Characterising weight loss in Vitis vinifera Shiraz berries at sub-optimal maturity Joanne Tilbrook

Transcription

1 Characterising weight loss in Vitis vinifera Shiraz berries at sub-optimal maturity Joanne Tilbrook Thesis presented for the degree of Doctor of Philosophy The University of Adelaide School of Agriculture, Food and Wine Discipline of Wine and Horticulture November 2009

2 Contents Chapter ! Introduction to the papers of the thesis... 1! Introduction... 2! Chapter ! Direct measurement of xylem hydraulics through berry development in Vitis vinifera cvs Shiraz and Chardonnay... 5! Abstract... 6! Introduction... 7! Materials and methods... 9! Fruit material... 9! Berry weight, Brix, ph and osmolality... 10! Pressure probe measurements... 10! Steady state pressure... 12! Elasticity... 13! Hydraulic conductance... 14! Results... 15! Pressure probe measurements... 15! Hydraulic conductance into berries... 17! Discussion... 20! Chapter ! Cell death in grape berries: varietal differences linked to xylem pressure and berry weight loss... 27! Abstract... 29! Introduction... 31! Materials and methods... 36! Fruit material... 36! Berry weight, Brix and osmolality... 37! Vital and non vital staining of pericarp tissue... 37! Analysis of vitality... 39! Xylem equilibrium pressures... 40! Results... 41! Pericarp cell death is evident in berries of Chardonnay and Shiraz late in development, but not Thompson Seedless... 41! Cell vitality relative to sugar and water relations of the berry... 43! Aberrant behaviour detected using the vitality assay... 45! Discussion... 48! Chapter ! Hydraulic connection of grape berries to the vine: varietal differences in water conductance into and out of berries, and potential for backflow ! Abstract... 59! Introduction... 61! Materials and methods... 65! Fruit material... 65! Deformability... 65! Berry weight and total soluble solids... 66! Flow into the peduncle of whole bunches... 66! Flow conductance for different directions of flow into berries via the pedicel... 67! Stem water potential... 68! Dye loading of berries to examine xylem flow to the vine... 69! Results... 70! ii

3 Berry ripening dynamics... 70! Whole bunch flow... 72! Hydraulic conductance of individual berries for inflow and outflow... 75! Lucifer Yellow CH visualisation of backflow... 77! Discussion... 79! Chapter ! Effect of molybdenum application on Shiraz berry development in late stages of ripening and the impact on the organoleptic properties of the wine... 85! Abstract... 87! Introduction... 89! Materials and Methods... 92! Fruit material... 92! Molybdenum treatment of vines... 93! Berry weight and sugar accumulation... 93! Petiole and shoot analysis... 94! Yield and pruning weights... 94! Abscisic acid in berries... 95! Wine making, molybdenum and sensory analysis ! Results... 97! Berry weight and sugar accumulation... 97! Petiole and shoot analysis ! Abscisic acid in berries ! Yield and pruning weights ! Wine making and sensory analysis ! Discussion ! Chapter ! General Discussion ! Summary of findings ! Varietal differences in berry xylem hydraulics ! Varietal differences in berry cell vitality and cell membrane competence ! Molybdenum effect on berry development ! Conclusions ! Future research ! Acknowledgements ! References ! Appendices Tyerman SD, Tilbrook J, Pardo C, Kotula L, Sullivan W, Steudle E (2004) Direct measurement of hydraulic properties in developing berries of Vitis vinifera L. cv Shiraz and Chardonnay. Australian Journal of Grape and Wine Research 10, Tilbrook J, Tyerman SD (2006) Water, sugar and acid: how and where they come and go during berry ripening. In Australian Society of Viticulture and Oenology: Finishing the joboptimal ripening of Cabernet Sauvignon and Shiraz. pp 4-12 Openbook Australia. Tilbrook J, Tyerman SD (2008) Cell death in grape berries: varietal differences linked to xylem pressure and berry weight loss. Functional Plant Biology 35, Tilbrook J, Tyerman SD (2009) Hydraulic connection of grape berries to the vine: varietal differences in water conductance into and out of berries, and potential for backflow. Functional Plant Biology 36, iii

4 Abstract Post-veraison and prior to reaching harvest maturity, Vitis vinifera cv Shiraz berries lose weight where other varieties such as Chardonnay and Thompson Seedless do not. The berry weight loss occurs in the later stages of ripening from days after anthesis. This defines a third phase of development in addition to berry formation and berry expansion. Berry weight loss is due to net water loss, but the component water flows through different pathways have remained obscure. A method of direct measurement was developed using a pressure probe to measure the pedicel xylem hydraulic conductance of single detached berries through development. The probe measured the pressure developed in the xylem of non-transpiring berries. Pre-veraison, negative xylem pressures of -0.2 to -0.1 MPa were measured, increasing to around zero between veraison and 90 days after anthesis. The pressures around zero were maintained until harvest when the berry juice osmotic potential was around -3 MPa for Chardonnay and -4 MPa for Shiraz. Since cell turgor is low in the berry, this indicates that the juice osmotic potential is not translated into negative xylem pressure. It may suggest that the reflection coefficient of cell membranes surrounding the berry xylem in both varieties changes from close to 1 pre-veraison, to about at veraison and decreases to 0 at harvest. Both varieties showed a ten-fold reduction in hydraulic conductance from veraison to full ripeness. Shiraz had conductances that were two to five fold larger than Chardonnay, and maintained higher conductance from 90 days after anthesis, the period where berry weight loss occurred. In both varieties the hydraulic conductance reduced in the distal and proximal portions of the berries from veraison. iv

5 Focusing on xylem hydraulic conductance into and out of berries from 105 days after anthesis and during berry weight loss in Shiraz, significant varietal differences in xylem hydraulic conductance were found. Both varieties showed flow rectification such that conductance for inflow was higher than conductance for outflow. For flow in to the berry, Chardonnay had 14% of the conductance of Shiraz. For flow out of the berry Chardonnay was 4% of the conductance of Shiraz. From conductance measurements for outflow from the berry and stem water potential measurements, it was calculated that Shiraz could lose about 7% of berry volume per day, consistent with rates of berry weight loss. Using a XYL EM flowmeter, flow rates of water under a constant pressure into berries on detached bunches of these varieties are similar until days after anthesis. Shiraz berries then maintain constant flow rates until harvest maturity while Chardonnay inflow tapers to almost zero. Thompson Seedless maintains high xylem inflows. These data are consistent with single berry measurements with the pressure probe. A functional pathway for backflow from the berries to the vine via the xylem was visualised with Lucifer Yellow CH loaded at the cut stylar end of berries on potted vines. Transport of the dye out of the berry xylem ceased prior to 97 days after anthesis in Chardonnay, but was still transported into the torus and pedicel xylem of Shiraz at 118 days after anthesis. Xylem backflow could be responsible for a portion of the post-veraison weight loss in Shiraz berries. These data provide evidence of varietal differences in hydraulic connection of berries to the vine that we relate to cell vitality in the mesocarp. The key determinates of berry water relations appear to be maintenance or otherwise of semi permeable membranes in the mesocarp cells and control of flow to the xylem to give variable hydraulic connection back to the vine. v

6 Because of the very negative osmotic potential of the cell sap, the maintenance of semipermeable membranes in the berry is required for the berry to counter xylem and apoplast tensions that may be transferred from the vine. The transfer of tension is determined by the hydraulic connection through the xylem from the berry to the vine, which changes during development. We assess the membrane integrity of the three varieties, Shiraz, Chardonnay and Thompson Seedless throughout development using the vitality stains, fluorescein diacetate and propidium iodide, on fresh longitudinal sections of whole berries. The wine grapes, Chardonnay and Shiraz, maintained fully vital cells after veraison and during berry expansion, but began to show cell death in the mesocarp and endocarp at or near the time that the berries attain maximum weight. This corresponded to a change in rate of accumulation of solutes in the berry and the beginning of weight loss in Shiraz, but not in Chardonnay. Continuous decline in mesocarp and endocarp cell vitality occurred for both varieties until normal harvest dates. Shiraz grapes classified as high quality and sourced from a different vineyard also showed the same death response at the same time after anthesis, but they displayed amore consistent pattern of pericarp cell death. The table grape, Thompson Seedless, showed near to 100% vitality for all cells throughout development and well past normal harvest date, except for berries with noticeable berry collapse that were treated with gibberellic acid. The high cell vitality in Thompson Seedless berries corresponded to negative xylem pressures that contrasted to the slightly positive pressures for Shiraz and Chardonnay. I hypothesise that two variety dependent strategies exist for grapevine berries late in development: (1) programmed cell death in the pericarp and loss of osmotically competent membranes that requires concomitant reduction in the hydraulic conductance via the xylem to the vine; (2) continued cell vitality and osmotically competent membranes that can allow high hydraulic conductance to the vine. vi

7 Weight loss in Shiraz berries before harvest maturity for winemaking has, to date, not been manipulable by viticultural practices such as irrigation. This work shows that foliar application of molybdenum to Shiraz vines changed the time course of berry weight accumulation regardless of the timing of the application in two vineyards over two seasons. Molybdenum treatment delayed the transition of berries from phase 2 (berry weight accumulation) to phase 3 (weight loss) of development for 2 to 7 days. It also slowed sugar accumulation relative to berry weight accumulation in phase 2. Allometric analysis of abscisic acid content of berries relative to weight accumulation in phase 2 and phase 3 showed no significant differences. Fruit yields from molybdenum treated and control vines were not significantly different when harvested at the same ºBrix rather than the same day after anthesis. Pruning weights of treated vines were significantly higher than control vines, suggesting increased vigour related to increased availability of the molybdoenzyme nitrate reductase, and therefore increased potential to reduce nitrate for assimilation. Wine made from fruit of treated vines contained five times higher molybdenum than wines made from control fruit, but were still at levels safe for human consumption. Sensory analysis of wines made from molybdenum treated and control fruit indicate that organoleptic differences may be perceived in the wines because of molybdenum treatment. In summary, significant varietal differences were found in how berries isolate from the vine, with strong evidence that weight loss from Shiraz berries is caused by xylem backflow to the vine, perhaps associated with changes in aquaporin or cell membrane function in xylem associated tissue. Differences were also found in cell vitality and membrane competence across the endocarp and mesocarp of berries through development, with distinct varietal differences between the wine varieties Shiraz and Chardonnay, and the table grape Thompson Seedless. The kinetics of berry weight accumulation in Shiraz is altered by the foliar application vii

8 of molybdenum to vines at anthesis and capfall, but molybdenum may affect the organoleptic qualities of wine made from the fruit. viii

9 Thesis declaration This work contains no material which has been accepted for the award of any other degree or diploma in any university or other tertiary institution and, to the best of my knowledge contains no material previously published or written by another person, except where due reference has been made in the text. I give consent to this copy of my thesis when deposited in the University Library, being made available for loan and photocopying, subject to the provisions of the Copyright Act The author acknowledges that copyright of the published works contained within this thesis (as listed below) resides with the copyright holders of those works. The author was unable to work over the season due to illness, however, using the author s method, hydraulic data was collected by Lukasz Kotula, a student visiting from the Department of Plant Ecology, University of Bayreuth. This data is clearly identified. Professor Steve Tyerman provided expert and technical advice when required, and editorial advice on drafts of papers and thesis. This thesis contains published work: Chapter 2: This chapter is the original work of the author. The data presented forms part of a published paper: Tyerman SD, Tilbrook J, Pardo C, Kotula L, Sullivan W and Steudle E (2004) Direct measurement of hydraulic properties in developing berries of Vitis vinifera L. cv Shiraz and Chardonnay. Australian Journal of Grape and Wine Research 10, Chapter 3: Awarded Best paper of 2008 by an early career researcher by the Australian Society of Plant Scientists and Functional Plant Biology, July Tilbrook J and Tyerman SD (2008) Cell death in grape berries: varietal differences linked to xylem pressure and berry weight loss. Functional Plant Biology 35, Chapter 4: Tilbrook J and Tyerman SD (2009) Hydraulic connection of grape berries to the vine: varietal differences in water conductance into and out of berries, and potential for backflow. Functional Plant Biology 36, ix

10 Chapter 6: The summary figure is modified from peer reviewed Proceedings. Tilbrook J and Tyerman SD (2006) Water, sugar and acid: how and where they come and go during berry ripening. In Australian Society of Viticulture and Oenology: Finishing the job-optimal ripening of Cabernet Sauvignon and Shiraz pp 4-12, Openbook Australia. Other Publications resulting from this work or published during candidature: Accepted for publication: This paper is the result of experiments planned by the author to explore varietal differences in loss of cell vitality during berry development. Fuentes S, Sullivan W, Tilbrook J, Tyerman S (manuscript accepted November 2009) A novel analysis of grapevine berry tissue vitality and morphology demonstrates a variety dependent correlation between tissue vitality and berry shrivel. Australian Journal of Grape and Wine Research. Published during PhD candidature: Vandeleur R, Niemietz C, Tilbrook J, Tyerman SD (2005) Roles of aquaporins in root responses to irrigation. Plant and Soil 274, Supervisor: Stephen D. Tyerman Date: 10/5/10 Signature: x

11 Acknowledgments I would like to recognise the broad range of knowledge and expertise of Professor Steve Tyerman. As a supervisor Steve was always receptive to ideas for experiments and creative in helping develop ways to use and test systems, and collect data. His enjoyment in seeing the results of his students, and helping direct their analyses and interpretation was clear. Thanks Steve, I appreciate the ongoing support and encouragement that I received. I extend my thanks to all members of the Tyerman and Kaiser Laboratory Group and the Discipline of Wine and Horticulture at the University of Adelaide. It was a delight to work within the Discipline. To my family, thankyou all for unconditional love and support shown over the time it has taken. xi

