0001 0.64 0.64 0.014 0.76

p-value 2010 Photoperiod (P): LD 226a 263a 271a 32 39a 41a 1b 4b 17b SD 179b 176b 183b 28 28b 29b 22a 50a 53a p-value 0.042 0.023 0.028 0.06 0.01 0.02 0.026 0.005 0.01

Cultivar (C): CF 215 228 228 35a 37a 39a 10 29 39 C-3309 202 217 227 31b 35b 36a 15 25 33 CD 190 215 226 24c 29c 29b 10 26 31 p-value 0.14 0.58 0.98 0.0001 0.0001 0.0001 0.47 0.69 0.16

(P x C) 0.65 0.55 0.74 0.77 0.69 0.91 0.64 0.25 0.003 p-value a,b,c, Means with different letters in columns of each factor are significantly different at p0.05 zPeriderm development recorded as percent of shoot internodes that changed color from green to brown. 71

D50BB %BB at 30 days 4wks 6wks 8wks 4wks 6wks 8wks 2009 Photoperiod (P): LD 16b 17b 20b 100a 100a 80.5a SD 28a 32a 32a 49b 16b 0b p-value 0.0001 0.001 0.0001 0.0001 0.0001 0.0001

Cultivar (C): CF 22b 25ab 26b 78a 75a 47a C-3309 18c 23b 22c 81a 50b 50a CD 25a 26a 30a 63b 45b 24b p-value 0.0005 0.12 0.0001 0.03 0.001 0.0001

(P x C) 0.07 0.01 0.0001 0.03 0.001 0.001 p-value

2010 Photoperiod (P): LD 15b 14b 14b 100a 99a 100a SD 19a 24a 32a 80b 60b 4b p-value 0.0008 0.0002 0.0001 0.0001 0.0001 0.0001

Cultivar (C): CF 12b 15b 23b 99a 85 55 C-3309 13b 18b 22c 97a 76 50 CD 25a 23a 25a 74b 79 50 p-value 0.0001 0.01 0.0005 0.0001 0.25 0.15

(P x C) 0.003 0.07 0.0005 0.0001 0.09 0.15 p-value a,b,c Means with different letters in columns of each factor are significantly different at p0.05

Table 3.3. Freezing tolerance (LT50 in oC) of Couderc-3309 (C-3309), Cabernet franc (CF) and Concord (CD) of basal buds after exposure to long day (LD and short day (SD) photoperiod at 4, 6 and 8 weeks.

LT50 4wks 6wks 8wks Photoperiod (P): LD -7.7b -7.7b -7.8b SD -8.4a -9.7a -10.5b p-value 0.05 0.0001 0.0001

Cultivar (C) : CF -7.5a -7.2a -7.3b C-3309 -8.2a -9.1a -8.9a CD -8.4a -8.7a -9.7a p-value 0.14 0.11 0.0007

P x C 0.02 0.007 0.01 p-value a,b,c Means with different letters in columns of each factor are significantly different at p0.05

Table 3.4. Freezing tolerance (LT50 in oC) of Couderc-3309 (C-3309), Cabernet franc (CF) and Concord (CD) of basal buds after exposure to long day (LD and short day (SD) photoperiod at 4, 6 and 8 weeks.

Photoperiod duration Cultivar LD SD 4 Weeks CF -7.6a -7.5a C-3309 -8.1a -8.4ab CD -7.3a -9.5b

6 Weeks CF -8.1ab -8.3b C-3309 -8.0ab -10.3c CD -7.0a -10.5c

8 Weeks CF -6.1a -8.5b C-3309 -6.9a -12.5c CD -7.4ab -10.5c a,b,c Means with different letters for each photoperiod duration are significantly different at p0.05

Table 3.5. Soluble sugar concentration (mg g-1 dry wt) in leaves of Cabernet franc (CF), Concord (CD) and Couderc 3309 (C-3309) grapevines exposed to 4, 6 and 8 weeks long day (LD) and short day (SD) photoperiod regimes

4 Weeks

Cultivar Fructose Glucose Myo-inositol Sucrose Galactinol Raffinose

2009 Photoperiod (P) LD 19.8b 24.1 8.7 37.7 1.1 0.1b SD 31.6a 35.1 8.6 44.3 1.3 1.5a p-value 0.04 0.06 0.8 0.11 0.31 0.0001

Cultivar (C) CF 24.8 28.3 11.7a 31.5b 0.8b 0.2b C-3309 26.3 28.9 7.3b 46.8a 1.1ab 1.3a CD 26.1 31.7 7.1b 44.6a 1.7a 0.9ab p-value 0.97 0.87 0.0001 0.01 0.03 0.01

(P x C) 0.21 0.19 0.91 0.02 0.99 0.07 p-value

2010 Photoperiod (P) LD 23.3 25.5 6.4 25.0b 0.9 0.3b SD 20.5 23.4 5.4 33.0a 0.6 1.9a p-value 0.06 0.1 0.18 0.05 0.06 0.0001

Cultivar (C) CF 20.6b 21.7b 9.2a 37.3a 0.2b 0.0c C-3309 30.0a 27.1a 4.1b 23.3b 1.2a 2.2a CD 15.1c 24.4ab 4.4b 32.4b 0.9a 1.1b p-value 0.0001 0.01 0.0001 0.02 0.002 0.0001

(P x C) 0.13 0.01 0.004 0.10 0.82 0.004 p-value a, b, c Means with different letters in columns of each factor are significantly different at p 0.05

Continued

Table 3.5. Continued

6 Weeks

Cultivar Fructose Glucose Myo-inositol Sucrose Galactinol Raffinose

2009 Photoperiod (P) LD 20.1 26.1b 9.6 43.1b 1.5 0.7b SD 25.4 31.5a 9.4 60.7a 1.7 3.9a p-value 0.08 0.03 0.67 0.0001 0.23 0.0001

Cultivar (C) CF 23.3ab 24.9b 12.7a 57.8a 1.7ab 0.7c C-3309 26.6a 29.6ab 7.6b 47.0b 1.3b 3.8a CD 18.3b 31.7a 8.1b 50.9ab 1.9a 2.4b p-value 0.05 0.05 0.0001 0.02 0.04 0.0001

