vitis vinifera shiraz berries at sub-optimal maturity · vitis vinifera shiraz berries at...
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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
ii
Contents
Chapter 1 ................................................................................................................ 1!
Introduction to the papers of the thesis ...................................................................... 1!Introduction .............................................................................................................................. 2!
Chapter 2 ................................................................................................................ 5!
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 3 .............................................................................................................. 27!
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 4 .............................................................................................................. 57!
Hydraulic connection of grape berries to the vine: varietal differences in water
conductance into and out of berries, and potential for backflow. .......................... 57!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!
iii
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 5 .............................................................................................................. 85!
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. ............................................................ 96!
Results .................................................................................................................................... 97!
Berry weight and sugar accumulation................................................................................ 97!
Petiole and shoot analysis ................................................................................................ 100!
Abscisic acid in berries .................................................................................................... 102!
Yield and pruning weights ............................................................................................... 103!
Wine making and sensory analysis .................................................................................. 105!
Discussion............................................................................................................................. 107!
Chapter 6 ............................................................................................................ 113!
General Discussion .................................................................................................... 113!
Summary of findings................................................................................................. 114!Varietal differences in berry xylem hydraulics .................................................................... 114!
Varietal differences in berry cell vitality and cell membrane competence .......................... 115!
Molybdenum effect on berry development .......................................................................... 116!
Conclusions .......................................................................................................................... 117!
Future research ..................................................................................................................... 119!
Acknowledgements.................................................................................................... 121!
References........................................................................................................... 123!
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, 170-181.
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 job-
optimal 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, 173-184.
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, 541-550.
iv
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
90-100 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 0.1-0.2 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.
v
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 90-100 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.
vi
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.
vii
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
viii
of molybdenum to vines at anthesis and capfall, but molybdenum may affect the
organoleptic qualities of wine made from the fruit.
ix
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 1968. 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 2003-2004 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, 170-181.
Chapter 3:
Awarded “Best paper of 2008 by an early career researcher” by the Australian
Society of Plant Scientists and Functional Plant Biology, July 2009.
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, 173-184.
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, 541-550.
x
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, 141-161.
Supervisor: Stephen D. Tyerman Date: 10/5/10 Signature:
xi
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.
1
Chapter 1
Introduction to the papers of the thesis
2
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 90 – 100 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
3
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.
4
5
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, 170-
181.
6
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 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. 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 0.1-0.2 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: Lo , hydraulic conductance; Ro , hydraulic resistance; daa, days
after anthesis; TSS, total soluble solids
Key words: berry ripening, pressure probe, hydraulic conductance, Vitis vinifera,
berry shrivel.
7
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 20-25 °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
8
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
9
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
2002-2003 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
10
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
11
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 395-440, 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.
12
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.
13
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
14
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).
15
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 2002-2003 and 2003-2004. 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 2002-2003 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).
16
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 85-90 daa. At this
time the osmotic potential of Chardonnay juice was about -3 MPa and Shiraz
17
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 70-110 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 2002-2003 season during method
development (open symbols) and the 2003-2004 season (solid symbols)
collected by Lukasz Kotula).
18
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 2002-2003 (open symbols) and 2003-2004 (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).
19
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 (Ro) 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 Ro at cut position 1 from the Ro at position 0, the Ro at cut position 1 minus the Ro 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 (Ro) for each section of the berry system was calculated as Ro at
position 0 minus Ro at cut position 1, Ro at cut position 1 minus Ro at cut position
2, and Ro at cut position 2 minus Ro at cut position 3 (Fig 6 d & e). Chardonnay
20
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, semi-
permeable 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 pre-
veraison to 0.1- 0.2 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
21
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 85-90
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
22
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
23
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
90-100 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 10-12 m3 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
24
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).
25
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
26
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.
27
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, 173-184.
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 2009.
28
29
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
30
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.
31
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
32
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.
33
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).
34
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
90-100 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
35
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
36
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
2005-2006 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.