12 Chapter 1 Introduction to the papers of the thesis 1

13 Introduction Vitis vinifera cv Shiraz, also known as Syrah, is a red wine grape variety that is grown predominantly in the Rhône Valley in France, and Australia. The fruit can be made into a range of wine styles, from light to full flavoured (Iland and Gago 1997). About a quarter of the annual 1.7 million tonne Australian wine grape crush is Shiraz (WFA 2009), which has a distinctive characteristic during berry development that is investigated in this thesis. The berries soften at veraison and berry weight increases until around days after anthesis. For the remainder of development until harvest maturity is reached, berries usually lose weight (Davies and Robinson 1996; McCarthy 1999; Rogiers et al. 2001). The degree of weight loss (or shrivel) of berries can vary, but in a four year irrigation study near Waikerie in South Australia, a weight loss of around 20% occurred regardless of the season or irrigation regime (McCarthy 1997). Shiraz berry weight loss also occurs in diverse climates (Davies and Robinson 1996; Rogiers et al. 2000; Smart et al. 1974). Berries have a high water content and water loss has been implicated in berry weight loss (McCarthy 1999; McCarthy and Coombe 1999). The water loss concentrates the sugars in the berries. The high sugar content of the juice results in wine with high alcohol content, which can be unpalatable to consumers. Water loss also has the potential to impact on berry sugar metabolism (McCarthy 2000) concentration of mineral nutrients (Rogiers et al. 2000) and vineyard yields (McCarthy 1999). Indirect measurement of water flows into berries revealed xylem dominant inflow pre-veraison, changing to phloem dominant inflow post-veraison (Greenspan et al. 1994). There was anatomical evidence to support this (Düring et al. 1987; Findlay et al. 1987; Rogiers et al. 2001) and some indirect evidence to suggest 2

14 that xylem hydraulic backflow may occur (Greenspan et al. 1996; Lang and Thorpe 1989). From this body of evidence, a three-strand approach was developed to investigate and characterise the weight loss in Shiraz berries that occurred at sub-optimal maturity. Firstly, xylem function in the pedicel and berry were quantitatively examined. Quantitative and qualitative hydraulic measurements of inflow and outflow via pedicel xylem were made on single berries and bunches. Secondly, the cell vitality and cell membrane competence of the berry mesocarp and exocarp were examined to establish whether they change during development and, if so, how those changes related to berry xylem hydraulics. Thirdly, a series of viticulture-based experiments were conducted to examine whether the developmental time course of weight loss in Shiraz berries could be manipulated or shifted with the foliar application of molybdenum. These experiments evolved from the idea that molybdenum availability may be a limiting factor in reactions where a molybdenum cofactor binds to enzymes, which catalyse reactions in abscisic acid synthesis and nitrogen reduction and assimilation (reviewed in Kaiser et al. 2005). The results of this three-strand approach are presented in the following four papers and the key findings summarised in Chapter 6, General Discussion. 3

15 4

16 Chapter 2 Direct measurement of xylem hydraulics through berry development in Vitis vinifera cvs Shiraz and Chardonnay This chapter is the original work of the author. The data presented forms part of a published paper: Tyerman SD, Tilbrook J, Pardo C, Kotula L, Sullivan W, Steudle E (2004) Direct measurement of hydraulic properties in developing berries of Vitis vinifera L. cv Shiraz and Chardonnay. Australian Journal of Grape and Wine Research10,

17 Abstract Post-veraison and prior to reaching harvest maturity, Shiraz berries lose weight where other varieties such as Chardonnay do not. A method of direct measurement was developed using a pressure probe to measure the pedicel xylem conductance of single detached berries through development. Both varieties showed a ten-fold reduction in conductance from veraison to full ripeness. Shiraz had conductances that were two to five fold larger than Chardonnay, and maintained higher conductance in the post 90 days after anthesis, the period where berry weight loss occurred. In both varieties the hydraulic conductance reduced in the distal and proximal portions of the berries from veraison. The probe measured the pressure developed in the xylem of nontranspiring berries. Pre-veraison, negative xylem pressures of -0.2 to -0.1 MPa were measured, increasing to around zero between veraison and 90 days after anthesis. The pressures around zero were maintained until harvest when the berry juice osmotic potential was around -3 MPa for Chardonnay and -4 MPa for Shiraz. As cell turgor pressure is low and the juice osmotic potential is not translated into negative xylem pressure, it suggests that the reflection coefficient of cell membranes surrounding the berry xylem in both varieties changes from close to 1 pre-veraison, to about at veraison and decreases to 0 at harvest. As Chardonnay berries do not lose weight prior to harvest, the data suggests that there is varietal difference in how berries isolate from the vine and xylem backflow is possible in Shiraz, perhaps associated with changes in aquaporin or cell membrane function in xylem associated tissue. Abbreviations: L o, hydraulic conductance; R o, hydraulic resistance; daa, days after anthesis; TSS, total soluble solids Key words: berry ripening, pressure probe, hydraulic conductance, Vitis vinifera, berry shrivel. 6

18 Introduction Vitis vinifera berries are grown for both edible fruit and wine, and exhibit a range of flavours, textures and appearances. The berries are fleshy fruits that accumulate water, sugars and other organic compounds during berry development. The generally accepted view of berry development has been of three phases; a rapid growth phase that commences with cell division and enlargement, a brief period where growth slows or pauses, then a second period of rapid growth and sugar accumulation in the mesocarp until maximum berry weight is reached (Coombe and McCarthy 2000). Berry content is mostly water, and the percentage water content can be estimated in seeded grape berries by subtracting the total Brix of the juice (total soluble solids) from 100 (discussed in McCarthy and Coombe 1999). For most wine making, berries are harvested in the range of Brix. Therefore, the berries are approximately 75-80% water at harvest maturity depending on the grape variety and the style of wine being made. Water enters berries via the xylem tracheids and phloem sieve elements into the central and peripheral vascular bundles within the berries to supply the mesocarp and seed tissue. Pre-veraison, water inflow is predominantly via the xylem, and post-veraison it is phloem dominant (Düring et al. 1987; Greenspan et al. 1996; Greenspan et al. 1994). Dye perfusion and microscope studies in the varieties Shiraz, Pinot noir, Chardonnay, Riesling and Merlot found that xylem tracheids stretched and broke around veraison or berry softening. It was hypothesised that this was the reason for the apparent cessation of xylem flow into berries (Creasy et al. 1993; Düring et al. 1987; Findlay et al. 1987; McCarthy and Coombe 1999; Rogiers et al. 2001). It was possible that while dye movement via xylem tissue ceased at veraison, water continued to be transported into the berry via the 7

19 xylem (Rogiers et al. 2001). This has been confirmed in recent work which established that while the older tracheids in berry peripheral xylem tissue are stretched and broken in the post-veraison growth phase, the younger xylem vessels remained intact and may maintain some function (Chatelet et al. 2008b). The pattern of berry weight accumulation in Shiraz berries has shown a distinct difference when compared to most other grape varieties. Over four years of irrigation experiments on Shiraz vines in the Riverland of South Australia, it was noted that regardless of the season or watering regime, Shiraz berries reached a maximum weight around 91 days after anthesis, then lost up to 30% of that weight before harvest maturity was reached (McCarthy 1999). This phenomenon has been noted to varying degrees in other regions such as Wagga in New South Wales (Rogiers et al. 2000) and McLaren Vale in South Australia (Davies and Robinson1996). Berry weight loss is likely to have an effect on sugar metabolism, development of flavours (Coombe and McCarthy, 2000) and the accumulation of minerals in berries (Rogiers et al. 2006; Rogiers et al. 2000). McCarthy and Coombe (1999) hypothesised that post-veraison Shiraz berry weight loss was due to a series of events: cessation of water inflow via the xylem at veraison, reduction in phloem inflow to zero at maximum berry weight, and a continuing loss of water across the berry cuticle due to transpiration. Cuticular transpiration for Shiraz berries is relatively low and reduces throughout berry development (Rogiers et al. 2004). To assess the contribution of xylem flow into Shiraz berries, Rogiers et al. (2000) examined the accumulation of xylem mobile calcium, and xylem and phloem mobile potassium during development. They found that calcium and potassium content both increased in post-veraison berries, but potassium increased at a faster rate therefore increasing the potassium:calcium ratio. This suggested that some xylem function was maintained in Shiraz berries. These results contrasted 8

20 with work on de Chaunac berries that indicated a cessation of calcium accumulation at veraison (Hrazdina et al. 1984; Lang and Thorpe 1989) and Pinot Noir berries where potassium was shown to increase rapidly post-veraison but calcium did not. Overall, the data suggested that there may be varietal differences in berry xylem function, particularly after veraison. It has been considered unlikely that backflow of water from berries to the vine could occur late in berry development because the osmotic pressures developed in post-veraison berries were significantly more negative than the negative water potential developed in the xylem of a transpiring vine (Lang and Düring 1991; McCarthy and Coombe 1999). This hypothesis assumes that cell membranes across the berry mesocarp maintain selectivity and a reflection coefficient at or near 1. There is no published direct measurement of xylem flow into grape berries. Therefore, it would be useful to directly quantify and characterise the hydraulic xylem flow into berries to examine how it changes through berry development and how it relates to weight loss in Shiraz berries. Materials and methods Fruit material Fruit was obtained from nine year old Vitis vinifera cv Shiraz,Barossa Valley Research Centre 12 (BVRC12) and cv Chardonnay I10V1 own rooted vines in the Coombe vineyard on Waite campus of the University of Adelaide. In the season, bunches of grapes from Shiraz were cut from twelve, vines that formed part of a randomised block rootstock trial, placed in a closed plastic bag in a polystyrene cool box on ice and taken to the laboratory (about 500 m). Similarly, bunches of grapes of Chardonnay were taken from twelve adjacent vines. Anthesis was defined as 50 % capfall from an inflorescence, and 9

21 inflorescences were labelled individually. Berry softening occurred at 54 days after anthesis (daa) for Shiraz berries and 60 daa for Chardonnay. Commercial irrigation and spray regimes were used in the vineyard. Berry weight, Brix, ph and osmolality At each time point, fifty berries were collected from ten of the replicate vines by taking five berry samples from the proximal, middle and distal part of one bunch on each vine and placed into a cliplock plastic bag and taken to the laboratory. The berries were weighed, crushed and the juice collected. Brix of the juice was measured using temperature compensated digital refractometer (ATAGO Model PR101) and ph with a Cyberscan 310 Series (Eutech Instruments). Pressure probe measurements The root pressure probe is an instrument designed to measure equilibrium pressures and hydraulic conductance in severed roots of plants (Steudle and Jeschke 1983; Steudle et al. 1993). The aim was to measure the hydraulic conductance of pedicel xylem tissue of single, detached berries, analogous with the measurements made on detached roots using the pressure probe. Immediately before an experiment, a single berry with the pedicel attached was cut from the bunch through the proximal end of the pedicel using a sharp razor blade while the pedicel was under Millipore filtered water additionally filtered (0.2 µm) and de-aerated by vacuum. A bead of water was maintained on the cut surface. Cyanacrylate glue (Loctite 401 or 406) was applied to the pedicel (avoiding the cut surface), and it was inserted into tubing that had been flared to fit (Tefzel-Schlauch tubing, ID 1.0 mm and OD 1.6 mm etched with Loctite 770 primer). The tubing was immediately backfilled with water prepared as above. The tubing with attached berry was sealed to the pressure probe ensuring that the system contained no air bubbles and was pressure tight. The system is 10

22 defined as the berry attached to the tubing plus the pressure probe and the seals (Fig 1). Figure 1 Pressure probe modified to measure berry hydraulic conductance. Berries are attached to tubing, which is sealed into the probe. Volume flow into the berry is measured by following the meniscus in the glass capillary or by the micrometer screw. The pressure transducer measures the pressure in the system. The berry was covered with wet tissue paper to stop transpiration. To determine whether the xylem of the berry pedicel was connected hydraulically to the probe, pre-veraison berries were mounted on the probe with the tube filled with a 0.01% aqueous solution of cellufluor (Sigma-Aldrich, initially dissolved in a drop of ethanol then made up to volume in Millipore filtered water). The probe and berries were left overnight then sectioned using a hand microtome, mounted in 70% glycerol and visualised under a Zeiss Axiophot fluorescence microscope with a filter cube inserted: excitation filter , beam splitter FT 460 and barrier filter LP 470. Digital images were obtained using a JVC 3CCD digital camera (Fig 2). Figure 2 Cellufluor perfused through the central and peripheral xylem tissue of preveraison berries. Shown are Shiraz brush and basal peripheral xylem (left) and Chardonnay peripheral xylem (right). Bar = 200!m. 11

23 Steady state pressure Once attached to the probe and the seals tightened, the berry system was allowed to relax to a steady state (Fig 3 a). It was assumed that the pedicel phloem would become blocked when the berry was cut from the rachis analogous with root pressure probe experiments in other plant species (Steudle et al. 1993), so that measurements were of the xylem and composite membrane system of the berry and pedicel. In pre-veraison berries the highly negative pressures generated in the xylem frequently caused cavitation in the pressure probe. In some cases this could be resolved by increasing the pressure in the system (and flow into the berry via the xylem) to force the air bubble back into solution, but generally this was not possible and the experiment was abandoned. 12

24 Figure 3 (a) Once sealed into the probe, the berry and probe system was allowed to equilibrate. Pre-veraison, cavitation or air bubbles frequently occurred in the system because of the extremely negative pressures developed; when this happened no measurements could be made. (b) A sequence of volumes were introduced into or withdrawn from the berry and the pressure responses used to calculate the elasticity in the system. (c) The pressure changes in response to volume changes for the calculation of elasticity are plotted and compare pre-veraison, post-veraison and closed system (where the tube was sealed and no berry attached) measurements. (d) An example of a pressure clamp experiment where pressure is maintained by continued injection of volume into a berry. (e) The flow rate injected into the pedicel xylem is plotted against the measured pressure. The slope calculated from this relationship is the hydraulic conductance into the berry. Elasticity The elasticity (!) of the system was measured by changing the volume in the system (! s = "P/"V) by shifting the micrometer screw and following the movement of the oil/water interface (meniscus) in the glass capillary of the probe (Fig 1). The resulting changes in pressure were recorded (Fig 3 b). The elasticity of the probe and a closed section of tubing (! p ) was measured to establish the 13