(P x C) 0.11 0.08 0.47 0.06 0.005 0.0001 p-value

2010 Photoperiod (P) LD 25.5 26.9a 7.1 51.6 1.2 0.1b SD 31.7 31.1b 6.2 51.4 1.2 2.8a p-value 0.08 0.03 0.08 0.95 0.98 0.0001

Cultivar (C) CF 26.6ab 24.9 8.2a 55.1 1.6a 0.6b C-3309 31.5a 38.5 5.9b 50.0 1.0b 1.3a CD 17.7b 23.8 5.8b 49.4 0.9b 1.9a p-value 0.05 0.17 0.003 0.31 0.02 0.02

(P x C) 0.66 0.52 0.19 0.01 0.01 0.01 p-value

Continued

Table 3.5. Continued

8 Weeks

Cultivar Fructose Glucose Myo-inositol Sucrose Galactinol Raffinose 2009 Photoperiod (P) LD 22.4b 29.1b 8.7 51.2 1.6b 1.1b SD 41.8a 43.5a 8.1 55.5 2.1a 2.8a p-value 0.05 0.05 0.15 0.50 0.05 0.0001

Cultivar (C) CF 20.8 25.5b 10.4a 59.5a 1.7ab 1.5b C-3309 42.9 40.7a 7.2b 36.6b 1.3b 2.2a CD 32.7 42.7a 7.6b 64.0a 2.5a 2.3a p-value 0.18 0.01 0.0002 0.008 0.03 0.005

(P x C) 0.01 0.004 0.02 0.19 0.47 0.0008 p-value

2010 Photoperiod (P) LD 27.6 28.7 7.4 52.5 3.9a 1.5b SD 37.9 38.8 7.4 51.4 2.4b 3.9a p-value 0.25 0.15 0.96 0.83 0.0005 0.0005

Cultivar (C) CF 24.9b 31.7ab 8.1 56.1 3.7a 0.8b C-3309 40.2a 47.1a 7.9 64.5 3.3ab 3.7a CD 33.1ab 22.6b 6.2 65.2 2.5b 3.6a p-value 0.005 0.03 0.24 0.26 0.02 0.0007

(P x C) 0.19 0.01 0.25 0.02 0.49 0.05 p-value

Table 3.6. Soluble sugar concentration (mg g-1 dry wt) in buds of Cabernet franc (CF), Concord (CD) and Couderc 3309 (C-3309) grapevines exposed to 4, 6 and 8 weeks long day (LD) and short day (SD) photoperiod regimes

4 Weeks

Cultivar Fructose Glucose Myo-inositol Sucrose Galactinol Raffinose

2009 Photoperiod (P) LD 18.2 17.9 4.5 12.2b 1.0 0.8b SD 17.0 18.1 3.8 22.0a 1.2 1.5a p-value 0.43 0.71 0.09 0.01 0.73 0.03

Cultivar (C) CF 17.9 13.7 3.1b 13.5b 0.5b 0.5b C-3309 14.8 19.1 3.0b 13.5b 0.9b 1.5a CD 20.2 21.2 6.3a 24.4a 2.0a 1.4a p-value 0.27 0.09 0.001 0.03 0.0006 0.01

(P x C) 0.25 0.03 0.64 0.01 0.20 0.02 p-value

2010 Photoperiod (P) LD 10.9 16.7 4.1 10.0 0.5 0.1b SD 17.9 19.6 2.8 12.0 0.4 1.1a p-value 0.29 0.29 0.08 0.27 0.92 0.02

Cultivar (C) CF 10.7 16.6 5.4a 24.0a 0.6 0.0b C-3309 18.8 15.9 2.3b 15.0b 0.3 1.2a CD 13.7 17.0 2.5b 17.0b 0.4 0.6b p-value 0.23 0.36 0.02 0.009 0.69 0.01

(P x C) 0.11 0.16 0.09 0.53 0.24 0.02 p-value a, b, c Means with different letters in columns of each factor are significantly different at p 0.05

Continued

Table 3.6. Continued

6 Weeks

Cultivar Fructose Glucose Myo-inositol Sucrose Galactinol Raffinose

2009 Photoperiod (P) LD 18.8b 15.9b 4.9a 10.0b 2.3a 1.2 SD 31.3a 31.7a 3.4b 27.4a 1.2b 1.3 p-value 0.001 0.0002 0.0003 0.0001 0.01 0.19

Cultivar (C) CF 20.7b 13.3b 3.2b 11.4b 1.4b 1.1 C-3309 24.1ab 28.2a 3.2b 23.7a 0.8b 1.2 CD 30.4a 30.0a 6.4a 20.9a 2.9a 1.5 p-value 0.05 0.001 0.0001 0.002 0.002 0.09

(P x C) 0.01 0.006 0.004 0.09 0.46 0.03 p-value

2010 Photoperiod (P) LD 13.9 12.2 5.3a 14.0 0.9 0.8 SD 18.2 17.5 1.7b 16.0 1.1 1.4 p-value 0.20 0.08 0.006 0.83 0.64 0.03

Cultivar (C) CF 16.2 14.1 2.6 12.0 0.3 0.7b C-3309 14.1 12.9 3.0 16.0 1.2 1.1b CD 17.8 17.5 5.0 18.0 1.6 2.5a p-value 0.21 0.13 0.19 0.23 0.31 0.02

(P x C) 0.99 0.90 0.59 0.30 0.65 0.03 p-value

Continued

Table 3.6. Continued

8 Weeks

Cultivar Fructose Glucose Myo-inositol Sucrose Galactinol Raffinose

2009 Photoperiod (P) LD 23.2 19.8 3.3a 14.3b 1.72a 1.7a SD 25.4 23.0 1.2b 24.0a 0.69b 2.5b p-value 0.1 0.09 0.0001 0.001 0.0001 0.0003

Cultivar (C) CF 18.9b 14.4b 1.6c 12.3b 1.0b 1.0b C-3309 30.7a 31.1a 2.2b 27.5a 0.8b 1.2b CD 23.3b 18.6b 3.0a 17.6b 1.8a 1.7a p-value 0.002 0.002 0.001 0.0007 0.001 0.006

(P x C) 0.02 0.61 0.003 0.13 0.004 0.01 p-value

2010 Photoperiod (P) LD 12.2b 11.5b 2.1a 14.7b 0.4 1.1b SD 21.4a 23.8a 0.8b 19.4a 0.4 2.6a p-value 0.01 0.002 0.001 0.02 0.89 0.02

Cultivar (C) CF 13.9 15.9 1.4 9.8 0.3 1.1b C-3309 15.9 16.4 1.6 13.3 0.3 2.0a CD 20.4 20.8 1.5 13.2 0.5 2.3a p-value 0.26 0.35 0.95 0.32 0.29 0.02

(P x C) 0.97 0.63 0.81 0.62 0.49 0.04 p-value

Figure 3.1. Shoot length progression of Cabernet franc (CF), Couderc 3309 (C-3309) and Concord (CD) grapevines exposed to long day (LD) and short day (SD) photoperiod regimes during an eight-week period in 2010. Measurements are means (n = 4).