37
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 = 8.3143 J/mol.K and T = absolute temperature in
K). Solutes per berry (g) was approximated by the product of berry weight and
oBrix/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
38
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 pre-
veraison 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.
39
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
40
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 post-
veraison 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
41
from the bunch while under water. To seal the pedicel with the pressure probe a
piece of tubing 15-20 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
42
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.
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.
a
a
b
c
a
43
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).
44
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
45
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 (KPa)
Range; days after anthesis
Shiraz 4.7 +/- 1.7 (20) 108-136
Chardonnay 11.8 +/- 2.2 (15) 108-136
Thompson Seedless -24.0 +/- 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.
46
Figure 13The pattern of cell death differs between grades of Shiraz fruit. Premium (a) and mid-
quality (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 mid-quality 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.
47
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
48
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.
49
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
50
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.
51
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.
52
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 90-100 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
53
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
54
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.
55
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.
56
57
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, 541-550
58
59
Abstract
Weight loss in Vitis vinifera cv Shiraz berries occurs in the later stages of ripening
from 90-100 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 90-
100 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
60
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; Lo , hydraulic conductance.
Keywords: Hydraulic conductance, berry xylem, backflow, Vitis vinifera, berry
shrivel, berry ripening, pressure probe, flow meter.
61
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).
62
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 pre-
veraison 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 post-
veraison 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
63
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
64
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 90-100 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
65
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 2004-2005 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 2006. 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 1.055 N. At each time point measurements
were made of five berries from proximal, mid and base of five bunches (n=25
66
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
67
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 (Lo) for different flow directions (Lo IN or
Lo OUT) through pedicels of single berries were made using a root pressure
68
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 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. 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. Lo IN and Lo OUT were
measured on the same berries. The mean post 105 daa equilibration pressures
of berries were slightly positive in Shiraz (0.006 MPa +/-0.0017 s.e.m) and
Chardonnay (0.009 MPa +/-0.0024 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 -0.01 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 Lo, 13-14 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.
69
Measurements were made between 12.30 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
7.30–8.30 am and ceased 12.30-1.30 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 395-440, beam splitter FT 460 and barrier
filter LP 470. Digital images were obtained using a Nikon DXM 1200F digital
camera.
70
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.
71
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).
72
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 5.5-6.3 %
deformability for the 90-101 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=75-100 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/berry100 kPa) during development (Fig 16
b). Maximum flow/berry100 kPa was observed in pre-veraison berries of all varieties.
Post-veraison berries showed declining flow/berry100 kPa reflecting the previously
documented decline in hydraulic conductance (Tyerman et al. 2004). Thompson
Seedless berries had a 40% reduction in flow/berry100 kPa comparing pre-veraison
73
data at 61 daa to post-veraison at 143 daa (4.05 x10-12 +/- 5 x10-13 to 2.4 x10-12
+/- 3.5 x10-13 m3s-1). In Shiraz flow/berry100 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/berry100 kPa we measured midday stem water potentials of the
field vines from which the bunches were collected. Stem water potential was
74
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/berry100 kPa (Fig 16 b). There was no
correlation between flow/berry100 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 post-
veraison at 143 daa (-0.93 MPa +/- 0.07).
Summarized data for berry deformability and flow/berry100 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/berry100 kPa and higher
deformability than Thompson Seedless. Comparing the two wine grape varieties,
Chardonnay had about half the flow/berry100 kPa and half the deformability than
those of Shiraz. The flow/berry100 kPa of Thompson Seedless during that period
was double that of Shiraz (Fig 17).
75
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<0.001. 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 (105 - 146 daa) on individual berries of Shiraz
and Chardonnay are shown in Fig 18.
76
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: (Lo) 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 Lo IN compared with Chardonnay Lo 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
77
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 (Lo IN and Lo OUT) were significantly
different between Shiraz and Chardonnay (P < 0.001). Within varieties there was
a higher conductance for Lo IN than for Lo 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).