25 elasticity of that part of the system so that it could be related to data with the berries attached (Fig 3 c). The data was also used to ascertain whether glue had blocked the cut surface of the pedicel and confounded data. For example, if! s was equal to or higher than! p, it showed that there was not hydraulic continuity into the berry. Other criteria were also used to confirm continuity. At the conclusion of each experiment, the pedicel was cut between the berry and the tubing and the cut surface allowed to dry. As water evaporated from the cut surface, negative pressure was generated in the xylem and measured with the probe. If this returned to zero, (atmospheric pressure) when a drop of water was placed on the cut surface, it was assumed that there was a hydraulic connection. It was also established that hydraulic conductance was high through discrete sections of berry pedicels, and when the exposed cut surface of the pedicel was sealed with cyanacrylate, no hydraulic flow was measurable (data not shown). Hydraulic conductance Initial experiments identified that the usual pressure probe method used on roots where pressure relaxation curves and! were used to calculate conductance (Steudle et al. 1993) were unsuitable because they overestimated conductance through cut pedicels. Instead, a pressure clamp procedure was used to calculate hydraulic conductance. A pressure was imposed and maintained on the system for a period of time (Fig 3 d) and the volume flow into the berry during that time was measured either by measuring the movement of the oil-water interface in the glass capillary of the probe or by the movement of the micrometer screw. Volume flow measurements were tested and found to be accurate by both methods. A range of pressures between 0.01 and 0.06 MPa were imposed and hydraulic flows measured. The relationship between pressure and flow rates into berries was established and hydraulic conductance calculated (Fig 3 e). 14

26 Portions of the berry were excised sequentially with a sharp razor blade and hydraulic conductance measured to identify whether it was limited at a particular location in the berry. Tissue was cut at the brush, receptacle or pedicel. Data was collected in and In the latter season it was collected by Lukasz Kotula (refer to Thesis Declaration) and this data is clearly differentiated. Results Pressure probe measurements The fluorescent dye experiments confirmed that a xylem hydraulic connection was maintained between the probe and the berry, with cellufluor transported from the tubing of the probe through the pedicel xylem and into berry xylem tissue in pre-veraison berries. Dye was clearly visible though the pedicel, torus, brush, central vascular bundle and all the peripheral vasculature to the distal tip of the berry (Fig 2). During the season, Shiraz berries softened at 55 days after anthesis (daa) and Chardonnay at 60 daa, indicating veraison. Once attached to the probe, berries were allowed to equilibrate. The extremely negative pressures generated in the xylem pre-veraison (< -0.1 MPa) meant that data was difficult to collect until several days before veraison or berry softening, xylem pressures became less negative and measurements could be made (Fig 4 a). Berry equilibration pressures are shown in the context of the osmotic potential, Brix and ph of the juice of berries (Fig 4 b & c). 15

27 Figure 4 The pedicel equilibration pressure of berries (a), osmotic potential of (b) and the TSS and ph (c) of Shiraz (solid symbols) and Chardonnay (open symbols) berry juice is shown relative to daa. Using the equilibration and juice osmotic potential data, an estimate of the reflection coefficient of the osmotic barrier within the berry was calculated to be approximately 0.15.Each data point in (a) represents a measurement on a single berry. In (b) and (c), n=mean of 50 berry samples, refer to Methods. Xylem pressure for both Shiraz and Chardonnay berries increased from negative pressures around veraison to around zero pressure at about daa. At this time the osmotic potential of Chardonnay juice was about -3 MPa and Shiraz 16

28 about -4 MPa, which increased to -4 MPa and -5 MPa respectively by 110 daa. Pedicel xylem pressures were maintained at, or just above zero until harvest maturity was reached in both varieties, with Chardonnay showing a trend towards slightly positive pressures post 90 daa. At harvest, total soluble solids were about 22 Brix and ph was 4 in both varieties. Hydraulic conductance into berries Hydraulic conductance declined gradually after berry softening until around 110 daa for whole berries of both Shiraz and Chardonnay (Fig 5 a & b). In Shiraz, conductance into berries reduced ten-fold over that period. For Chardonnay the reduction in conductance into whole berries was not as distinct with flows around one third of Shiraz in the early post-veraison period, and trending downwards towards 110 daa. Conductance into whole Shiraz berries was about double that of whole Chardonnay at all points during the period. That same trend of a two-fold higher conductance into Shiraz compared to Chardonnay was maintained in berries cut through the mesocarp at the base of the seeds and through the base of the berries (Fig 5 c-f). While Shiraz conductance data through the cut receptacles was more widely spread and trended down in the day period compared to Chardonnay, no clear difference in conductance through the cut receptacles was observed (Fig 5 g-h). Data is combined for the season during method development (open symbols) and the season (solid symbols) collected by Lukasz Kotula). 17

29 Figure 5 Berry hydraulic conductance for Shiraz (a, c, e, g on the left) and Chardonnay (b, d, f, h on the right) measured over (open symbols) and (solid symbols) and linear regressions fitted. Each data point represents a measurement made on an individual berry. Conductance was measured for whole berries, and berries with tissue portions excised sequentially (refer to Figure 6 for excision locations). Using the regression equations of hydraulic conductance into berries in Fig 5, resistance (1/conductance) in whole and cut berry systems, and the individual elements of resistance at 70 and 100 daa were calculated (Fig 6). 18

30 Figure 6 To identify the location in the berry where hydraulic conductance changed between 70 daa (solid symbols) and 100 daa (open symbols), Shiraz (triangles, b& d) and Chardonnay (circles, c & e) were cut at various positions (a) and measurements made. The whole berry is represented as position 0. Hydraulic resistance (R o) is presented as a function of the cut position at the two time points for Shiraz (b) and Chardonnay (c). The hydraulic resistance for each section of the berries is calculated by subtracting the R o at cut position 1 from the R o at position 0, the R o at cut position 1 minus the R o at position 2 and so on. Data points are taken from the linear regressions performed in Figure 5. Hydraulic resistance increased in both varieties from 70 to 100 daa in whole berries and when those cut through the pericarp below the seeds, but no effective change was seen at the brush and receptacle cut positions (Fig 6 b &c). The resistance (R o ) for each section of the berry system was calculated as R o at position 0 minus R o at cut position 1, R o at cut position 1 minus R o at cut position 2, and R o at cut position 2 minus R o at cut position 3 (Fig 6 d & e). Chardonnay 19

31 berries show a much larger increase in resistance of the distal and proximal portions of berries compared to Shiraz. Discussion Direct evidence of the pressures developed in pedicel xylem in detached developing grape berries is presented. Pre-veraison, the equilibration pressures generated in the berry system were sufficiently negative that they could not be measured with the probe until the berries were close to softening at 55 and 60 daa for Shiraz and Chardonnay respectively. The negative pressures in the system imply that cells in the mesocarp were functioning with perfect, semipermeable membranes that had a reflection coefficient at or close to 1. This means that the membranes were probably selective and maintained a significant pressure gradient across them. During the pre-veraison period, negative pressures generated in the apoplast of the berry were in excess of what was generated in the vine xylem, resulting in hydraulic inflow into the berry. Around berry softening in both varieties, the juice osmotic potentials were around -1 MPa, and continued to decrease to -4 to -5 MPa as berries approached harvest maturity at 110 daa. In the post-veraison berries it is highly significant that the very negative osmotic pressures indicated by the osmolarity of the berry juices were not translated into negative pressure in the pedicel xylem during that period. It is likely that the cell membranes within the mesocarp ceased to function selectively. From the berry equilibration pressures, it is estimated that the reflection coefficient of the mesocarp cell membranes shifted from close to 1 preveraison to at veraison, and around zero at harvest maturity. This could suggest leakage between the symplast of the cells and the apoplast. Another option is that compartmentation between the apoplast and symplast breaks down at veraison in the mesocarp of grape berries. Berry softening at veraison with 20

32 consequent increasing deformability of the berries has been offered as circumstantial evidence that supports this (Lang and Düring, 1991). In Riesling berries detached from the rachis and pressurised at different stages through berry development, the solute content of xylem exudate from the pedicels was directly related to that of the juice from the same berries when crushed (Lang and Düring 1991). Measurements on individual post-veraison cells in the mesocarp of Shiraz and Chardonnay berries using a cell pressure probe found that the cells had close to zero turgor (J. Tilbrook, unpublished results). This has also been observed in Pinot Noir, Merlot and Cabernet Sauvignon (Thomas et al. 2006). More recent work has found that at or just before berry softening in grapes, phloem unloading of photosynthates in berries changes from symplastic to apoplastic (Zhang et al. 2006). This is consistent with our data showing a rapid increase in berry xylem equilibration pressures from berry softening until daa on berries attached to the pressure probe, which would reflect an increasing solute concentration in the apoplast. It is during this period that berry equilibration pressures increase until they are about zero, which implies the loss of an effective osmotic gradient across the cell membranes of the mesocarp. Equilibration pressures then stay relatively stable for the remainder of berry development. Using the linear regressions of hydraulic conductance into berries through development, resistance (1/conductance) was examined at 70 and 100 daa for whole and cut berries (Fig 6), either side of the ~90 daa nominated by McCarthy (1999) as the time weight loss commenced for Shiraz. Resistance is plotted against the cut positions for Shiraz (Fig 6 b) and Chardonnay (Fig 6 c). Resistance into Shiraz and Chardonnay berry xylem increased from 70 to 100 daa in a similar pattern with Chardonnay resistances being greater than Shiraz at all cut positions except through the pedicel. The resistances were measured as a 21

33 series and therefore the components could be examined individually by subtracting the intermediate series resistances through the berry. The hydraulic resistance for the distal section of a berry is calculated by subtracting the resistance of the berry measured at cut position 1 from the resistance of the whole berry, for example (Fig 6 d & e). Chardonnay exhibited about double the resistance through the distal and proximal (brush) sections of the berry compared to Shiraz, while the receptacle and pedicel sections did not change significantly. The high resistances measured in Chardonnay berries indicate that they isolate efficiently from the vine at 100 daa, consistent with dye uptake experiments in Merlot, Pinot Noir and Muscat Gordo (Creasy et al. 1993; Findlay et al. 1987). Shiraz berries however exhibit much lower resistances which conflicts with dye uptake studies in that variety (Rogiers et al. 2001). Quantitative evidence of significant differences in hydraulics between the wine grape varieties is presented; in Shiraz, a variety that exhibits berry weight loss and Chardonnay, a variety that maintains berry weight until harvest. The data also suggests that, contrary to the long accepted view of a sudden cessation of xylem hydraulic inflow at veraison or berry softening (Coombe and McCarthy 2000; Creasy et al. 1993; Findlay et al. 1987), there is in fact a gradual reduction or tapering of inflow via the xylem into berries from berry softening until harvest. Shiraz show higher xylem hydraulic conductance into berries throughout development compared to Chardonnay. This may be linked to why Shiraz berries lose weight. If Shiraz berries do not isolate from the vine as efficiently as other varieties, it poses the questions: is backflow via the pedicel xylem a significant issue in Shiraz weight loss before harvest and if it is, to what degree? In Cabernet Sauvignon berries, xylem backflow has been considered insignificant (Greenspan 22

34 et al. 1996) but in Italiaberries it was calculated that 36 % of berry water loss was due to backflow (Lang and Thorpe 1989). For post ~90 daa weight loss in Shiraz berries, it has been hypothesised that almost all the weight lost is water (Rogiers et al. 2000). Another explanation is that the berry weight loss is a cessation of inflow into berries via the phloem at daa (McCarthy and Coombe, 1999) as well as inflow via the xylem ceasing or slowing. Transpiration of water from berries has also been examined. Grape berries have very few stomata on their cuticles (Blanke and Leyhe 1987), and Shiraz is no exception (Rogiers et al. 2004). Despite the stomata becoming blocked with waxes and the wax platelets on the berry cuticle thinning significantly in the post-veraison period, transpiration across the cuticle of Shiraz berries reduced to 16% of pre-veraison values and was estimated to account for up to 15 mg loss of fresh weight per berry per day (Rogiers et al. 2004). Potential backflow from Shiraz berries can be calculated using the hydraulic conductance into berries measured at 100 daa (5 x m 3 s -1 MPa -1 ) at a pressure gradient of 0.1 MPa, which estimates a water loss of approximately 43 mg per day. Together backflow and transpiration could account for more than 30 % weight loss in Shiraz berries over a seven day period. The reduction in hydraulic conductance into berries during development may also suggest a change in cell membrane permeabilities, perhaps associated with aquaporin function. Aquaporins are membrane bound proteins that transport water and low molecular weight compounds across various membranes in plant cells (Katsuhara et al. 2008). They have a significant role in various fruits, including grape berries (Delrot et al. 2001; Picaud et al. 2003) because grapes accumulate sugars in cell vacuoles and high osmotic pressures are generated (reviewed in Katsuhara et al. 2008). It is also possible that the parenchyma cells associated with the berry xylem have a role in regulating water transport to and 23

35 from the xylem. Our data suggests that the post-veraison pressures developed in the berry xylem are insufficient to counter the pressures developed in the vine xylem. This means that berries need to have some mechanism to prevent backflow into the vine, and whatever that is, it appears to function more effectively in Chardonnay berries compared to Shiraz. Taking the hypothesis of McCarthy and Coombe (1999) which proposes a tapering of inflow via the phloem at maximum berry weight in Shiraz and the new data presented, an updated working hypothesis of how this may occur is shown in Fig 7. Pre-veraison the cells of the berry mesocarp have competent membranes and a concentration gradient is maintained, resulting in rapid uptake of solutes into the cells (Fig 7 a). After veraison the mesocarp cells lose selectivity, or the concentration of solutes in the berry apoplast increases so that the effective pressures measured in the berry xylem increase and plateau around zero, which can not counter the xylem tensions developed in the vine xylem. Phloem unloading is maintained or enhanced, possibly with increased aquaporin function to replace the reducing inflow via the xylem (Fig 7 b). Finally, when phloem translocation has ceased, Shiraz berries are not sufficiently isolated from the vine and lose weight (Fig 7 c). 24