Figure 3.2. Number of nodes of Cabernet franc (CF), Couderc 3309 (C-3309) and Concord (CD) grapevines exposed to long day (LD) and short day (SD) photoperiod regimes during an eight-week period in 2010. Measurements are means (n = 4).

Figure 3.3. Percent budburst for Cabernet franc (CF), Couderc 3309 (C-3309) and Concord (CD) grapevines grown under long day (LD) and short day (SD) photoperiod regimes at 8 weeks in 2010. Measurements are means (n = 4).

Figure 3.5. Raffinose concentrations of Couderc-3309 (C-3309), Cabernet franc (CF) and Concord (CD) grapevines basal leaves after exposure to long day (LD and short day (SD) photoperiod at 4, 6 and 8 weeks in 2010. Measurements are means +/- standard error (n = 4).

R= -0.588 p= 0.01 A

R= -0.793 B p0.001

R= -0.666 p= 0.003 C

Figure 3.6. Relationship of bud FT and leaf raffinose concentrations of Couderc-3309 (C- 3309), Cabernet franc (CF) and Concord (CD) grapevines basal leaves after exposure to long day (LD and short day (SD) photoperiod at (A) 4, (B) 6, and (C) 8 weeks.

R= -0.309 p= 0.213 A

R= -0.199 B p= 0.428

R= -0.331 C p=0.180

Figure 3.7. Relationship of bud FT and bud raffinose concentrations of Couderc-3309 (C- 3309), Cabernet franc (CF) and Concord (CD) grapevines basal leaves after exposure to long day (LD and short day (SD) photoperiod at (A) 4, (B) 6, and (C) 8 weeks.

Chapter 4: Seasonal changes of freezing tolerance and soluble sugars among field-grown Vitis genotypes

Trudi Grant and Imed Dami

The purpose of this study was to characterize seasonal changes in freezing tolerance (FT,

LT50) and the corresponding changes of specific soluble sugars, especially raffinose, in leaves and buds of three grape cultivars, Vitis riparia x V. rupestris ‘Couderc 3309’ (C-

3309, cold hardy), V. labruscana ‘Concord’ (CD, cold hardy), and V. vinifera, ‘Cabernet

Franc’ (CF, cold sensitive). The influence of genotype and bud position on FT and sugar concentrations also investigated as well as the influence of artificial de-acclimation. The seasonal changes in FT was similar in both years and the minimum LT50’s recorded in

January of both years ranged from -27.8 to -30.8oC and -24.4 to -31.9oC, respectively.

Generally basal buds were more FT than middle and apical buds. The cold-hardy cultivars CD and C-3309 had the lowest LT50 compared to the cold sensitive CF. There was variation in sugar concentrations in the three cultivars, but leaf raffinose content showed a cultivar dependent response associated with early acclimation. In August to

September, CF had no raffinose accumulation until exposure to low non-freezing temperature in October. The cold hardy cultivars, however, showed early response to SD

88 before low temperatures with CD accumulating the highest concentration of 1.64 mg.g-1 and C-3309, 1.16 mg.g-1 by October. In buds, among all sugars, fructose, glucose and sucrose, raffinose, and stachyose had strong correlation with LT50. There were distinctive responses associated with bud position and cultivar that were also related to raffinose. Basal buds accumulated the most raffinose showing maximum values of 5.3 mg.g-1 while middle tissues and apical tissues were much less. Raffinose accumulation was also two to three times higher in the cold hardy cultivars than in cold sensitive cultivars. These results suggest that raffinose accumulation might be an early step in the process of cold acclimation that coincides with early development of FT. Leaf raffinose concentration might also be useful as a detection tool to distinguish various Vitis genotypes with contrasting FT.

Key words: freezing tolerance, cold acclimation, buds, leaves, sugars, raffinose, Vitis

Grapevines adapt to cold climates by undergoing seasonal changes that result in a transition from a cold-sensitive to a cold-hardy state, a process known as cold acclimation. This process leads to an increase in FT or cold hardiness, which is the ability of dormant grapevine tissues to survive freezing temperature stress (Zabadal et al. 2007).

The adaptability of varying species to FT involves several mechanisms to deal with freezing stress. Plants either survive ice in their tissues or they avoid freezing altogether.

Grapevines, like many other woody plants, avoid freezing particularly in their bud tissues by supercooling, a mechanism that allows water to remain in the liquid phase below subfreezing temperatures. The quantification of the limiting temperature for supercooling of several species including grapes is determined using thermal analysis (TA) (Andrews et al. 1984, Wample et al. 1990). TA is based on detecting the latent heat of fusion, called low temperature exotherm (LTE) that is given off when ice is formed in the tissues using thermocouples. The lethal temperature calculated is based on the mean LTE and is expressed as LT50 (lethal temperature that kills 50% of the bud population), and corresponds to the intracellular ice formation that leads to cell injury (Wample et al.

1990, Wisniewski 1995). Genetic experiments have shown that the inheritance of the capacity for FT induced by cold acclimation is a quantitative trait that is controlled by a number of additive genes, and gene expression leads to multiple changes from the molecular level to the organ level (Guy 1990). Cold responsive genes would probably include genes that encode enzymes responsible for the synthesis of sugars or sugar derivatives, proteins associated with the dehydrative aspects of freezing stress or proteins that have antifreeze or cryoprotective properties (Wisniewski et al., 2003).