78
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) Mid-peduncle 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 -0.68 to -
0.75 MPa in the pre-veraison vines (Table 2).
79
Table 2 A varietal difference was apparent in LYCH dye studies of xylem water backflow from the
cut berry tip (refer to Methods). The presence or absence LYCH in xylem tissue of glasshouse grown Shiraz and Chardonnay fruit, rachis and vine was observed. The berry-vine xylem hydraulic connection was maintained in Shiraz berries post 105 daa, whereas Chardonnay berry xylem appeared to be functionally isolated from the vine at 97 daa.
At 97 daa, no dye uptake was observed moving from Chardonnay berries back
into torus, pedicel or rachis xylem. In Shiraz, LYCH was noted in the torus,
pedicel and up to a quarter of the way up the rachis from the bunch tip at 101 daa
(Table 2). By 118 daa, LYCH was visible in the Shiraz bunch xylem in the torus
and pedicel xylem only.
Discussion
We have examined the differences in the xylem water flow into and out of grape
berries across varieties that show distinct differences in cell death in the
mesocarp, differences in deformability, and differences in weight loss in the post
veraison period of berry development. Varietal differences were apparent in the
flow/berry100 kPa measured on detached bunches in the post 105 daa period with
the order from greatest to least being Thompson Seedless, Shiraz and
Chardonnay. These differences in flow/berry100 kPa are attributed to varietal
differences in hydraulic conductances of the berry. In the two wine varieties that
show cell death in phase 3 of development we have shown that Lo IN and Lo OUT
of berries is significantly different between varieties during the post 105 daa
period when Shiraz is losing weight and Chardonnay is not. In contrast,
Shiraz Chardonnay Days after anthesis 52 101 118 52 97
Torus of cut berry + + + + - Pedicel of cut berry + + + + - Distal tip of rachis + + - + - " up rachis + + - + -
Mid rachis + - - + - # up rachis + - - + - Peduncle + - - + - Stem below node + - - - - Stem above node - - - - -
Stem # (MPa) -0.75 -0.49 -0.61 -0.68 -0.65
° Brix 4.1 22.3 22.7 6.6 22.7
80
Thompson Seedless shows very different behaviour, maintaining a relatively high
post-veraison flow/berry100 kPa (high conductance) along with low deformability,
continuing cell vitality in phase 3, and negative xylem pressures (Tilbrook and
Tyerman 2008).
The post 105 daa measurements with the pressure probe on individual berries of
Shiraz and Chardonnay showed that the Lo IN to Shiraz berries was 7.6-fold
higher than that of Chardonnay which is consistent with our previous work
(Tyerman et al. 2004). Extending our previous work, we show evidence here of
flow rectification to and from the berry. For Shiraz berries, Lo IN and Lo OUT were
significantly different with Lo IN being 1.8 fold higher than Lo OUT. This indicates
some kind of rectification of flow via the berry xylem and apoplast that may be
related to a membrane component in the pathway. Chardonnay showed a greater
difference in Lo between flow directions but it should be noted that Lo OUT was
very small (Lo IN= 6.4 x Lo OUT, paired t-test, p=0.056). Comparing Lo OUT
between varieties shows a much greater difference than previously observed with
a 26-fold greater Lo OUT for Shiraz compared to Chardonnay. The conductance
for flow out of the berry is more relevant to examination of backflow from the
berry.
A significant volume of water could leave a berry of the wine grape varieties via
the xylem because the berry does not develop negative pressures that would
counteract the negative pressures generated in the vine from transpiration. We
have previously shown that the osmotic potential of berry juice in the post-
veraison period does not translate into a negative pressure measured in pedicel
xylem of Shiraz and Chardonnay (Tyerman et al. 2004). To maintain a constant
berry volume under conditions of negative pressures in the apoplast of berries
(and therefore in the xylem), the cell plasma membranes must maintain semi-
permeability with a solute reflection coefficient near 1. Tilbrook and Tyerman
81
(2008) showed that the cell membranes of a significant portion of the mesocarp
of Shiraz and Chardonnay berries become leaky, and this correlates with loss of
negative pressure in the apoplast-xylem continuum of the berry. This inability to
generate a negative pressure in the wine varieties, in contrast to Thompson
Seedless, is an important consideration for the examination of the potential for
xylem backflow to occur from berry to the vine.