36 Figure 7 A working hypothesis of berry hydraulics (a) Pre-veraison berries have low solute concentration in the apoplast and mesocarp cells are osmotically competent with cell membranes having a reflection coefficient of around 1. Hydraulic inflow is predominantly via the xylem to support transpiration and cell enlargement, with aquaporins in the xylem parenchyma cells functioning. (b) Post-veraison the mesocarp cells have lost selectivity and the reflection coefficient is zero or the solute concentration in the apoplast is increased as phloem unloading increases and changes from symplastic to apoplastic (Zhang et al. 2006). A combination of both is also possible. The water potential in the apoplast increases and the ability of the xylem/composite membrane system to counter the negative pressures in the vine xylem is reduced. Hydraulic conductance may be reduced by a loss of aquaporin activity in xylem associated cells and increased by activation of aquaporins in phloem associated cells. (c) During the period where shrinkage occurs, phloem translocation into the berry has ceased. If the xylem/composite membrane hydraulic conductance has not reduced to the point of sufficient isolation from the vine, the berry loses weight by a combination of backflow and continued transpiration. The data presented strongly suggests that Shiraz berries lose weight prior to achieving harvest maturity because of a varietal difference in the efficiency of 25

37 berry isolation from the vine. This needs further characterisation. Another issue to consider is how membrane competence in mesocarp cells changes through development. Does it vary with variety and is it linked to changes in berry xylem conductance? Understanding these processes will be the next step in identifying what is different about Shiraz and why the berries have such significant water loss late in development. 26

38 Chapter 3 Cell death in grape berries: varietal differences linked to xylem pressure and berry weight loss. This chapter is published: Tilbrook J, Tyerman SD (2008) Cell death in grape berries: varietal differences linked to xylem pressure and berry weight loss. Functional Plant Biology 35, The paper received an award: Best paper of 2008 by an early career researcher awarded by the Australian Society of Plant Scientists and Functional Plant Biology, July

39 28

40 Abstract Some varieties of Vitis vinifera L. can undergo berry weight loss during later stages of ripening. This defines a third phase of development in addition to berry formation and berry expansion. Berry weight loss is due to net water loss, but the component water flows through different pathways have remained obscure. Because of the very negative osmotic potential of the cell sap, the maintenance of semipermeable membranes in the berry is required for the berry to counter xylem and apoplast tensions that may be transferred from the vine. The transfer of tension is determined by the hydraulic connection through the xylem from the berry to the vine, which changes during development. Here we assess the membrane integrity of three varieties of V. vinifera berries (cvv. Shiraz, Chardonnay and Thompson Seedless) throughout development using the vitality stains, fluorescein diacetate and propidium iodide on fresh longitudinal sections of whole berries. We also measured the xylem pressure using a pressure probe connected to the pedicel of detached berries. The wine grapes, Chardonnay and Shiraz, maintained fully vital cells after veraison and during berry expansion, but began to show cell death in the mesocarp and endocarp at or near the time that the berries attain maximum weight. This corresponded to a change in rate of accumulation of solutes in the berry and the beginning of weight loss in Shiraz, but not in Chardonnay. Continuous decline in mesocarp and endocarp cell vitality occurred for both varieties until normal harvest dates. Shiraz grapes classified as high quality and sourced from a different vineyard also showed the same death response at the same time after anthesis, but they displayed amore consistent pattern of pericarp cell death. The table grape, Thompson Seedless, showed near to 100% vitality for all cells throughout development and well past normal harvest date, except for berries with noticeable berry collapse that were treated 29

41 with gibberellic acid. The high cell vitality in Thompson Seedless berries corresponded to negative xylem pressures that contrasted to the slightly positive pressures for Shiraz and Chardonnay. We hypothesise that two variety dependent strategies exist for grapevine berries late in development: (1) programmed cell death in the pericarp and loss of osmotically competent membranes that requires concomitant reduction in the hydraulic conductance via the xylem to the vine; (2) continued cell vitality and osmotically competent membranes that can allow high hydraulic conductance to the vine. Abbreviations: FDA; fluorescein diacetate, PI; propidium iodide, daa; days after anthesis. Additional keywords: grape berry development, cell death, berry shrivel, berry weight loss. 30

42 Introduction There has been much interest recently in the water budget of the developing grape berry (Bondada et al. 2005; Keller et al. 2006; Rogiers et al. 2006; Thomas et al. 2006; Tilbrook and Tyerman 2006; Zhang et al. 2006). This is for two reasons: First the berry is a good model for other fleshy fruits and can provide some generalisations of how water movement into and out of the fruit is regulated through changes in solute partitioning, phloem and xylem transport, and transpiration. Second, the water content of the harvested berries and the way in which water is retained in the berry has a large impact on quality; whether this be via concentration of soluble solids and flavour molecules in wine grapes, or through crispness (turgidity) of the fruit in the case of table grapes. Yield is also determined largely by water content since water makes up the major component of berry mass. Loss of water from the berry can be substantial in some varieties and this can reduce yield by over 25%, for example in Shiraz (McCarthy 1999; McCarthy and Coombe 1999). It is generally agreed that before veraison during the first phase of berry development (berry formation, phase 1 Fig 8) the berry transpires and water inflow occurs via the phloem and the xylem (Lang and Thorpe, 1989; Greenspan et al. 1994; Dreier et al. 2000; Rogiers et al. 2004; Rogiers et al. 2006). After a lag in expansion and just after the berry softens, sugar and water accumulation rapidly increase via phloem import, here referred to as phase 2 (Fig 8). After the berry reaches maximum weight a third phase is apparent in some varieties, when berry weight loss begins (Sadras and McCarthy 2007). This can occur before grape flavour development for winemaking is evident (Coombe and McCarthy 1997), and sugar concentration can be further increased by a combination of volume decrease of the berry and further sugar import, although further sugar 31

43 import to the berry seems to be plastic between seasons (Sadras and McCarthy 2007). Fig 8 shows these phases of berry development in context with other changes in xylem function and berry water relations taken from the literature and our own data. It should be noted that phase 1 incorporates Stages I and II of Coombe (1992). During phase 2 the berry appears to become less hydraulically connected to the vine (Greenspan et al. 1994; Greenspan et al, 1996; Tyerman et al. 2004; Tilbrook and Tyerman 2006). From earlier dye uptake studies and calcium uptake into the berry (as a xylem tracer)(findlay et al. 1987; Creasy et al. 1993; Rogiers et al. 2001), the hydraulic isolation was proposed to be due to discontinuity of xylem vessels in the berry. However, our previous quantitative measurements of the xylem pathway to the berry showed that the pathway remained functional, though hydraulic conductance was reduced in magnitude depending on variety (Fig 1, Tyerman et al. 2004). Bondada et al. (2005) qualitatively confirmed that xylem hydraulic conductance continued in post veraison berries by demonstrating that dye uptake could still occur provided the appropriate driving force on water flow to the berry could be sustained. Tyerman et al. (2004) also showed that the xylem pressure changed from being negative to slightly positive during veraison, supporting the view that the nature of the driving force for water movement to the berry in the xylem changes during phase 2 (Fig 8). These discoveries have focussed attention on the solute partitioning between apoplast and symplast in the berry because this will determine how the very negative osmotic potential of the berry juice, largely mesocarp cell sap, is translated into the driving force for water movement, both through the phloem and the xylem. 32

44 Figure 8 Summary of the development of grapevine berries with various physiological changes associated with berry water relations. Three phases can be identified based on changes in the rate of change of berry weight (see also Sadras and McCarthy, 2007). These phases are delineated by a vertical dashed line in each panel. (a) Berry weight and deformability. Shown are fits to data for Shiraz (Tilbrook, unpublished data). (b) Xylem equilibrium pressure and turgor pressure of berry pericarp cells. Shown are fits to data sourced from Tyerman et al. (2004) (xylem pressure) and Thomas et al. (2006) (turgor). (c) Hydraulic conductance (relative to maximum in pre-veraison berries) of the xylem pathway into Shiraz berries (fitted curves from Tyerman et al. 2004; and Tilbrook unpublished data), and Shiraz berry transpiration relative to the maximum in pre-veraison berries (Rogiers et al. 2004). 33

45 The demonstration by (Zhang et al. 2006) that phloem unloading switches from symplastic to apolastic during veraison, suggests that osmotic potential of the apoplast decreases. This could drive greater flow through the phloem (Patrick 1997) and may explain the transition to slightly positive xylem pressure measured by Tyerman et al. (2004). An important issue remains however, and this is whether or not the osmotic competence of the cell membranes, particularly of the large mesocarp cells, changes in the post veraison berry. This is important because normal cell membranes will show semipermeability, and for large solutes like sugars, an osmotic potential difference across the membrane is reflected by an equivalent hydrostatic pressure difference. The large negative osmotic potential of the mesocarp cells could only balance negative apoplast pressures and xylem tensions if the membranes remain semipermeable. There is indirect evidence that cell membranes become leaky in the mesocarp of post veraison Riesling (Lang and Düring 1991; Dreier et al. 1998) and cell compartmentation can break down in Thompson Seedless (Dreier et al. 1998). However, turgor at low pressures (0.05 MPa), and therefore membrane semipermeability and cell vitality, was maintained in cells to a depth of 1.5 mm below the cuticle until 100 days after anthesis (daa) in several wine grape varieties, including Chardonnay (Thomas et al. 2006). The reduction in turgor pressure at veraison (transition from phase 1 to phase 2) of the mesocarp closely corresponds to increased deformability of the berry (Fig 8) and suggests that the apoplast osmotic potential declines. The turgor pressure observations, like most, do not continue into the final 2-3 weeks before harvest, because the fruit becomes very deformable (Fig 8), and berry contents can be extremely difficult to work with. It is during phase 3, post daa in varieties like Shiraz, that considerable loss of weight can occur to the point where the berries may shrivel (Fig 8). This loss of weight has been 34

46 proposed to be due to a combination of reduced phloem inflow and continued transpiration (McCarthy and Coombe 1999; Rogiers et al. 2004; Tyerman et al. 2004; Keller et al. 2006) though backflow to the vine via the xylem may also contribute (McCarthy and Coombe 1999; Rogiers et al. 2004; Tyerman et al. 2004; Keller et al. 2006). Backflow was directly demonstrated by dye loading at the stylar end of post veraison berries and observing the dye in the xylem of the vine (Keller et al. 2006). Though the apoplasmic water of the berry is available for movement back to the vine, Keller et al. (2006) conclude that the berry cell membranes remain semipermeable, thereby making it difficult for the leaves to extract water from the berry cells because of the large negative osmotic potential of the cell sap. Thus if backflow alone were to account for a loss of up to 30% of maximum weight, as is often observed in Shiraz, there would have to be a loss of membrane semipermeability for a large proportion of the cells in the berry. The aim of this work was to test the hypothesis that cells across the pericarp of berries maintain membrane competence and vitality until harvest maturity is achieved. It formed part of a project to ascertain the cause of weight loss in Shiraz berries. Shiraz, Chardonnay and Thompson Seedless fruit were compared, because Chardonnay does not normally show weight loss and Thompson Seedless was found by us to have very different xylem pressures compared to the other varieties. We used two vital stains, fluorescein diacetate (FDA) and propidium iodide (PI). These dyes are a well documented and reliable method of determining cell vitality in cell suspensions and flow cytometry (Jones and Senft 1985). FDA is a non-polar, non-fluorescent molecule that crosses cell membranes. Once in the cytoplasm, esterases cleave the acetate groups from the molecule and it becomes highly polar and fluorescent green. If a cell does not have an active metabolism, it will not fluoresce. PI is a membrane impermeant dye that enters cells only when cell membranes are disrupted or not intact. It 35

47 binds non-covalently, intercalating in a stoichiometric manner with single and double stranded DNA and RNA. When intercalated it fluoresces red (Cosa et al. 2001; Bernas et al. 2004; Kral et al. 2005). The loss of cell membrane integrity in plants is considered to mark the end of homeostasis and indicates cell death (Noodén, 2004). Using these vital stains we examined the vitality of cells in medial longitudinal sections of fresh berries through development. The data were analysed to provide a new insight into cell vitality and cell membrane competence through berry development that we relate to measurements of xylem pressure using the pressure probe attached to individual berry pedicels. Materials and methods Fruit material Experimental fruit used in the time course experiments was from the Coombe vineyard (Shiraz BVRC12 and Chardonnay I10V1, 12 years old) and Alverstoke vineyard (Thompson Seedless M12, not treated with gibberellic acid, 4 years old) on the Waite Campus of the University of Adelaide. Bunches of fruit were labelled individually when an estimated 50% of the flower caps from that bunch had dehisced. The day this occurred was designated as anthesis. Anthesis was complete over 1-2 days. Premium quality Shiraz (unknown clone, about 30 years old) was obtained on the day of harvest courtesy of Hardys, McLaren Vale from a McLaren Flat site. Thompson Seedless fruit treated with gibberellic acid and showing signs of berry collapse was from CSIRO Merbein, courtesy of Mike Treeby and Tori Nguyen. All data from vineyard fruit was obtained during the season. The pre-veraison Shiraz BVRC12 berries in the fluorescent dye control experiment were from glasshouse grown vines at the Plant Research Centre in the Waite Campus precinct. 36