In both herbaceous and woody plants it has been indicated that soluble sugars play an important role in the maintenance of FT (Sakai and Larcher, 1987, Levitt, 1980).

Proposed roles associated with the accumulation of sugars include the decrease in the crystallization of water which reduces freeze induced dehydration, cryoprotection of cellular constituents, providing metabolic energy, glass formation which may stop all biochemical and most physical activity, and freezing point depression (Burke, 1986,

Burke et al., 1976, Caffrey et al., 1988, Crowe et al., 1988, Franks, 1985, Jeffrey and

Huang, 1990, Koster and Leopold, 1988, Lineberger and Steponkus, 1980, Santarius,

1973). The correlation has not only been quantitative but also qualitative. Plants may accumulate glucose, sucrose, fructose, raffinose and stachyose. Raffinose and stachyose were shown to accumulate in Alfalfa genotypes (Cunningham et al., 2003), all sugars but especially sucrose was shown in red raspberry (Palonen and Junttila, 2002) and fructans in the bluegrass P. annua L. (Dionne et al., 2001). Among sugars, the raffinose family of oligosaccharides (RFO) appears to be the most important to cold hardiness as it has been frequently observed to change exclusively with cold acclimation, and has been reported to provide cryoprotection to cell membranes and stabilization of proteins (e.g. enzymes) during freezing stress. It has generally been observed that RFO concentrations increase in the fall as a response to low temperature, reach a maximum during the coldest months in mid-winter, and decrease in the spring (Stushnoff et al. 1993). Several of the woody plant species investigated by Stushnoff et al. (1993) had only raffinose and stachyose correlating strongly with cold hardiness. Other researchers have also found that RFO correlates better than total soluble sugars with cold hardiness in herbaceous and woody

91 plants (Bachmann et al. 1994, Hamman et al. 1996, Dami 1997, Castonguay and Nadeau

1998, Stushnoff et al. 1998, Taji et al. 2002). There is also differential accumulation of

RFO’s in sensitive and hardy grape cultivars. It was found that cold hardy cultivars such as Frontenac, Couderc 3309 (C-3309), and Concord accumulated the highest amount of raffinose in both bud and leaf tissues and cold sensitive cultivars such as Cabernet Franc accumulated the least (Grant et al. 2009). These results corroborate with previous findings indicating differences between RFO accumulation and cold hardiness in alfalfa cultivars (Medicago sativa), (Castonguay et al. 1995, Castonguay and Nadeau 1998) and

Hydrangea species (Pagter et al. 2008) with hardier cultivars accumulating the highest

RFO amounts.

The development of FT in grapes begins during the autumn and culminates in the winter with maximum supercooling capacity (Hamman et al. 1996, Jones et al. 1999).

Exposure to short-days leads to the first stage of cold acclimation where plants show decreased shoot growth and the onset of bud dormancy. The second phase begins with below freezing temperatures after leaf fall and thus is photoperiod independent. Cold acclimation occurs rapidly with exposure to low non-freezing temperatures (Sakai and

Larcher, 1987). Though all species of grapes supercool, the level of FT achieved varies.

Generally, they are divided into groups based on the maximum FT of their primary dormant buds achieved in mid-winter: very tender (sensitive), tender (sensitive), moderately tender (sensitive), moderately hardy, hardy and very hardy (Zabadal et al.

2007). Given that all species of grapes express the same phenomenon of supercooling, it is unknown why they don’t survive to the same temperatures. It is the purpose of this

92 study to evaluate grape genotypes with known contrasting FT and characterize the respective changes of soluble sugar concentrations during the dormant season. The specific objectives of this study were to: 1) determine the seasonal change in FT (LT50) of three grape cultivars under field conditions; 2) determine the corresponding changes of specific soluble sugars, especially raffinose, in leaves and buds; 3) Investigate FT and sugar accumulation in relation to genotype and bud position on the cane; and 4) investigate the influence of artificial de-acclimation on FT and sugar concentrations in buds.

Materials and Methods

Plant materials and treatments: Grape cultivars of contrasting FT, as rated previously

(Dami et al. 2005, Zabadal et al. 2007) were used in this study: Vitis riparia x V. rupestris ‘Couderc 3309’ (cold hardy), V. labruscana ‘Concord’ (cold hardy), and V. vinifera, ‘Cabernet Franc’ (cold sensitive). Grapevines were grown at the Horticultural

Research Unit 2, Ohio Agriculture Research and Development Center (OARDC),

Wooster, OH (lat.: 40o47’N; long.: 81o55’W, elevation: 311m asl, soil type: silt-loam).

Cabernet Franc (CF) vines were planted at a spacing of 1.8m by 3.1m (vine by row), trained to bilateral low cordon training system with upward shoot positioning. Couderc

3309 (C-3309) vines were planted at a similar spacing, 1.8m by 3.1m and trained to bilateral mid cordon system with downward shoot positioning. Concord (CD) plants were planted at a spacing of 2.4m by 3.1m, and trained to bilateral high cordon with downward

93 shoot positioning. Bud and leaf samples were collected from basal (node positions three to seven), middle (node positions eight to 12), and apical (node positions 13 to17) cane positions. Collection dates were from August to April, for growing seasons 2008-2009 and 2009-2010. Shoots were also visually assessed for periderm (lignified shoots) development during the early acclimation stages (August to November).

Weather data: Average monthly day length (sunrise to sunset) and daily maximum and minimum temperature (oC) were monitored through the duration of the study. Day length data was calculated from data obtained at

(http://www.timeanddate.com/worldclock/astronomy). Temperature data were collected from the OARDC weather system (http://www.oardc.ohio-state.edu/newweather). The killing frost dates were determined as the date of first sub-zero temperature recorded.

Freezing tolerance: FT was determined by thermal analysis by monitoring the low temperature exotherms (LTE) detected at the ice nucleation temperature of the primary buds. Canes were collected and placed in plastic bags to prevent moisture loss. To determine changes in FT, five buds from each replication, were excised from collected canes in the laboratory and loaded onto thermoelectric modules (MELCOR, Trenton, NJ).