Taking the midday stem water potential of the vines as the maximum driving
force for water removal from the berry, and ignoring the small positive pressures
developed by the berry, the measured Lo OUT allows calculation of flow rates
from the berries. For a midday stem water potential of -1.25 MPa, flow from
Shiraz berries would be 1.5 x 10-2 mL h-1 while from Chardonnay the flow would
be 5.8 x 10-4 mL h-1. If this occurred over a period of say 5 hours per day then a
Shiraz berry could loose 0.076 mL per day via backflow compared to only 0.003
mL from a Chardonnay berry. It is likely that water loss would be larger than this
because stem water potentials are always more negative (even pre-dawn) than
the xylem pressures measured on detached berries. The rate of weight loss that
we observed in Shiraz berries was 0.011 gm per day, which is in broad
agreement with the calculated water loss by backflow. The rate of weight loss of
Chardonnay over the same daa period was 0.001 gm per day which was not
statistically significant. Hydraulic isolation from the vine is clearly more effective
in Chardonnay berries when compared to Shiraz.
Dye loading studies confirmed varietal differences in berry isolation from the vine.
Pre-veraison, the potential for water to move from berry xylem back into potted
vines was almost identical in Shiraz and Chardonnay, but different in the post-
veraison berries. No tracer was detected moving from the xylem of Chardonnay
berries back into the rachis at 97 daa, suggesting isolation of berry xylem from
the vine. In contrast, at101 daa tracer moved freely through Shiraz berry xylem
82
into the rachis xylem a quarter of the way up the bunch and was still able to move
out of Shiraz berry xylem back into the torus and pedicel of a berry at 118 daa. It
is acknowledged that excising the tip of the berries changes the pressure
generated in the berry xylem to zero, but as the berry xylem generates close to
zero pressure (slightly positive) in both varieties post-veraison in this paper and
(Tyerman et al. 2004), the data gives useful information about varietal
differences. The distances the LYCH moved in pre-veraison experiments may be
a reflection of the sudden loss of pre-veraison negative pressure in the xylem of
the berry when the stylar end is excised for dye loading. The Shiraz data is
consistent with that of Keller et al. (2006) in finding that the tracer travelled from
the peduncle into stem xylem below the node, rather than up towards the
transpiring vine canopy.
The negative pressures generated in the pedicel xylem of post-veraison
Thompson Seedless berries meant hydraulic conductance for flow out of the
berry could not be measured with the pressure probe method. Backflow may be
prevented in Thompson Seedless because the berry can generate negative
apoplastic pressures (Tilbrook and Tyerman 2008) that may balance the negative
xylem pressures in the vine. In fact, anecdotally, the problem with Thompson
Seedless can be excess berry uptake of water and berry splitting indicative of
continued xylem function and maintenance of a large osmotic gradient across
mesocarp cell membranes. It is interesting to put this data into the context of the
work on a table grape variety by Zhang et al. (2006) and a wine variety by Wada
et al. (2008). In both types of fruit the concentration of soluble solutes in the berry
apoplast was ~50 mM before veraison. In the post-veraison period, the wine
variety reached ~1 M solutes in the apoplast while the table grape peaked at
under 200 mM. While the data is from only two varieties, it may suggest that
there are also varietal differences in post-veraison phloem unloading. We
83
reiterate caution when extrapolating data and function between table and wine
grape varieties in the physiology of berry development and ripening.
Other work has indicated that berry weight loss in Shiraz may be due to a
reduction of water inflow (Rogiers et al. 2004) perhaps via the phloem (Rogiers et
al. 2006). Berry transpiration may also differ between varieties and comparisons
between varieties late in development remain to be examined. Reduced phloem
translocation and high transpiration may be contributing factors to berry weight
loss. However, the data presented here strongly suggests that Shiraz berries
maintain a significant xylem connection, and that berry xylem did not hydraulically
isolate from the vine as seen in Chardonnay. Thus xylem backflow may be
contributing to berry weight loss in Shiraz after their maximum weight has been
achieved and before harvest maturity for winemaking.