48 Berry weight, Brix and osmolality Berry weight and Brix data reflect whole vineyard berry development. Weights are the means of 50 berry samples. For Shiraz, samples of five berries from proximal, mid and base of two random bunches on separate vines from each of five replicate panels in separate rows that formed part of a randomised block rootstock trial were collected. For Chardonnay, five berry samples from proximal, mid and base of ten random bunches on separate vines in a row of twenty four adjacent vines. These data were not collected for Thompson Seedless as insufficient experimental fruit was locally available. Berries collected for weight samples were crushed, juice collected and briefly centrifuged to settle any solids. Brix of the juice was measured using a temperature compensated digital refractometer (ATAGO Model PR101). A Wescor (Model 5500) water vapour pressure osmometer measured juice osmolality which was converted to osmotic potential (# $ = -RTC, where R = J/mol.K and T = absolute temperature in K). Solutes per berry (g) was approximated by the product of berry weight and o Brix/100 (McCarthy and Coombe, 1999). Vital and non vital staining of pericarp tissue Bunches (all clones of Shiraz and Chardonnay) or clusters (Thompson Seedless) were cut from within the vine canopies, placed in plastic bags on ice and taken to the laboratory (~500 m). Berries were sectioned longitudinally between the seeds (where present). One half of each berry was pooled and crushed for juice to measure Brix and to calculate osmotic pressure (as above). The FDA section of the method was developed from that kindly shared by Professor Ken Shackel, U. C. Davis. From a 4.8 mm FDA (Sigma-Aldrich) in acetone stock solution, an aqueous 4.8 µm FDA solution (Oparka and Read, 1994) was prepared to the same osmotic pressure as the berry juice with sucrose, then applied to excess on the cut surface of the half berries. After 15 minutes incubation, sectioned berries 37

49 were viewed with a Leica M-Z FL111 dissecting microscope at minimum magnification under ultra violet light with a green fluorescent protein filter in place. Images were promptly obtained using a Leica DC 300F camera and Image Pro Plus 5.1 (MediaCybernetics) with consistent settings and exposure times. The FDA solution was blotted from the cut berry surfaces, then a counterstain of aqueous 190 µm PI solution (Sigma-Aldrich) (Oparka and Read 1994) freshly prepared from an aqueous stock solution and made to the same juice osmotic pressure with sucrose. It was applied to excess, incubated for 10 minutes, viewed and imaged as before. To confirm that the method was a reliable indicator of cell membrane competence and cell vitality, fresh and microwaved preveraison berries were prepared and imaged according to the described method (Fig 9). The pre-veraison fruit for the set of control sections was prepared as above for the fresh sections or microwaved for 15 s at 650 watts before the dyes were applied. Exposure times were consistent for the set. The fresh, sectioned berries showed a vivid fluorescent green response to FDA indicating that the cells across the pericarp had competent membranes and living cytoplasm. Berries that had cell membranes disrupted by microwaving had no fluorescent response to FDA (Fig 9). The results of the PI application to the sections were a direct contrast. No red fluorescence was visible in the fresh sections (with the exception of a sliver of seed coat) however it was intensely visible in the pericarp cells of the microwaved, cell membrane disrupted samples. PI is known to stain cell walls, but this was not visible, nor was auto-fluorescence or artifacts at the cut surface of the pericarp using this method at low magnification. 38

50 Figure 9 Pre-veraison berries sectioned longitudinally and either left fresh or microwaved to disrupt cell membranes. A comparison is shown between fresh and microwaved berries with no dyes applied (controls), with FDA only, with PI only, and with both FDA and PI applied. Note that no autofluorescence is visible. Although we refer here to the pericarp (comprising exocarp, mesocarp and endocarp), the microscopy technique used in this study prevented cellular details in the exocarp region to be delineated. The vitality observations are weighted more to the large volume of the mesocarp and endocarp. However, vitality/death of cells of the exocarp can be seen as thin green outlines or red regions in the sections. Analysis of vitality Pixel analysis of Shiraz and Chardonnay sections stained only with FDA was performed using GLOBAL LAB % Image/2 version 2.5 (Data Translation). Comparative analysis of pixel blocks or line transects within images showed that pixels with arbitrary values designated as > 75 were vital and reflected living cytoplasm in cells (Fig 10). Two methods were compared: 1) The berry cuticle was outlined and pixels within the outline (ie the cut surface) analysed. Vital pixels were expressed as a percentage of the total number of pixels; 2) Line transects were taken across the distal, mid and mesial (DMM) of berry longitudinal sections and the percentage of total pixels with values > 75 were averaged between the three sections. A separate transect was taken through obviously vital regions of the pericarp where cell cytoplasm surrounding large 39

51 vacuoles could be delineated. The percentage of total pixels with values > 75 was recorded and the ratio of the averaged DMM over the pericarp transect value was converted to a percentage. This is referred to as relative cell vitality. The second method yielded qualitatively similar results to the first method, but was deemed more suitable because it could account for the expansion of living pericarp cells, which resulted in a reduced count of vital pixels due to dilution by the large central vacuoles. The second method also accounted for slight variations in exposure between preparations since it was self referencing within a berry. To quantify the structural variability in location of living and dead cells between high quality and mid-quality fruit, DMM transects were drawn and pixel values graphed for each transect. Figure 10Comparisons of blocks of pixels in images of berry sections stained with FDA indicate that pixels with a value of greater than 75 arbitrary units (with the highest frequencies between 150 and 200 arbitrary units) corresponded to vital, fluorescing tissue. The background of the image and non-vital tissue had pixels with values of less than 75 arbitrary units. Example shown is a postveraison Chardonnay berry. Bar = 5 mm. Xylem equilibrium pressures Xylem equilibrium pressures of berries attached to the pressure probe via the pedicel were measured as detailed in Tyerman et al. (2004). Briefly, whole shoots with bunches attached were cut from the field grown vines described above and were transported immediately to the laboratory with the cut end immersed in water. Once in the laboratory an individual berry and pedicel was cut 40

52 from the bunch while under water. To seal the pedicel with the pressure probe a piece of tubing mm long (Tefzel-Schlauch tubing ID 1.0 mm OD 1.6 mm or ID 1.6 mm OD 3.2 mm depending on pedicel diameter) was flared at one end in order to snugly fit the pedicel. The pedicel was sealed into the flared end with a cyanoacrylate glue (Super Glue TM or Loctite TM 401 or 406 adhesive with tubing treated with 770 primer). The water used to fill the tubing and probe was millipore purified water de-aerated by vacuum treatment and filtered to 0.2 µm. As the seal was tightened around the tubing the pressure was elevated in the system and was allowed to relax to a steady state level. Results Pericarp cell death is evident in berries of Chardonnay and Shiraz late in development, but not Thompson Seedless. At 73 days after anthesis (daa), Shiraz and Chardonnay berries moved from the lag phase of berry development (late part of phase 1) into the rapid phase of weight and sugar accumulation (phase 2). The staining of sections of both varieties with FDA indicated that cells across the pericarp maintained vitality through veraison and continued as the fruit approached maximum weight. In Shiraz, the berries were still enlarging at 98 daa when counterstaining with PI showed an intense red fluorescent response across the pericarp, which was repeated approaching maximum weight at 103 daa (Fig 11a). In Chardonnay the berries reached maximum weight at 108 daa and exhibited a PI response at 110 daa that was sustained at 117 daa (Fig 11b). Yellow fluorescence was visible where tissue showed a mixed response living and dead cells in proximity. While the period of sudden PI response did not occur at the same number of days after anthesis for the wine grape varieties, it was contemporaneous in terms of the calendar date. No weather, water or other stresses were noted over this period. We also observed similar death events in berries from glasshouse grown vines 41

53 grown during winter of 2006 and this also did not correspond to any particular climate change. More cells adjacent to the central and peripheral vasculature maintained cell membrane competence compared to the body of the mesocarp. a b c Figure 11 Changes in berry cell vitality with development. A developmental time series where Shiraz (a) Chardonnay (b) and Thompson Seedless (c) berries were longitudinally sectioned and stained with FDA then counterstained with PI to test vitality and membrane competence. Three separate berries from a bunch are shown at the time point indicated as days after anthesis (daa).this is representative of a larger dataset used for Fig 5. The total soluble solids measured at timepoints is shown; values with an asterix were calculated from measured osmolarity of juice using a standard curve between osmolarity and Brix (R 2 = 0.999). Veraison occurred at 73 daa for Shiraz and Chardonnay, and 78 daa for Thompson Seedless berries. 42

54 Cell vitality relative to sugar and water relations of the berry Relative cell vitality as a function of days after anthesis is plotted in Fig 12 a for each of the grapevine varieties. This data is shown in the context of juice osmotic potential for all the varieties (Fig 12 b), and the weight and sugar accumulation in the berries of Chardonnay and Shiraz (Fig 12 c & d). Figure 12 Changes in berry cell vitality is associated with other developmental changes in berry physiology. (a) Relative cell vitality as a function of days after anthesis for each of the varieties tested, n=6-7. The inset shows the linear decline in relative cell vitality in Shiraz and Chardonnay. The slopes are significantly different (p < 0.05). For Thompson Seedless a linear fit to the entire data set yielded a slope not significantly different from zero. (b) Juice osmotic potential of the juice of pooled and crushed opposing halves of sectioned berries for each of the varieties versus days after anthesis. Linear fits of osmotic potential with time for each variety were not significantly different (P > 0.05), so a fit to the combined data is shown with 95% confidence interval. (c) Mean berry weight for Chardonnay and Shiraz versus days after anthesis. A 2 nd order polynomial was fitted to the data from veraison (solid lines). The fitted curves were significantly different between varieties (P<0.05). (d) Mean solutes per berry versus days after anthesis for Chardonnay and Shiraz. The fitted curves (solid lines) were significantly different between varieties (P < 0.05). For Shiraz the best fit was obtained with a 2 nd order polynomial, while for Chardonnay the best fit was with a straight line. Shown in each set of data are the points at which the PI response was observed as an indicator of the first sign of cell death in the pericarp (Chardonnay, solid line; Shiraz, dashed line). 43

55 At or just after veraison all varieties showed 100% relative cell vitality. This was maintained throughout the entire measurement period for Thompson Seedless and until maximum berry weight for Chardonnay and Shiraz. The measurements above 100% for Thompson Seedless are ascribed to the higher density of cells adjacent to central and peripheral vascular bundles of the berry when compared to the mesocarp cells in the calculation of relative cell vitality. The range of days after anthesis over which the PI fluorescence was observed in Shiraz and Chardonnay, are indicated on Fig 12 as horizontal bars. These events corresponded with the onset of a linear decline in relative cell vitality measured with FDA in both varieties (inset in Fig 12 a). The linear regressions for this phase of declining vitality indicated that Shiraz had a significantly higher rate of decline compared to Chardonnay. The initial death events also corresponded to the period just before maximum berry weight in Shiraz and just after maximum berry weight in Chardonnay (Fig 12 c). The death event and onset of decline in cell vitality also corresponded to a change in slope of accumulation of solutes per berry in Shiraz and a plateau in Chardonnay (Fig 12 d). Xylem pressure measured using the pressure probe attached to the pedicel of individual berries is shown in Table 1. These measurements were taken in the period when cell vitality was declining. Despite the very negative osmotic potentials of the berry juice of Chardonnay and Shiraz (Fig 12 b) the xylem pressures of detached berries were rather small and positive (note that values given are in KPa). Contrasting behaviour was observed for Thompson Seedless berries that had higher osmotic potentials (Fig 12 b) but which developed negative xylem pressures that would cavitate the pressure probe. In this case the measurements given in Table 1 are extrapolations of the exponential 44

56 approach to equilibrium after the berry was attached to the probe (Tyerman et al. 2004). Table 1 Xylem equilibrium pressures of berries connected to the pressure probe. Shiraz and Chardonnay berries were measured after maximum berry weight and after the first sign of pericarp cell death. For Thompson Seedless berries, pressures were determined from exponential extrapolations from the pressure equilibrium time course before cavitation was evident. Data are means ± s.e (n). In each case the pressures were significantly different from zero (one sample t- test, P < 0.05) and were significantly different between varieties (one way ANOVA with post tests, P < 0.05) Variety Xylem equilibrium pressure Range; days after anthesis (KPa) Shiraz 4.7 +/- 1.7 (20) Chardonnay /- 2.2 (15) Thompson Seedless /- 4.4 (5) 136 Aberrant behaviour detected using the vitality assay Transects across the pericarps of premium Shiraz fruit from 30 year old vines in McLaren Flat, South Australia were compared to the mid-quality fruit from 12 year old Shiraz vines in the Coombe vineyard. The premium fruit showed a consistent and distinct pattern of vital and non-vital cells across the pericarp which can be graphically represented (Fig 13 a). This structural pattern was consistent in the sections examined. At harvest maturity the cells in the premium fruit around the peripheral and central vasculature clearly maintain competent membranes while the mesocarp at maximum distance from the vascular tissue shows no response to FDA. This suggests that there are distinct regions within the pericarp where there are no vital cells at harvest. 45

57 Figure 13The pattern of cell death differs between grades of Shiraz fruit. Premium (a) and midquality (b) berries stained with FDA on the day of the harvest, 30 and 29.9 Brix respectively. Proximal, central and distal transects were drawn and pixels analysed. The premium fruit shows a distinctive structure with vital cells being maintained adjacent to central and peripheral vascular bundles with semi-defined regions of non-vital cells in the body of the pericarp (n=7). The midquality fruit showed varied and less organized patterns of cell death across the pericarp, n=6. While the different quality fruit showed structural differences, analysis of % vital pixels (Fig 5) found no significant difference between the two at harvest. Bar = 5 mm. The mid-quality Shiraz showed more structural variation in the distribution of cells with and without a functioning cytoplasm compared to the premium Shiraz. The apparent loss of membrane competence in cells, or cell death, was spread across the pericarp in a less organised manner (Fig 13 b), although some berry sections did show similarities to the premium fruit pattern of cell death. Field grown premium and the mid-quality Shiraz were harvested for winemaking on the same day at 29.6 and 30 Brix respectively. While the apparent vitality structure of the two sets of berry sections are quite different on the day of harvest (Fig 13) there was no significant difference in the relative cell vitality measurement (unpaired t-test, n=6, p = 0.49, Fig 12 a). The fruit from both locations appeared similarly shrivelled. 46