The loaded modules were placed in a Tenny programmable freezer (Tenny Inc., New

Columbia, PA) and subjected to controlled freezing tests by lowering the temperature from -2oC to -45oC at a rate of 4oC/hour. The FT of each cultivar was determined by identifying the median or mean LTE, and expressed as LT50 (oC).

De-acclimation treatment: De-acclimation of basal buds was conducted on January 27,

2010 (minimum air temp = -6.9oC) and repeated on February 24, 2010 (minimum air temp = -2.7oC). Treatment consisted of collecting canes from the field and immediately placing them in plastic bags with moistened paper towels to prevent moisture loss. To induce artificial de-acclimation, six bud cuttings were placed in plastic bags and placed in a growth chamber (Conviron, Winnipeg, Manitoba, Canada) at 25oC with high relative humidity ( 75%) for seven days. Buds were then excised for FT using TA as described above. A sub sample was used for soluble sugar analysis.

Qualitative and quantitative analyses of soluble sugars: Analyses of soluble sugars were conducted on basal leaves; and basal, middle and apical buds. Leaves and buds were collected and immediately placed at -80oC. The frozen leaves were lyophilized (Vitis

Freezemobile, New York, NY) and ground to pass through a 20-mesh screen. Buds were excised, plunged in liquid nitrogen and pulverized with mortar and pestle, and then lyophilized. The dry bud tissue was mixed with 75% ethanol in a tube, vortexed and allowed to stand at room temperature for two to four hours vortexing occasionally. The tubes were centrifuged for five minutes at 10 000 rpm. The supernatant was transferred to a vial then dried under air at 45oC. The extraction was repeated twice. The extracted bud metabolites and approximately five mg of leaf tissue were derivitized using the method by Streeter and Strimbu (1998). This involved mixing the ground leaf tissue or extracted bud metabolites with pyridine containing hydroxylamine and an internal standard, phenyl

-D glucopyranoside (Sigma-Aldrich, St. Louis, MO). After incubation at 70oC, the

95 oxime derivatives were reacted with hexamethyldisilazane (HMDS) and trifluoroacetic acid (TFA) (Sigma-Aldrich, St. Louis, MO) to form the tri-methylsilyl (TMS) derivatives of the oximes. The derivatives were injected into a gas chromatograph (Hewlett Packard

5890 Series II, Hewlett Packard, Boulder, CO) with a 30 m capillary column (HP5-MS,

250 m inner diameter and 0.25 m thickness). Injection temperature was 280oC and oven ramp was: 180oC hold for two minutes, 6oC/minute ramp to 215oC hold one minute,

40oC/minute ramp to 320oC, hold for 22 minutes. Helium, the carrier gas, was at a constant flow rate of 1.0 ml.min-1. Soluble sugars were identified and peaks quantified

(Chemstation Quantitation Process Program, Agilent Technologies, Wilmington, DE) by comparison to standard sugars and the internal standard, phenyl -D glucopyranoside.

Statistical Analysis: Statistical analysis was conducted using SAS (SAS Institute, Cary,

NC) and LSD was used to compare means at p  0.05. Regression analyses were also conducted to determine if leaf raffinose could be a prediction of bud raffinose concentrations (early acclimation stage) and correlation analyses were conducted between LT50 and sugar concentrations for both leaves and buds and average minimum temperature and also between LT50 and average monthly (maximum and minimum) temperature for each month.

Results

Weather data: Minimum and maximum air temperatures for August to April growing season are summarized for 2008 - 2009 and 2009 - 2010 (Figure 4.1). The first killing fall frost dates occurred on October 23, 2008 and October 1, 2009. In 2009, the lowest temperature recorded at the vineyard was -26.6oC on January 16. In 2010, the lowest temperature recorded was -18.0oC on February 10. Average monthly day length (sunrise to sunset) data is depicted in Figure 4.2. Day length in August was 13 hrs then decreased to 10 hrs in November and December and was shortest in January and February (9hrs).

Bud freezing tolerance: The seasonal change of FT was similar in both seasons (Figure

4.1, Table 4.1). The profile of FT of buds from different positions and cultivar can be divided into three stages of cold acclimation as previously reported (Figure 4.3). The first stage of acclimation begins in the fall, the second stage or maximum hardiness ranges from the time of first killing frost and reaches a maximum in January, and the third stage or de-acclimation begins in late February through to the spring. In August the average killing temperature (LT50) was between -6.9 and -8.0oC for 2008 and -5.7 to -

7.7oC for 2009 (Table 4.1 and Figure 4.1). As the buds began to acclimate, there was a decrease in the LT50. Killing frost dates were recorded in October of both years and

LT50 values were reduced to between -13.8 to -21.2oC in 2008 and -13.9 to -22.6oC in

2009. The minimum LT50 was recorded in January of both 2009 and 2010 ranging from -

27.8 to -30.8oC and -24.4 to -31.9oC, respectively. This corresponded to the time of the

97 lowest temperatures recorded in the vineyard (Figure 4.1). As air temperatures began to increase, the LT50 also increased. In April of both years, LT50 rose to between -11.6 and

-19.3oC.

Generally basal buds were able to withstand freezing stress at lower temperatures than middle buds then apical buds (Table 4.1). Initial LT50’s for all three bud positions were very low with apical tissues (-5.7 to -7.0oC) significantly higher than middle or basal buds (-7.5 and -7.9oC) for both years, with middle and basal buds generally not differing between each other. This was the general pattern observed during the early stages of acclimation for both years. As the buds reached their maximum LT50 the pattern of differences between all three bud positions was consistent with basal tissues being the most FT compared to middle and apical tissues except in Jan of 2010 when there were no significant differences. The minimum LT50 recorded for basal buds were -

30.4oC in 2009 and -28.4oC in 2010. Middle buds had similar LT50 for both years.

Apical buds were however, significantly lower reaching only -27.8oC in 2009 and -

28.1oC in 2010. The general trend observed for FT was that apical tissues were always the least FT compared to middle or basal buds, which were always the most FT. FT began to decrease in all tissues in February. In April of both years, LT50 values increased to between -12.0 and -14.8oC for apical buds, -14.9 and -15.5oC for middle buds and -

17.6 and -19.3oC for basal buds.