The question remains as to what the mechanism is in the berry that reduces
hydraulic conductance for outflow. The berry xylem exhibits a qualitatively low
resistance pathway (Bondada et al. 2005), but that doesn’t necessarily mean that
Lo doesn’t reduce as the berry approaches harvest as our data quantitatively
shows. One possibility is that there is a barrier between the berry apoplast and
the xylem that reduces flow back into the xylem when the berry is vulnerable, that
is when there is loss of membrane semipermeability across the mesocarp. Since
it has been shown that cells surrounding vascular bundles in berries remains vital
(Krasnow et al. 2008; Tilbrook and Tyerman 2008), it is quite possible that an
apoplastic barrier similar to those in roots and some leaves is functioning late in
berry development to halt backflow via the apoplast and/or to allow control by the
cell membranes of the vascular associated cells. In Shiraz the barrier could be
incomplete or not functional so that backflow could occur. It is quite possible that
flow through a membrane barrier could be regulated by aquaporins as in other
tissues (Tyerman et al. 2002). For example, two grape berry aquaporins with
84
varying transport capacities have increased expression post-veraison in
Chardonnay, Ugni Blanc and at harvest in Pinot meunier (Picaud et al. 2003).
Together, quantitative and qualitative data presented here provide evidence to
accept our hypothesis that there are varietal differences in water conductance in
and out of grape berries, and that there are varietal differences in the potential for
backflow from berries into the vine. Chardonnay berries hydraulically isolate more
effectively from the vine than Shiraz berries, which may contribute to preventing
pre-harvest weight loss. We propose that grape berries generally have two
strategies to control water balance through the final stages of development: 1. A
reduction in hydraulic conductance out of the berry via the xylem to counter the
loss of a significant portion of osmotically competent cells across the mesocarp;
2. Continuing osmotically competent cells that maintain a gradient across the
plasma membranes of the mesocarp, which allows hydraulic conductance into
the berry xylem to continue through to maturity as seen in Thompson Seedless. If
the mechanism that reduces hydraulic conductance into the berry fails or is mis-
timed in the first strategy, a water deficit may occur in the berry. The risk inherent
in the second strategy is that the berries may split at high water potentials. It will
be important to investigate how berry transpiration changes with changes in
hydraulic conductance and if there are varietal differences. In Shiraz
transpiration appears to decrease approximately in proportion to berry hydraulic
conductance (Tilbrook and Tyerman 2008), but different data sets from different
studies were used in this comparison. It also remains to be determined what
structures or mechanisms underlie reduced hydraulic conductance of berries late
in maturity.
85
Chapter 5
Effect of molybdenum application on Shiraz berry
development in late stages of ripening and the
impact on the organoleptic properties of the wine
This chapter is a manuscript in preparation.
86
87
Abstract
Weight loss in Vitis vinifera cv 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 by 2 to 7 days. It also slowed sugar
accumulation relative to berry weight accumulation in phase 2. Analysis of
abscisic acid content of berries relative to weight accumulation reflected the
developmental shift resulting from molybdenum treatment. 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.
Abbreviations: molybdenum, Mo; molybdenum cofactor, Moco, daa: days after anthesis.
88
89
Introduction
Plants and animals require the micronutrient molybdenum (Mo) for growth. While
only minimal quantities of the transition element Mo are needed (Gupta 1997a), it
has structural and catalytic functions in oxidation and reduction reactions in
plants (Marschner 1995). It is not generally active on its own, with a single
exception in bacteria, but is incorporated into a pterin complex referred to as the
molybdenum cofactor (Moco). Moco binds to molybdo-enzymes, which are found
to catalyse several reactions in plants: the final step in abscisic acid synthesis by
an aldehyde oxidase, catabolism of purine and stress reactions by xanthine
dehydrogenase, detoxification of sulfite by sulfite oxidase,nitrate reduction and
assimilation by nitrate reductase and nitrogenase respectively (Kaiser et al. 2005;
Mendel and Hänsch 2002; Schwarz and Mendel 2006).