58 The FDA method was applied to Thompson Seedless berries (21 Brix, unknown daa) that had been treated with gibberellic acid, and had signs of early stages of berry collapse (Fig 14 a) Figure 14 Longitudinal sections of Thompson Seedless berries with (a) and without (b & c) berry collapse, FDA applied. (a) Gibberellic acid treated berry with visible collapse (23 Brix, days after anthesis (daa) unknown) shows a dark region at the distal end of the berry reflecting cell death associated with collapse, and disruption of central and peripheral vasculature. Pericarp cells appear elongated, semi-rectangular and loosely stacked through the distal two thirds perhaps indicating the spread of berry collapse. (b) Healthy berry treated with gibberellic acid (20.2 Brix, daa unknown) shows more compact, functioning cells across the pericarp and intact vasculature. (c) Healthy berry not treated with gibberellic acid (24.3 Brix 124 daa) also has compact, functioning cells across the entire pericarp and intact vasculature. Remnants of the locules, one with a vestigal seed, are visible. (Fruit treated with gibberellic acid courtesy of Mike Treeby and Tori Nguyen, CSIRO, Merbein) Bar = 5 mm. Regions of cells with no living cytoplasm were observed in the distal half of the pericarp, with adjacent distal regions appearing to show smaller areas of apparent cell death. Distinct differences were noted when this fruit was compared to fruit that was treated with gibberellic acid but had no signs of berry collapse (20 Brix, Fig 14 b), and untreated fruit (24.3 Brix, Fig 14 c). The berries showing signs of berry collapse had elongated, semi-rectangular cells that appeared to be very loosely stacked in rows across the pericarp between the central and peripheral vascular bundles, and cell death had already occurred in some regions. The central and peripheral vascular bundles appear disrupted 47

59 around the regions of cell death. The gibberellic acid treated fruit that was sound, and untreated berries (Fig 14 b and 14 c) had dense, compact, irregularly shaped cells across the pericarp, with some compact elongated cells in the mesocarp between the central and peripheral vasculature. The gibberellic acid treated fruit was much larger than untreated fruit, as would be expected. Discussion Veraison is the beginning of the ripening phase (phase 2 in Fig 8) of grape berry development in Vitis vinifera. It is indicated by softening of the berry, a rapid accumulation of hexose sugars, and in some varieties, colour development. The fleshy, carbohydrate rich fruit is thought to have selectively evolved as a high value food reward for animals, birds in particular, that consume it and disperse the seeds widely without physical damage (Hardie and O'Brien 1988). At veraison, seeds have reached full size (Hardie et al. 1996) and can germinate with cold treatment (reviewed in Pratt 1971). Unpalatable phenolic compounds in berries reduce from veraison (Adams 2006) as sugars are accumulating. A second phase of softening and further sugar concentration is evident in some wine grape varieties (phase 3 in Fig 8). The selection by human kind of cultivated varieties of grapevines for wine making or table grapes probably enhanced or reduced some of these characters over time, depending on the desired end use of the fruit. It may not be so surprising that contrasting behaviour is exhibited between the wine grape and table grape varieties observed in this work. We show here the first direct evidence of loss of membrane competence in the mesocarp in two wine grape varieties that corresponds to the phase 3 stage of development. This occurs at or near maximum berry weight and clearly defines the third phase of berry development independently of whether berry weight loss occurs or not. This third phase of development defined by loss of cell vitality was not evident in the berries of the table grape variety Thompson Seedless. 48

60 From the fluorescent dye test series it is clear that autofluorescence, damaged cells on the cut surface and other artifacts do not interfere at this macro level of microscopy. In the second phase of development in Chardonnay, Shiraz and Thompson Seedless berries, the dye studies initially showed vital cells across the pericarp and no visible response to PI. On the same day (14 February, 2006), the wine grape sections showed a dramatic, vivid red response to PI, while the Thompson Seedless sections had a slight blush of red fluorescence. The response was the same a week later in the wine varieties, but was not repeated in the table grape. No weather, water or other stresses were noted over this period. We believe that the contemporaneous calendar timing of the PI response was not an artefact of any treatment we imposed on the sampling regime for the following reasons: (i) The strong PI response in the wine grapes corresponded to the beginning in the decline of cell vitality using the FDA method that was observed over the following weeks. It also corresponded to other developmental changes including maximum berry weight and slowing of sugar accumulation. (ii) The same degree of cell death measured with FDA was observed in fruit sourced from a different region and vineyard, and measured at the same time after anthesis. Why the PI response was not sustained in the wine varieties when the FDA studies show increasing cell death is not clear. Perhaps the nucleic acids denature to a degree that a structural change interferes with the PI intercalating into a conformation that is fluorescent. Compounds in the cytosol of other plants have been found to reduce PI accessibility of DNA when cells are lysed (Price et al. 2000; Noirot et al. 2003; Loureiro et al. 2006). If intercalation with RNA was responsible for the fluorescence seen, RNA is rapidly degraded during senescence (Jones 2004) and there may be insufficient quantities to fluoresce after the initial response. This needs follow up work, but it is likely that the strong 49

61 PI response observed in the two wine grapes is part of the cell death process in the fruit because it mirrors the increasing, subsequent pattern of cell death across the pericarp indicated by the FDA data. While PI can stain cell walls it was not evident in the control experiments at the magnification used. Total DNA in the pericarp of Shiraz berries has been found to peak at 35 daa, then be maintained until 100 daa on a per berry basis (Ojeda et al. 1999). The data set stops several days after maximum berry weight was achieved at 95 daa, which is probably the period where loss of membrane competence has started to occur so it cannot be related to data in this paper. Anecdotal evidence of increasing difficulty in extracting DNA and RNA as harvest maturity of berries approaches may be related to cell death and subsequent catabolism of nucleic acids. This is certainly an area of research that warrants further investigation. Cells surrounding the central and peripheral vasculature of berry sections maintained vitality compared to the rest of the pericarp, although this reduces as harvest approaches. This pattern is highly evident in the transects of the premium Shiraz berries (Fig 13). While the relative vitality was not significantly different at harvest, a difference was found in the distribution or pattern of vital and non-vital cells across the sectioned pericarp of premium Shiraz fruit from 30 year old vines in McLaren Flat when compared to the mid-quality fruit from 12 year old Shiraz vines in the Coombe vineyard. The premium fruit showed a consistent and distinct pattern of vital and non-vital cells across the pericarp. At harvest maturity the cells in the premium fruit around the peripheral and central vasculature clearly maintain competent membranes (and living cells) while the mesocarp at maximum distance from the vascular tissue shows no response to FDA. This suggests that there are distinct regions within the pericarp where there are no vital cells at harvest. The mid-quality fruit showed variation in pattern and less organised cell death across the pericarp. 50

62 Visible disintegration of cell membranes is described as terminal or late events in the cell death process in plants (Noodén 2004). In later stages of berry development the mesocarp cells of Traminer have lost internal membranes and cell contents including lipids, starches and polyphenols were mixed within (Hardie et al. 1996). Autolysis of vacuoles are likely to be indicative of cell death (Thomas et al. 2003) and no functioning cytosol clearly means a cell is dead. Both mitochondria and plasma membranes appeared intact in mesocarp cells late in the ripening of fruit of the table grape Vitis vinifera x Vitis labrusca cv. Kyoho (Zhang et al. 1997). Mitochondrial integrity is often maintained until late in the senescence process (Noodén 2004). The continuing vitality we observed in Thompson Seedless indicates that cell death is variety dependent and therefore vitality may also be maintained in the Kyoho grape. In contrast to the two wine grape varieties, membrane competence and vitality of pericarp cells in Thompson Seedless berries was maintained throughout development and into the post harvest period. This may explain why Thompson Seedless grapes maintain turgidity, crispness and consumer appeal post harvest. The continuing cell vitality data corresponds with pedicel xylem pressure measurements showing that this variety generates negative pressures in the pedicel xylem until harvest. This indicates a continuing, functioning xylem connection to the apoplast, and cells with osmotically competent membranes that sustain a pressure gradient across them as the berry matures. This observation is consistent with anecdotal evidence of growers severing fruiting canes of Thompson Seedless from the vine to prevent water uptake and berry splitting if there is a rain event as harvest approaches. The data from Thompson Seedless berries in early stages of collapse warrants further investigation using the methods described here, which provide a quick visual way of identifying cell death and patterns of aberrant cell death. 51

63 Recent work shows a change of phloem unloading from a symplastic to an apoplastic pathway at or just prior to veraison (Zhang et al. 2006). Both Zhang et al. (2006) and Lang and Düring (1991) presented direct evidence that the apoplastic solute concentration rises from 65 and 60 daa respectively in a table grape and a wine grape variety. Lang and Düring (1991) found that there was no significant difference in the osmotic potential of berry pedicel xylem exudate and berry juice between 60 and 110 daa. These data clearly separate the increase in apoplastic solute concentration in berries from the later loss of cell membrane competence that is shown in this paper. This means that the leaky membrane theory described by Lang and Düring (1991) as an explanation of the rise in apoplast solute concentration at veraison is not supported, but it is apt later in berry development in Chardonnay and Shiraz. The loss of cell vitality in Chardonnay and Shiraz compared to its maintenance in Thompson Seedless suggests that their senescence processes are significantly different, so care needs to be taken when applying research results from one variety to another. The image analysis showed that Shiraz and Chardonnay had a significantly different time course of loss of cell vitality in the pericarp relative to days after anthesis. Regions in the pericarp of Shiraz entered the senescence or cell death phase earlier in berry development compared to Chardonnay, and this difference increases significantly because relative vitality declines more rapidly in Shiraz berries. This difference is exacerbated by later harvesting for winemaking (127 daa) compared to the Chardonnay fruit that was harvested at 117 daa. Our data suggests an hypothesis for the mechanism of weight loss observed in Shiraz berries that occurs generally after daa (McCarthy 1999; Tyerman et al. 2004). Since the membranes of the pericarp cells begin to lose semipermeability at this point, as judged by loss of vitality, the large negative osmotic potential of the berry sap is no longer effective in opposing the xylem 52

64 tensions developed by the leaves. We have shown previously that Shiraz berries late in development maintain higher hydraulic conductance back to the vine compared to Chardonnay berries (Tyerman et al. 2004), therefore significant volume may leave the berry back to the vine in Shiraz. Reduced phloem inflow and continued transpiration would all combine to cause a net loss of water from Shiraz berries. Chardonnay berries also maintain some finite conductance back to the vine (Tyerman et al. 2004), but the quantitative conductance is much less than in Shiraz and drops to very low levels by 90 daa, that is, at about the same time that cell death begins to be evident in Chardonnay. Recent work of Bondada et al. (2005) and Keller et al. (2006) demonstrated qualitatively that backflow can occur, however our previous work demonstrated large varietal differences in a quantitative measure of the pathway for this flow, the hydraulic conductance. More generally we propose that there is a balance in development between the programmed cell death of the pericarp cells and the decline in hydraulic conductance from the berry to the vine. If the hydraulic conductance to the vine is reduced sufficiently when the pericarp cell membranes lose semipermeability (cells non vital), backflow would be reduced. This would be the case for Chardonnay. For Shiraz, there is an apparent miss-timing where the pericarp cells lose vitality and osmotic competence earlier in development, but the hydraulic conductance remains high back to the vine later in development. For Thompson Seedless where cell vitality is maintained through development, the xylem tension can be resisted by the osmotic potential of the pericarp cell sap. In this case the hydraulic conductance to the vine could be kept high. This is supported by our measurements of xylem pressure which remains negative in Thompson Seedless. The question remains how the hydraulic conductance from berry to vine via the xylem is regulated. Tyerman et al. (2004) showed that the largest change in 53

65 hydraulic conductance through development occurred within the distal part of the berry. The dye studies have shown that the xylem remain qualitatively connected (Bondada et al. 2005; Keller et al. 2006) late in development, but it is possible that the hydraulic conductance of vessels and tracheids is still reduced. Another possibility is that the cells surrounding the xylem vessels function as an effective membrane barrier from the apoplast of the berry to the xylem lumens. Variable hydraulic conductivity of these cells could be via regulated activity of aquaporins. Our work has demonstrated that despite extensive cell death in the mesocarp and endocarp, the vascular tissue remains conspicuously vital, indicating that regulation at the membrane level in these cells is possible. The processes of ripening and senescence are often inter-related (Jones 2004). During ripening, a variety of physical and biochemical mechanisms change the structure of cell walls in fleshy fruits with textural results ranging from crisp to melting or soft and deformable (Brummell et al. 2004; Brummell 2006). The deterioration of plant membranes during senescence is well documented and a number of enzymes are involved (Paliyath and Droillard 1992). Research has focussed on the post harvest period, but interestingly, enzymes of the lipoxygenase family have been associated with membrane lipid peroxidation in ripening saskatoons (Rogiers et al. 1998) and volatile aroma or flavour development in tomatoes (Chen et al. 2004) and kiwifruit (Zhang et al. 2006). It has been noted that aroma compounds in wine grapes increase late in ripening when sugar accumulation has slowed or stopped and non- anthocyanin glycosides rise sharply at around 100 daa in Shiraz (Coombe and McCarthy 1997). In this context another important issue raised by our results is whether flavour development in Shiraz grapes can be attributed to the significant loss of cell integrity within the body of the pericarp and the ensuing degradation processes. 54