There were also variations in the FT among the three cultivars (Table 4.1, Figures

4.1 and 4.3). Generally C-3309 was more FT than CD and CF was the least FT of the three cultivars tested for both years (Figure 4.3). During the fall as vines began to

98 acclimate, LT50 values were high. C-3309 had the lowest LT50 of -6.1oC and -6.9oC in

August of 2008 and 2009 respectively. CF and CD were not different from each other with LT50 ranging between of -7.3 and -8.0oC. As acclimation proceeded, CD and C-

3309 LT50 values began to decrease at a faster rate compared to CF for both years. In

September, CD had the lowest LT50 below -14oC while CF and C-3309 ranged between -

7.2 and -11.0oC. In October, C-3309 LT50 values showed a decrease reaching values comparable to or lower than CD, both cultivars having LT50 values below -20.0oC compared to CF that only dropped -13.8oC. C-3309 continued to maintain significantly lower LT50 values compared to the other two cultivars for both years. In 2008 and 2009 the minimum LT50 in January for C-3309 ranged from -30.8 to -31.9oC and CD buds between -28.5 to -30.4oC with significant differences between the two cultivars. CF, which was the least cold hardy of the three cultivars survived to only -23.1oC in

December 2008 and -23.5oC in 2009. A major freezing event occurred on January 16,

2009 where vineyard air temperature recorded was -26.6oC. This temperature was much lower than the LT50 for CF and this resulted in a major loss of grapevines. Buds were collected subsequent to the freezing event and no LTE’s were detected indicating that the buds were dead. In January 2010 the minimum air temperature (-18oC) remained above the computed LT50 of -28.1oC (Table 4.1). The vines then began de-acclimating as temperatures increased in the vineyard (Figure 4.1). CD and C-3309 were slower at losing their cold hardiness compared to CF, which had an increased LT50 from March (-

19.3oC in 2010) while, CD and C-3309 still had very low LT50’s of around -23 to -25oC

99 for both years. In April, LT50 values then dropped to below -17.0oC for C-3309 and CD, and CF had an LT50 of -11.6oC, significantly higher than the other two cultivars.

There were significant interactions between node position and cultivars for all sampling dates except February, 08 to 09 season and August, 09 to 10 season (Table 4.1).

This significant interaction indicated that generally, LT50 values were dependent on bud position and cultivar. While basal buds were generally more FT compared to the other node positions, C-3309 basal buds were the most FT, followed by CD and CF basal buds were the least FT (Figure 4.3).

Correlation analysis between LT50 and air temperature showed a direct relationship between LT50 and both mean air temperature (R = 0.98, p 0.005 for 2009 and R = 0.95, p 0.005 for 2010) and minimum air temperature (R = 0.97, p 0.005 for

2009 and R = 0.95, p 0.005 for 2010) indicating that the pattern of FT is directly related to the change in air temperature.

Qualitative and quantitative analyses of soluble sugars: Sugars quantified in all cultivars were fructose, glucose, myo-inositol, sucrose, galactinol, raffinose and stachyose (buds only) (Tables 4.2, 4.3 and 4.4). There were no consistent differences in basal leaf sugar concentrations over time among the cultivars for some of the sugars analyzed (Table 4.2). Sucrose concentration was significantly higher in CF (34 mg.g-1 dry weight), compared to C-3309 and CD for August sample dates. After September however, CD had the highest concentrations increasing to over 40 mg.g-1 dry weight compared to CF and CD. CD had the lowest concentrations among cultivars for fructose,

100 glucose and myo-inositol. Raffinose concentrations were also cultivar dependent with CF having no accumulation until October when samples had only 0.15 mg.g-1 (Table 4.2).

CD had the highest concentration increasing from 1.19 to 1.64 mg.g-1 in October and C-

3309 increasing from 0.62 to 1.16 mg.g-1.

In bud tissues, there was a general pattern for sugar accumulation. Sugar concentrations showed increases over time for fructose, glucose, sucrose and raffinose until maximum concentrations were achieved in January and February and then concentrations decreased in March and April (Table 4.3, Figures 4.4 and 4.5). Galactinol varied with time but showed no consistent pattern. Myo-inositol also decreased during the acclimation stage, reached a minimum in December to January, then increased during the de-acclimation stages. Glucose, fructose and sucrose were the most predominant sugars present accounting for 24.8 to 40.1%, 22.2 to 35.9% and 20.0 to 45.0% of total soluble sugar concentration, respectively for both years. The other sugars were present at relatively very low concentrations (Tables 4.3).

Sugar concentrations differed in the bud tissues collected from apical, middle and basal positions. For glucose, fructose and sucrose, basal tissues generally had the highest concentrations, though not always significantly different from middle and/or apical tissues, which generally had the lowest concentrations. Throughout the growing season for both years basal tissues ranged from 17.8 to 18.1 mg.g-1 for glucose, 15.1 to 16.9 mg.g-1 for fructose and 14.3 to 27.6 mg.g-1 for sucrose. Middle and apical tissues were similar or sometimes less. This variation in tissue concentration continued over time. The concentrations of these sugars reached a peak during January or February. In basal

101 tissues, glucose increased to 65.9 and 71.3 mg.g-1 in January for 2009 and 2010, respectively. Fructose increased to 55.1 mg.g-1 in February of 2009 but was 61.9 mg.g-1 in

January of 2010. Sucrose also had high concentrations at the peak of winter increasing from 14.3 and 27.6 mg.g-1 in August of 2009 and 2010 to 45.2 and 63.0 for both years, respectively. There were some collection dates that showed some interaction between bud position and cultivar. These interactions were however, random and not consistent with any time point. For example, interaction p-values of 0.0007 were calculated for glucose in 2009 in October, but in 2010, the only significant interaction was in March (p=0.05).

There were variations also for fructose and sucrose.