Soil pH and drainage have been identified as the most significant factors in Mo
availability for plant growth, along with organic matter, the nature of the parent
rock and interactions with other nutrients in the soil (Gupta 1997b). As soil pH
increases, more Mo becomes available for uptake, usually as the anion form
MoO4-. In poorly drained soils Mo accumulates in an available form, and plants
growing on them take up sufficient Mo to cause Mo toxicity or molybdenosis in
sheep and cattle. Other factors in soil Mo availability are the concentration of
adsorbing oxides and the amount of organic compounds (reviewed in Gupta
1997b). There is a link with sulphate uptake, and Mo transport can occur via a
plant sulphate transporter (Fitzpatrick et al. 2008). Mo deficiency in plants is not
often seen (reviewed in Kaiser et al. 2005; Gupta 1997b), but in Vitis vinifera cv
Merlot it is characterised by shortened, zig-zag internodes and pale green
cupped leaves that have marginal leaf necrosis, and millerandage (hen and
chicken) in the bunches (Kaiser et al. 2005; Robinson 2000).
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Foliar application of molybdate to own rooted Merlot vines at particular times in
development significantly reduces the incidence of millerandage (Kaiser et al.
2005; Williams et al. 2004). This results in improved yields in commercial
vineyards of own rooted Merlot vines where the initial petiolar concentration of
molybdenum (Mo) is in the range 0.05-0.09 mg/kg, (Longbottom 2007; Williams
et al. 2004). The activity of nitrate reductase can be induced in leaves of Merlot
when molybdate is directly applied (Kaiser et al. 2005). Molybdenum may have
other effects on berry development since a visual difference in the degree of
berry shrivel in fruit at harvest on Mo treated and untreated vines has been
observed during trials on Merlot vines in the McLaren Vale region of South
Australia (Longbottom 2007).
Grape berry development occurs in three phases (Sadras and McCarthy 2007;
Tilbrook and Tyerman 2008): phase 1 corresponds to the rapid growth from fruit
set to berry softening or veraison; phase 2 corresponds to the period of sugar
accumulation and increase in berry weight where the relative rate of sugar
accumulation is greater than the relative rate of increase in fresh mass; and,
phase 3 occurs after peak berry weight where berries lose fresh weight offsetting
the change in sugar per berry (Sadras and McCarthy 2007). Sadras and
McCarthy (2007) used an allometric analysis of berry sugar and weight
accumulation to account for size dependent effects on sugar concentration and to
remove differences in timing of the onset of the different phases (ontogenetic
drift). An allometric coefficient was obtained from the slope of the regression
between the logarithm of sugar per berry and the logarithm of berry weight.
phase 2 is distinguished by an allometric coefficient greater than 1, while phase 3
has an allometric coefficient < 1. These two phases are clearly differentiated in
the log-log relationship. Here we use this analysis to examine the effects of
molybdenum sprays on berry sugar and weight accumulation.
91
In most seasons, Shiraz berries reach a maximum weight around 90-100 days
after flowering (McCarthy 1999; Rogiers et al. 2000; Tyerman et al. 2004)and the
berries lose weight before harvest maturity is achieved (McCarthy 1999; Rogiers
et al. 2000; Tyerman et al. 2004). The onset of phase 3 corresponds to the
beginning of cell death across the mesocarp in Shiraz and also in Chardonnay
which normally does not show weight loss (Tilbrook and Tyerman 2008). The
weight loss observed in Shiraz may be due to backflow to the vine since the
hydraulic connection to the vine in Shiraz continues at this stage and is
substantially higher than in Chardonnay (Tilbrook and Tyerman 2009). The
weight loss and cell death in Shiraz is well advanced by the time the berries have
a shrivelled appearance. Given the yield loss that occurs due to weight loss (up
to 30%) and the probable impact on quality, management options that allow
manipulation of the onset and degree of weight loss would be practically
significant.