66 According to this data it is more than loss of compartmentation in the mesocarp; it is extensive lysing and mixing of cell contents. The trend for hang time while the winemakers wait for ripe flavours in this variety would also contribute to the loss of cell vitality by harvest. The highly organised pattern of cell death in the premium Shiraz fruit suggests that this idea should be examined. In conclusion, the variety dependent vitality changes in the pericarp of grapevine berries late in maturity seems to be linked to the strategy of berry water balance in the particular variety. We hypothesise that there are two strategies: 1) Cell death in the mesocarp and loss of osmotically competent membranes requires concomitant reduction in the hydraulic conductance of the pathway via the xylem back to the vine; 2) Continued cell vitality and osmotically competent membranes that can allow continued hydraulic conductance to the vine. A miss-match in the timing of strategy 1 could cause substantial backflow to the vine and loss of berry weight. For strategy 2 there is a greater danger of berry splitting upon the vine attaining high water potentials. Our data also strongly suggests that closer examination of the cell death processes in wine grapes would contribute significantly to understanding the final phases of flavour development. 55

67 56

68 Chapter 4 Hydraulic connection of grape berries to the vine: varietal differences in water conductance into and out of berries, and potential for backflow. This chapter has been published: Tilbrook J, Tyerman SD (2009) Hydraulic connection of grape berries to the vine: varietal differences in water conductance into and out of berries, and potential for backflow. Functional Plant Biology 36,

69 58

70 Abstract Weight loss in Vitis vinifera cv Shiraz berries occurs in the later stages of ripening from days after anthesis. This rarely occurs in varieties such as Chardonnay and Thompson Seedless. Flow rates of water under a constant pressure into berries on detached bunches of these varieties are similar until days after anthesis. Shiraz berries then maintain constant flow rates until harvest maturity while Chardonnay inflow tapers to almost zero. Thompson Seedless maintains high xylem inflows. Hydraulic conductance for flow in and out of individual Shiraz and Chardonnay berries was measured using a root pressure probe. From 105 days after anthesis, during berry weight loss in Shiraz, significant varietal differences in xylem hydraulic conductance were found. Both varieties showed flow rectification such that conductance for inflow was higher than conductance for outflow. For flow in to the berry, Chardonnay had 14% of the conductance of Shiraz. For flow out of the berry Chardonnay was 4% of the conductance of Shiraz. From conductance measurements for outflow from the berry and stem water potential measurements, it was calculated that Shiraz could lose about 7% of berry volume per day, consistent with rates of berry weight loss. A functional pathway for backflow from the berries to the vine via the xylem was visualised with Lucifer Yellow CH loaded at the cut stylar end of berries on potted vines. Transport of the dye out of the berry xylem ceased prior to 97 days after anthesis in Chardonnay, but was still transported into the torus and pedicel xylem of Shiraz at 118 days after anthesis. Xylem backflow could be responsible for a portion of the post-veraison weight loss in Shiraz berries. These data provide evidence of varietal differences in hydraulic connection of berries to the vine that we relate to cell vitality in the mesocarp. The key determinates of berry water relations appear to be maintenance or otherwise of semi permeable membranes 59

71 in the mesocarp cells and control of flow to the xylem to give variable hydraulic connection back to the vine. Abbreviations: LYCH; Lucifer Yellow CH dipotassium salt, daa; days after anthesis; L o, hydraulic conductance. Keywords: Hydraulic conductance, berry xylem, backflow, Vitis vinifera, berry shrivel, berry ripening, pressure probe, flow meter. 60

72 Introduction Grape berry development can be considered to have two or three phases (Sadras and McCarthy 2007; Tilbrook and Tyerman 2008). During the first phase of berry formation, water enters via the xylem, solutes and water enter via the phloem and transpiration occurs across the berry cuticle (Dreier et al. 2000; Greenspan et al. 1994; Lang and Thorpe 1989; Rogiers et al. 2004). Veraison marks the beginning of the second phase as berries soften and xylem water transport reduces, partly because of reduced transpiration coincidental with a decline in berry hydraulic conductance (Greenspan et al. 1994; Tilbrook and Tyerman 2006; Tilbrook and Tyerman 2008; Tyerman et al. 2004). Inflow of solutes and water into the berries is then predominantly via the phloem (Greenspan et al. 1994; Rogiers et al. 2006). A third phase has been described where berries of some varieties begin to lose weight before winemaking flavour and maturity is reached (Sadras and McCarthy 2007). In Shiraz for example, phase 3 involves a loss of up to 30% of the maximum weight (McCarthy 1999; Rogiers et al. 2006; Tyerman et al. 2004). This third phase may also be defined by cell death in the mesocarp of varieties that do not show weight loss, for example, Chardonnay (Tilbrook and Tyerman 2008). There appears to be plasticity in sugar accumulation between seasons in this third phase as sugar concentration rises in berries from a combination of further sugar import and weight (water) loss in berries of some varieties (Sadras and McCarthy 2007). The onset of weight loss in Shiraz correlates with the beginning of loss of vitality in mesocarp cells (Tilbrook and Tyerman 2008). Loss of vitality also occurs in Chardonnay at about the same number of days after anthesis (daa) as for Shiraz, but Chardonnay normally does not show weight loss. Therefore the onset of decreasing cell vitality may be a more robust indicator of the onset of the third phase of development (Tilbrook and Tyerman 2008). 61

73 Weight loss in berries will be the net result of reduced water inflow via the phloem, the net flow through the xylem (in or out of the berry) and loss by transpiration. Flow out of the berry via the xylem is referred to as backflow. Transpiration across the berry cuticle post-veraison reduces to 16 % of the preveraison value, therefore it has been hypothesized that Shiraz berry weight loss is the result of reduced phloem inflow (Rogiers et al. 2004). Dye studies initially suggested that water flow into berries via the xylem ceased at veraison as it was observed that peripheral xylem tracheids in the berry stretched and broke (Creasy et al. 1993; Findlay et al. 1987), and xylem tracers accumulated in the brush tissue when loaded via the pedicel xylem (Findlay et al. 1987; Greenspan et al. 1996; Rogiers et al. 2001). However, recent work has provided evidence of continuing xylem function (Bondada et al. 2005; Chatelet et al. 2008b; Keller et al. 2006). By applying a hydrostatic gradient to a number of varieties of postveraison grape berries, the axial and peripheral xylem was found to be intact and able to conduct water (Bondada et al. 2005). This was confirmed by transport of xylem mobile dye from the stylar end of post-veraison Merlot berries and Vitis labrusca cv Concord berries back into the bunch rachis and vine shoot (Keller et al. 2006). In post-veraison Chardonnay berries, later developing or younger tracheary elements in the peripheral xylem bundles were found to maintain integrity while older elements stretched and broke (Chatelet et al. 2008a; Chatelet et al. 2008b). Collectively, the evidence suggests that berry xylem maintains capacity to transport water in the post-veraison period though quantitative measurements of water flow into Shiraz and Chardonnay berries demonstrated a decline in hydraulic conductance of berries during and after veraison (Tyerman et al. 2004). It has been shown that phloem unloading in the berry changes from symplastic to apoplastic at or just before veraison (Zhang et al. 2006). It is postulated that this 62

74 maintains a pressure difference in the phloem between the leaf source and the berry sink tissue to maximize the accumulation of sugars during ripening (Patrick 1997; van Bel 2003; Zhang et al. 2006). It has been noted that sinks that accumulate very high concentrations of sugars have apoplastic phloem unloading, and may or may not have an apoplastic barrier at the phloem/storage cell interface (Patrick 1997). Also, high solute concentration in the apoplast would increase the volume of water leaving the phloem (if not impeded), and contribute to the fast accumulation of water and sugar in the second rapid growth phase of berries (Lang and Thorpe 1986). High osmolarities in the apoplast would mean a loss of turgor in companion cells (van Bel 2003) and mesocarp cells (Wada et al. 2008), and thus the sudden softening of berries as veraison begins. Fruit softening may not be the result of a single event and can be related to changes in cell wall structure in peach (Brummell et al. 2004) and grapes (Nunan et al. 1998), and cell turgor in tomatoes (Shackel et al. 1991) and grapes (Thomas et al. 2006; Wada et al. 2008). The transition from symplastic to apoplastic unloading may also explain why the pressure measured in pedicel xylem, as a surrogate for berry apoplast pressure, changes over veraison from negative to slightly positive in Chardonnay and Shiraz (Tyerman et al. 2004). This however does not seem to occur in Thompson Seedless (Tilbrook and Tyerman 2008), which has other characteristics discussed below, that distinguish it from the wine grape varieties. Xylem backflow has been suggested as a way of returning excess phloem derived water to the vine (Rogiers et al. 2004; Tyerman et al. 2004; Keller et al. 2006). It is a possible cause of loss of berry weight in Shiraz berries prior to harvest if phloem inflow slows or ceases (Rogiers et al. 2004; Tyerman et al. 2004). Keller et al. (2006) demonstrated that berry xylem could allow backflow, but they considered that this would be unlikely to cause significant weight loss 63

75 based on the assumption that berry cell membranes maintain semi-permeability until harvest. If membranes remain semi-permeable, the large negative osmotic potential of the cells could balance the tension generated in the vine xylem by leaf transpiration. However, recent work found that while the cells surrounding the central and peripheral vascular bundles maintained intact membranes, 20-40% of the cells in the mesocarp of post-veraison Shiraz and Chardonnay berries lost membrane competence (defined as cell death) in the final stages of berry development post daa (Tilbrook and Tyerman 2008). A similar observation was made for Chardonnay, Cabernet Sauvignon and Nebbiolo (Krasnow et al. 2008). Thompson Seedless contrasts with these varieties in maintaining cell vitality well beyond full sugar ripeness (Tilbrook and Tyerman, 2008). Shiraz, Chardonnay and Thompson Seedless present an interesting comparison for the purposes of investigating the cause of weight loss in berries. Shiraz and Chardonnay show a similar degree of cell death in the mesocarp, yet Chardonnay berries do not normally lose weight. Thompson Seedless maintains cell vitality and does not loose weight. We hypothesise that these differences between varieties is reflected by differences in the magnitudes of xylem hydraulic conductance. When cell death occurs the xylem conductance should become restricted to prevent backflow. We have previously shown that Chardonnay has a lower xylem hydraulic conductance than Shiraz at the time when berry weight loss begins in Shiraz, however these measurements were only made for flow into the berry, when the relevant direction for backflow is in the opposite direction. In Thomson Seedless berries backflow may be prevented by the maintenance of osmotically competent membranes, negating the requirement for reduced hydraulic conductance of the xylem. Therefore we have compared the berry xylem hydraulic conductance of these varieties in greater detail and have 64

76 attempted to measure water flow through the pedicel to the berry in both directions. We also re-examined the changes in flow into whole detached bunches through development using a more sensitive flowmeter. Taking into account the quantitative differences in hydraulic conductance between Shiraz and Chardonnay, we used a dye loading technique on berries in intact bunches to compare the potential for backflow between the two varieties. Materials and methods Fruit material Experimental fruit for the single berry and whole bunch hydraulic data and the deformability data was from Coombe vineyard (Vitis vinifera Shiraz BVRC12 and Chardonnay I10V1 on their own roots, 11 years old) and Alverstoke vineyard (Vitis vinifera Thompson Seedless M12 on own roots, not treated with gibberellic acid, 3 years old) at the Waite Campus of the University of Adelaide, South Australia, during the season. For the dye studies where backflow was visualised, fruiting Shiraz BVRC12 and Chardonnay I10V1 vines were pot grown in a glasshouse in UC potting mix at the Plant Research Centre, Waite Campus of the University of Adelaide in All bunches of fruit used in experiments were labelled individually when an estimated 50% of the flower caps from that bunch had fallen off. The day this occurred was designated as anthesis and data is presented in terms of days after anthesis (daa). Veraison was defined as the commencement of berry softening. Deformability Berry deformability was measured using Harpenden skin fold callipers (Coombe and Bishop 1980). The berry diameter was measured, then remeasured with the calliper springs applying a force of N. At each time point measurements were made of five berries from proximal, mid and base of five bunches (n=25 65

77 berries) for Shiraz and Chardonnay. Shiraz measurements were on a random bunch on one random vine in each of five replicate panels in separate rows that formed part of a randomised block trial. Chardonnay measurements were on random bunches from five separate vines in a row of twenty-four adjacent vines. For Thompson Seedless, measurements were taken as above on four random bunches on four separate vines in a row of ten vines (n=20 berries). Deformability data is presented as the percentage reduction in berry diameter when the force is applied across the maximum diameter of the berries (Fig 15). Berry weight and total soluble solids Berry weight and total soluble solids ( Brix) data reflect whole vineyard berry development. Weights are the means of 50 berry samples. For Shiraz, samples collected were of five berries from proximal, mid and base of two random bunches on separate vines from each of five replicate panels in separate rows that formed part of a randomised block trial. For Chardonnay, five berry samples from proximal, mid and base of ten random bunches on separate vines in a row of twenty-four adjacent vines were collected. Similar data was not collected for Thompson Seedless berries as insufficient experimental fruit was available on the young, heavily pruned vines. Berries collected for weight samples were crushed, juice collected and briefly centrifuged to settle any solids. Brix of the juice was measured using a temperature compensated digital refractometer (ATAGO Model PR101). In the backflow experiments, juice of the berries in the experimental bunch was prepared and Brix measured as described. Flow into the peduncle of whole bunches Flow rates into the peduncle of whole bunches of fruit was measured using a XYL EM& flowmeter (Instrutec).The XYL EM apparatus was designed to measure xylem embolism by measuring hydraulic conductance of the xylem in stem segments of plants before and after flushing (Cochard et al. 2000). It 66