Raffinose was the only sugar to show a consistent pattern over time when comparing both node position and cultivars (Figure 4.5). There were consistently significantly higher concentrations of raffinose in basal tissues compared to middle and apical buds. Concentrations increased during the acclimation stage from August to

November, peaked, then decreased with de-acclimation for all positions. Basal buds increased from 2.0 to 2.5 mg.g-1 in August, reached a maximum in January of 5.3 mg.g-1 then declined to 0.8 to 1.4 mg.g-1 in 2009 and 2010, respectively. Middle buds accumulated a maximum of over 4.0 mg.g-1 and apical 2.7 to 3.1mg.g-1 for 2009 and

2010. Apical buds consistently had the lowest concentrations reaching only 1.8 to 2.8 mg.g-1 in January of 2009 and 2010, respectively. There was also variation in raffinose concentrations that was cultivar dependent. CF consistently had the lowest concentrations of raffinose and this trend continued throughout both growing seasons. In 2009, CF accumulated up to 2.5 mg.g-1 in December and 3.6 mg.g-1 in January of 2010. C-3309 and

102

CD had much higher concentrations of up to 3.8 to 4.9 mg.g-1 and 4.1 to 5.3 mg.g-1, respectively. Cultivar variation in the raffinose concentrations was node position dependent as is indicated by significant interaction for all time points except March and

April. All cultivars had higher concentrations in their basal tissues then middle and the lowest in apical buds and this was consistent for both 2009 and 2010.

The relationship between sugar concentrations and LT50 was examined by determining the correlation between each sugar and LT50 (Table 4.4). The sugars that had significant correlation (p 0.05) were glucose, fructose, sucrose, raffinose and stachyose. These sugars showed an inverse relationship with LT50, all increasing as

LT50 decreased. Leaf and bud raffinose concentrations for samples collected during acclimation also had a direct relationship for all cultivars combined (Figures 4.6 and 4.7).

De-acclimation: FT can be artificially lost when vines are subjected to warm temperatures. When vines were artificially de-acclimated at 25oC with high relative humidity ( 75%) for seven days there was a reduction in the FT of all the cultivars

(Table 4.5). In, CF LT50 increased from -25.1oC to -18.1oC in January, a 27% increase.

In CD buds, LT50 increased from -32.1 to -20.1, a 37% increase and C-3309 increased

22% from -28.1 to -21.7 oC in January. In February the increase in LT50 was similar ranging between 23 and 39%. The increase in LT50 of the de-acclimated buds is comparable to the LT50 observed for buds collected between March and April. Under field conditions in 2010, CF had 23% increase in LT50 in March (-19.3oC) indicating a start in de-acclimation while, CD and C-3309 still had very low LT50’s of around -23 to -

103

25oC, only an 18 to 22% increase, for both years (Table 4.1). In April LT50 values increased to below -17.0oC for C-3309 and CD and CF had an LT50 of -11.6oC, significantly higher than the other two cultivars. The increase in LT50 for natural de- acclimation was 37 to 48% by April, somewhat comparable to the 27 to 39% increases for artificially de-acclimated buds. Sugar concentrations were also reduced by the de- acclimation treatment (Table 4.6). Glucose concentrations were reduced by 67%, fructose by 78%, sucrose by 73% and raffinose by 88%. Sugars that had an opposite response included myo-inositol, which increased by more than 100% and galactinol, which increased by 72% in response to the de-acclimation treatment. These are comparable to natural de-acclimation values in April, which also saw a 73% to 82% reduction in sugar concentrations.

Discussion

In this study, the seasonal changes of FT proceeded as expected and were similar to previous reports (Zabadal et al. 2007). Grapevines increased in FT as shown by a reduction in LT50, had peak values in January, and then had decreased FT in March to

April. In January and February when buds were at their maximum hardiness, CF had the highest LT50 followed by CD then C-3309. This pattern coincides with previous ratings by Dami et al. (2005) and Zabadal et al. (2007) of C-3309 and CD as cold hardy (LT50 =

-26 to -31oC) and CF as cold sensitive or moderately cold tender (LT50 = -17 to -23oC).

The development of FT was closely related to changes in photoperiod and the average

104 low temperature. The first stages of acclimation from August to September, relate firstly to decreasing photoperiod and later to low non-freezing temperatures, which function in preparing plants for freezing temperatures (Weiser, 1970, Zabadal et al. 2007). At this stage, while LT50 values for CF was high, LT50 for CD decreased to approximately -

14oC, significantly lower than C-3309 and CF. This drop in LT50 occurred before average minimum air temperatures were below 10oC, indicating a photoperiod response.

C-3309 also had some early changes in 2009, LT50 decreasing to -11oC. In October when mean air temperatures dropped below 10oC, CD and C-3309 had significant reductions in

LT50 and then CF also had LT50 reduced to -13oC. At this early stage of cold acclimation, the leaves are important as sites of photoperiod signal reception and later of low temperature stimulus. The distinct events that occur during the early phases include the perception of changes in these environmental stimuli and result in growth cessation and the initial synthesis of metabolites that bring about changes in various tissues of the plant (Weiser, 1970, Levitt, 1980, Sakai and Larcher, 1987). In this study, there were distinct changes in soluble sugar concentrations in the leaves. Of all the sugars analyzed, for the three cultivars tested, the change in raffinose concentrations coincided and had strong correlation with FT development. In CD and C-3309, there was an early response related firstly to changes in photoperiod and as the temperature decreased, changes were observed in CF. It has generally been shown that in grapes RFO correlates with cold hardiness (Dami, 1997, Hamman et al. 1996, Stushnoff, et al. 1993). These findings are related to bud tissues, but this study has shown that leaf tissues showed an increase in raffinose concentration during the early stages of cold acclimation. It has been

105 demonstrated that signal transduction pathways starting from the perception of either SD or low-temperature stimulus and leading to cold acclimation are operational in birch

(Betula pendula) leaves and this early development is a possible protective mechanism during autumn, enabling prolonged periods for photosynthesis (Li et al. 2002). There were also differences in leaf concentrations that were cultivar dependent. CF, the cold sensitive cultivar, accumulated the least concentration of raffinose compared to CD and

C-3309 and was also the last cultivar to begin accumulating raffinose. Leaves of cold hardy cultivars, V. spp ‘Frontenac’, V. riparia x V. rupestris ‘C-3309’, and V. labruscana

‘Concord’, were previously shown to accumulate higher concentrations of raffinose than cold sensitive cultivars like V. vinifera ‘Cabernet Franc’ when exposed to low non- freezing temperatures in controlled temperature experiments (Grant et al. 2009). V. riparia x V. rupestris ‘C-3309’, and V. labruscana ‘Concord’ also had differential response to photoperiod compared to V. vinifera ‘Cabernet Franc’, both accumulating raffinose in their leaf tissues earlier and at higher concentrations (Grant 2012, dissertation chapter 2). This pattern of accumulation is confirmed from our observation of field- grown grapevines. The genotypes adapted to very low freezing temperatures began accumulating raffinose earlier in their leaf tissues and consistently had relatively higher amounts of raffinose levels compared to sensitive genotypes. It is possible that these cold hardy cultivars are able to initiate changes needed at an earlier stage and that allows these cultivars to be more FT and this trait may possibly be linked to SD perception. Monthly average day length for this early stage was 10 hours. It has been shown that in Vitis labruscana and Vitis riparia, very cold hardy cultivars, SD initiated growth cessation and

106 dormancy development as well as an increase in FT without low temperature stimuli

(Fennell and Hoover 1991, Grant 2012, dissertation chapter 2).

In addition, this study demonstrated that the pattern of sugar accumulation in leaves during the early acclimation stages coincides with sugar accumulation in buds tissues. Bud tissues from all three cultivars began accumulating raffinose early in the fall and continued to accumulate raffinose until the peak of winter then decreased with de- acclimation. Raffinose concentrations in bud tissues are also not uniform throughout the plant. Basal tissues often had higher concentrations of raffinose compared to middle or apical tissues, which indicates differences in hardiness progression. Our observations corroborate those of Badulescu and Ernst (2006) who reported that V. vinifera basal bud tissues had the highest sugar concentration. The reasons for RFO differences in apical, middle and basal tissues are still uncertain and it still needs to be investigated as differences may be associated with the varying tissue responses to the acclimating stimuli or to development progression, but it is likely the latter as cold acclimation of bud and stem tissues is known to proceed from the base to the shoot tip with the basal buds being the most developed and cold hardy. Furthermore, dehydration of basal stem tissues leads to periderm formation (Zabadal et al. 2007). The pattern of raffinose accumulation follows that of periderm formation and tissue dehydration in grape shoots and that pattern also follows responses in leaves following cold acclimation (Grant et al. 2009) and SD photoperiod (Grant 2012, dissertation chapter 2). Therefore, it is suggested that the high amount of raffinose accumulated in leaf and bud tissues plays a role on protecting plant

107 tissues during dehydration, which takes place prior to exposure to subfreezing temperatures.

Organic compounds such as sugars have been implicated in the hardening process of plants (Burke et al. 1976, Sakai and Larcher 1987, Levitt 1980, Crowe et al. 1988). In both herbaceous and woody plants it has been indicated that soluble sugars increase in the fall to winter when plants acclimate and decrease in the spring when deacclimation occurs (Sakai and Larcher, 1987, Levitt, 1980). The correlation has not only been quantitative but also qualitative. Plants may accumulate glucose, sucrose, fructose, raffinose and stachyose. Raffinose and stachyose was shown to accumulate in Alfalfa germplasms (Cunningham et al., 2003), all sugars but especially sucrose was shown in red raspberry (Palonen and Junttila, 2002) and fructans in the bluegrass P. annua L.

(Dionne et al., 2001). In this study, of all the sugars, glucose, fructose, sucrose, raffinose and stachyose were shown to correlate with LT50. Glucose, fructose and sucrose, however, did not show any consistent pattern related to bud position and cultivar.

The raffinose family of oligosaccharides (RFO) have been proposed to play varying functions ranging from transport and storage of carbon, to providing protection against abiotic stress such as desiccation and cold (Hannah et al., 2006, Peterbuer and Ricter,

2001). Accumulation in raffinose may also decrease the osmotic potential, which depresses the freezing point of cell water possibly through a colligative effect where the sugars change the bulk properties of the cell solution (Burke et al. 1976, Levitt, 1980). At low temperatures, raffinose may function by delaying the crystallization of sucrose

(Caffrey et al. 1988, Koster and Leopold, 1988). Also with decreasing temperatures,

108 raffinose does not change its configuration allowing it to have structure-preserving effect upon binding to proteins and membranes (Santarius 1973, Jeffrey and Huang 1990,

Lineberger and Steponkus 1980). The molecular structure of raffinose allows it to function by forming hydrogen bonds with these macromolecules and may substitute for water during desiccation stress, thus allowing the macromolecules to maintain their hydrated orientation (Crowe et al. 1988). It may also protect cells by forming intracellular glass, an undercooled liquid with the viscosity of a solid and its formation would ensure stability during periods of dormancy by preventing further desiccation and stabilizing cell structures (Burke 1986). Glass forms at the glass transition temperature - Tg. Raffinose is a trisaccharide, therefore has a higher molecular weight than monosaccharides and disaccharides and therefore it is more effective because it has a higher Tg and will form glass more readily (Franks 1985). In other words, raffinose is more protective than the disaccharide sucrose or the monosaccharides glucose and fructose. It is possible that the characteristics of raffinose allow it to function by any or all of the above mechanisms.

The exposure to low temperature and photoperiod during the early stages of acclimation has an affect on soluble sugar concentration including raffinose concentration and these early steps are important for the development of maximum FT. In this study we demonstrated there are differences in the response of grape genotypes under field conditions that leads to variation in soluble sugar accumulation particularly, raffinose concentrations which, showed differential response when bud positions and genotypes were compared, with basal tissues and the more cold hardy cultivars accumulating the highest concentration. The responses related to genotype may explain changes that make

109 a grape cultivar more FT than another. In this field study it was concluded that cold hardy grapevines responded to the environmental cues (SD and low temperature) differentially with cold hardy cultivars responding earlier to SD prior to any exposure to sub freezing temperatures and the photoperiod response resulted in the more cold hardy cultivars resulting in the earlier accumulation of raffinose in leaves and buds of grapevines and impacting a decrease in LT50. Our previous research has also shown that raffinose concentration in the leaf tissues before completion of dormancy and hardiness is developed could be used as an indication of hardiness variation among Vitis genotypes

(Grant 2010). The preparation for cold acclimation is evident in non-dormant plant part

(i.e. leaves) as was demonstrated in this study as changes in raffinose concentration occurred during the early stage.

110