78 measures water flow rates using a high precision flowmeter. Testing showed that this equipment was suitable to measure water flow into bunches via the peduncle xylem using a 0-5 g/hour flowmeter. For whole bunch measurements of Shiraz and Chardonnay, a fruiting cane with two bunches was chosen randomly (refer to Fruit material above), cut adjacent to the main stem and the cut end placed immediately into deionised water. For Thompson Seedless, a fruiting cane with a single bunch of fruit was similarly prepared. In the laboratory the proximal bunch (or only bunch) was cut from the cane with a sharp razor blade while the peduncle was immersed in 10 mm potassium chloride solution made with Millipore filtered (0.2 µm) water de-aerated by vacuum. The system was filled with this solution in accordance with XYL EM operating instructions. A bead of solution was maintained on the cut surface and the peduncle of the bunch was immediately sealed into the XYL EM tubing ensuring that no air bubbles were present in the system and it was pressure tight. A protocol was developed where there was an initial two minute flush of solution into the peduncle at 150 kpa to eliminate potential embolisms in the xylem of the bunch (analogous with the pressures imposed on berry pedicels when initially sealed to the pressure probe) then measurements were made with the system in a steady state ie at a constant pressure of 100 kpa. Calculations included compensation for temperature variation as specified in the XYL EM manual. Numbers of berries on each bunch were counted and whole bunch data calculated on a per berry basis. For Shiraz and Chardonnay, n=4-6 bunches and for Thompson Seedless, n=3-4 bunches at each time point. Flow conductance for different directions of flow into berries via the pedicel Measurements of hydraulic conductance (L o ) for different flow directions (L o IN or L o OUT) through pedicels of single berries were made using a root pressure 67

79 probe (Steudle et al. 1993) modified to attach a single berry (Tyerman et al. 2004). A whole bunch was cut from a vine and placed in a closed plastic bag in a polystyrene cool box on ice and taken to the laboratory (about 500 m). Immediately before use, a single berry with the pedicel attached was cut from the bunch through the proximal end of the pedicel using a sharp razor blade while the pedicel was under Millipore filtered water additionally filtered (0.2 µm) and deaerated by vacuum. A bead of water was maintained on the cut surface. Cyanacrylate glue (Loctite 401 or 406) was applied to the pedicel (avoiding the cut surface), and it was inserted into tubing that had been flared to fit (Tefzel- Schlauch tubing, ID 1.0 mm and OD 1.6 mm etched with Loctite 770 primer). The tubing was immediately backfilled with water prepared as above. The tubing with attached berry was sealed to the pressure probe ensuring that the system contained no air bubbles and was pressure tight. Flow out of a berry was defined as flow that was measured at pressures negative to the pressure that the berry equilibrated to. Flow in was defined as flow measured into the berry at pressures positive to the equilibration pressure of the berry. L o IN and L o OUT were measured on the same berries. The mean post 105 daa equilibration pressures of berries were slightly positive in Shiraz (0.006 MPa +/ s.e.m) and Chardonnay (0.009 MPa +/ s.e.m.), consistent with Tyerman et al. (2004). Pressure clamps negative and positive to the equilibration pressures were imposed step wise on the berry system down to MPa or up to 0.04 MPa respectively, and hydraulic conductance calculated from the flow rates (Tyerman et al. 2004). For pressure probe measurements of L o, berries were measured. Stem water potential To measure stem water potential (#), leaves were sealed in a foil covered plastic zip lock bag for at least an hour prior to excision at the petiole with a razor blade. 68

80 Measurements were made between and 1.30 pm, central standard time, using a pressure chamber (PMS Instrument Company). Measurements were made on the same vines that fruit was taken for hydraulic conductance or backflow visualisation (refer to Fruit material in this section). Dye loading of berries to examine xylem flow to the vine A dye-loading study was conducted to determine potential backflow from the apoplast of berries back into a transpiring vine. The experimental design was modified from a method described in Keller et al. (2006) and used Lucifer Yellow CH dipotassium salt (LYCH, Sigma Aldrich) as an apoplastic tracer (Oparka and Read 1994). A 1% aqueous solution of LYCH was prepared with Millipore water and filtered (0.2 µm). Two berries at the distal end of a bunch were identified and with the stylar end of each berry immersed in the LYCH solution, a 1 mm thick slice was cut from the tip with a sharp razor blade. The cut surface of the berry was immersed in 1% aqueous LYCH solution in an unlidded microfuge tube, and the tube of dye sealed around the berry with Parafilm. Dye uptake commenced am and ceased pm when the bunch was cut from the vine. To relate the plant water status of each vine to the uptake of LYCH, the stem water potential (#) was measured immediately prior to removal of the bunch. A separate vine and bunch was used for each experiment on a particular day after anthesis. Transverse hand sections were cut of berry tori, pedicels, rachis, peduncle and stem with a sharp razor blade. After mounting in 70% (v/v) glycerol the sections were examined for the presence or absence of LYCH fluorescence in the xylem vessels under UV light using a Zeiss Axiophot microscope with a filter cube inserted: excitation filter , beam splitter FT 460 and barrier filter LP 470. Digital images were obtained using a Nikon DXM 1200F digital camera. 69

81 Results Berry ripening dynamics Grape berry developmental parameters of berry weight, deformability (Coombe and Bishop 1980), total soluble solids and ph, provided reference data to relate the xylem hydraulic measurements to the stage of berry development (Fig 15). Veraison, defined as softening of berries, occurred at 65 daa for Shiraz and 70 daa for Chardonnay across the vineyard and is indicated with vertical lines on the graphs. 70

82 Figure 15Changes during development of berry weight (a), berry deformability (b), and total soluble solids and ph (c) of grape berry juice. Veraison occurred at 65 daa for Shiraz (dotted vertical line) and 70 daa for Chardonnay (dashed vertical line). Analysis of changes in slope of fitted curves to berry weight and deformability for Shiraz showed that maximum weight and the sudden increase in deformability occurred at the same time (100 daa, vertical dotted-dashed line). (a) Weight of berries during development (n=50 at each time point). (b) Percent deformability was measured on individual berries during development (n=25). (c) Measurements of TSS and ph of berry juice during fruit development (n=50 at each time point). 71

83 Berry weight showed the typical two phases of growth in Chardonnay while Shiraz displayed a third phase of berry weight loss. Differentiation of a smooth curve fitted to the Shiraz berry weight data shown in Fig 15 a indicated that maximum weight occurred at 100 daa (indicated as a vertical line in each panel). Shiraz and Chardonnay berries in this case ended with a similar total soluble solids concentration and ph despite the large weight loss in Shiraz. Pre-veraison Shiraz and Chardonnay berries showed 1-2% deformability (Fig 15 b). From the commencement of veraison, the deformability of berries increased concurrently with the rapid accumulation of solutes in the fruit (Fig 15 c). The sudden increase in deformability at the commencement of veraison reflected the rapid softening of the berries. Both wine grape varieties reached % deformability for the daa period. The sudden increase in deformability after 101 daa (Fig 15 b) was contemporaneous with the rapid berry weight loss in Shiraz berries (Fig 15 a). Deformability of the three grape varieties post 105 daa was significantly different (ANOVA, p<0.0001, n= for Shiraz and Chardonnay, n=20 for Thompson Seedless. Tukey s post-tests indicated a significant difference in all paired tests, p<0.0001) (Fig 3). Thompson Seedless berries maintained turgidity and showed a low deformability post 105 daa of 3.1 % +/- 0.3 s.e.m. (Fig 17). Whole bunch flow The flow rate into bunches at a constant pressure (100 kpa) was proportional to the number of berries on the bunch (Fig 16 a). This allowed a calculation of flow (@ 100 KPa) on a per berry basis (flow/berry 100 kpa ) during development (Fig 16 b). Maximum flow/berry 100 kpa was observed in pre-veraison berries of all varieties. Post-veraison berries showed declining flow/berry 100 kpa reflecting the previously documented decline in hydraulic conductance (Tyerman et al. 2004). Thompson Seedless berries had a 40% reduction in flow/berry 100 kpa comparing pre-veraison 72

84 data at 61 daa to post-veraison at 143 daa (4.05 x /- 5 x10-13 to 2.4 x /- 3.5 x10-13 m 3 s -1 ). In Shiraz flow/berry 100 kpa was maintained from 80 daa, contrasting with a continuous decline in Chardonnay. Figure 16 Measurements of flow through whole bunches and vine water relations during development. (a) The relationship between flow and number of berries on a bunch was established using the flowmeter. Example is measurements (n=24) made on Shiraz bunches (n=6) 74 daa (refer to Methods). (b) At a fixed pressure the flow rates into Chardonnay berries declined from 93 daa for the remainder of development, while the Shiraz flow rate per berry stayed relatively similar as weight loss commenced, (n=4-6 bunches). (c) Stem water potential measurements of field grown Shiraz and Chardonnay vines show a generally negative trend with fluctuations related to rain events and irrigation as the fruit develops to harvest maturity, (n=4). To examine if water stress of the sampled vines was related to observed changes in flow/berry 100 kpa we measured midday stem water potentials of the field vines from which the bunches were collected. Stem water potential was 73

85 consistently more negative in Shiraz vines compared to Chardonnay vines until the end of the season (Fig 16 c). The greatest difference in stem water potential between Chardonnay and Shiraz vines occurred between 90 and 100 daa. This corresponded to almost identical flow/berry 100 kpa (Fig 16 b). There was no correlation between flow/berry 100 kpa measured on detached bunches and stem water potential of the sampled vines. The Thompson Seedless stem water potential was similar pre-veraison at 61 daa (-0.99 MPa +/- 0.03) and postveraison at 143 daa (-0.93 MPa +/- 0.07). Summarized data for berry deformability and flow/berry 100 kpa in late post-veraison is compared between the three varieties in Fig 17. Late post veraison is defined as after 105 daa when Shiraz berries begin to lose weight (phase 3). Shown above the graph is the percentage of cell death for the three varieties as determined by Tilbrook and Tyerman (2008). Shiraz and Chardonnay, which show substantial cell death at this time, had lower flow/berry 100 kpa and higher deformability than Thompson Seedless. Comparing the two wine grape varieties, Chardonnay had about half the flow/berry 100 kpa and half the deformability than those of Shiraz. The flow/berry 100 kpa of Thompson Seedless during that period was double that of Shiraz (Fig 17). 74

86 Figure 17Summarised data of flow into berries on whole bunches and deformability for Chardonnay, Shiraz and Thompson Seedless for the post 105 daa period. Flow measured with the flowmeter into bunches of Shiraz, Chardonnay and Thompson Seedless at 100 kpa via the peduncle was significantly different on a per berry basis (number of bunches: Shiraz, n=13, Chardonnay, n=7, Thompson Seedless, n=3; (ANOVA, p< Tukey s post-tests indicate significant differences between Shiraz and Chardonnay, p<0.01, and Thompson Seedless and the two wine varieties, p<0.001). Berry deformability was significantly different, Shiraz and Chardonnay n=75-100, Thompson Seedless, n=20 (ANOVA, p<0.0001, Tukey s post-tests, all pairs p<0.001). Data is presented in the context of percent loss of berry cell vitality across the mesocarp at the same stage of berry development (shown above the graph, Tilbrook and Tyerman, 2008). Hydraulic conductance of individual berries for inflow and outflow Late post veraison measurements ( daa) on individual berries of Shiraz and Chardonnay are shown in Fig

87 Figure 18 Hydraulics of single berries for flow in and out of the berry measured using the pressure probe. (a) Linear regressions of flow rates into and out of Chardonnay and Shiraz berries relative to berry equilibration pressure post 105 daa shown with 95% confidence intervals (n=13-14 berries). (b) Hydraulic conductance measured with the pressure probe: (L o) IN and OUT (relative to berry equilibration pressure) of berry pedicels post 105 daa was significantly different in Shiraz and Chardonnay (ANOVA, p<0.0001, n=13-14). Tukey s post-tests showed all data sets were significantly different (p<0.05) with the exception of Chardonnay L o IN compared with Chardonnay L o OUT. Paired t-test of Chardonnay data, p=0.056). The combined regressions of flow as a function of applied pressure are shown with 95% confidence limits in Fig 18 (a) to illustrate the pressure ranges over which these measurements were made as well as the differences between inflow (positive values) and outflow (negative values). Note that the intercepts of the regressions on the x-axis (corresponding to the equilibrium pressure) do not exactly correspond for inflow and outflow because different berries were used for 76

88 the two directions of flow. The hydraulic conductances measured for inflow and outflow are summarised in Fig 18 (b). It was not possible to undertake conductance measurements for outflow in Thompson Seedless berries because they continued to generate negative pressures in the pedicel xylem that were sufficient to cause cavitation in the pressure probe system. The hydraulic conductances for inflow and outflow (L o IN and L o OUT) were significantly different between Shiraz and Chardonnay (P < 0.001). Within varieties there was a higher conductance for L o IN than for L o OUT, the ratio being 1.8 fold in Shiraz and 6.4 fold in Chardonnay. However, this difference was only significant for Shiraz (p<0.05). Lucifer Yellow CH visualisation of backflow Pre-veraison dye uptake via the berry xylem showed that water was highly mobile within the bunch xylem of Shiraz and Chardonnay in pot grown vines (Fig 19). Dye translocated from the cut berries through the central stem of the rachis and into the peduncle of both varieties (Table 2) with dye visible in pedicel, central and peripheral xylem of berries adjacent to those cut, and in berry pedicel xylem up to a quarter of the way up the rachis from the bunch tip (not shown). 77

89 Figure 19Visualisation of dye loading into xylem from berries still attached to pot grown vines. LYCH dye uptake through the cut tip of two pre-veraison berries at the distal end of a bunch (52 daa, Shiraz and Chardonnay) is visible as fluorescence in the cross-sectioned xylem of respective bunches and vines. Bar = 200 µm. (a) Pedicel of cut Chardonnay berry. (b) Pedicel of cut Shiraz berry. (c) One quarter of the distance along the rachis (distal) of Chardonnay bunch. (d) Midpeduncle of Shiraz bunch. (e) Mid-rachis of Shiraz bunch. (f) Mid-internode of cane, below the bunch node on a Shiraz vine. In Shiraz, LYCH was found in the xylem below the cane from which the bunch arose (Fig 19 f), but not above. Stem water potential was measured at to MPa in the pre-veraison vines (Table 2). 78