We do not presently understand what the physiological triggers are for phase 3
(cell death and weight loss) in berry development, but more information is
available for the transition from phase 1 to phase 2. An increase in
brassinosteroids, a steroidal hormone usually involved in plant growth and
development, has been associated with the berry softening and the
commencement in ripening of Cabernet Sauvignon (Symons et al. 2006). A peak
in abscisic acid (ABA) concentration corresponds to berry softening (Coombe
and Hale 1973; Goktürk Baydar and Harmankaya 2005), but the signalling
pathway is not clear (Yu et al. 2006). Changes in ABA concentration have not
been specifically examined with respect to the onset of phase 3. Since the onset
of phase 3 and weight loss is linked to berry water relations and the hydraulic
connection between the berry and the vine (Tilbrook and Tyerman 2008; Tilbrook
and Tyerman 2009), and given that ABA generally has strong effects on water
92
flows in plants, it would be worthwhile examining any association between ABA
and phase 3. Furthermore since the final step of ABA synthesis is catalysed by a
molybdo-enzyme (aldehyde oxidase), ABA synthesis may be affected by
changes in Mo status.
In view of the physiological links discussed above and the tantalising observation
made by Longbottom (2007) that Mo spray impacted on berry shrivel, we
investigated the impact on late stages of berry development of foliar spray of
sodium molybdate applied at various times during berry development. We
investigated Mo treatments on Shiraz because it is renowned for having the most
significant weight loss. The allometric analysis of Sadras and McCarthy (2007)
was applied because we found that timing of the onset of phases was affected by
the treatments and relative rates of sugar accumulation and berry weight change
were affected by Mo application. Wine was also made from the fruit in order to
assess if Mo changed any detectable organoleptic properties of the finished wine
and to quantify how much of the applied Mo was carried into the wine.
Materials and Methods
Fruit material
Two experimental sites were used for random block experiments over two
seasons. In 2004-2005 at the Coombe vineyard on Waite campus of the
University of Adelaide, three replicate blocks of four BVRC12 Vitis vinifera cv
Shiraz, own rooted, eleven year old vines were used for control and molybdenum
treatments. For the 2005-2006 trials, four replicate blocks of six, ten year old
BVRC12 Vitis vinifera cv Shiraz, own rooted vines at the Nuriootpa vineyards of
the South Australian Research and Development Institute (SARDI) were used for
control and molybdenum treatments. Both vineyards used vertical shoot
positioning with a single cordon. In the Coombe vineyard the cordon was 1.3 m
93
from ground level with two foliage wires that were lifted as the canes developed
during the season. The Nuriootpa vineyard cordon was 1 m from ground level
and no foliage wire was used. Both vineyards were under conventional drip
irrigation and commercial spray regimes.
Molybdenum treatment of vines
The leaf canopies of vines were sprayed using a knapsack sprayer to the point of
run-off (~270 mL per vine) from the leaves with either water (control) or an
aqueous solution of sodium molybdate (treatment) applied at a rate of 0.1 g of
sodium molybdate (Sigma Aldrich) per vine. This extrapolated to 300g of sodium
molybdate per hectare. Therefore, with two treatments, 0.2 g per vine or 600 g/ha
was applied. Inflorescences were not protected or singled out for treatment and
received spray incidental to the leaf canopy treatment. No adjuvant was added to
the solution to assist adherence to the leaves.
In the Coombe vineyard trial of 2004-2005, blocks of vines were sprayed with
water or sodium molybdate solution as described at 45 and 68 daa. At Nuriootpa
in 2005-2006, two trials were conducted, one with blocks of vines sprayed at 0
daa and one week later, and another with blocks of vines sprayed as berries
softened at 62 daa and one week later.
Berry weight and sugar accumulation
For the Coombe vineyard trial, samples of berries were collected from ten
random bunches on three replicate panels of four vines, five berries from
proximal, mid and base of a bunch. For the Nuriootpa trials, samples of berries
were collected from ten random bunches on four replicate panels of six vines,
five berries from proximal, mid and base of a bunch. The fifty berry samples from
replicates of all trials were placed on ice and transported to the laboratory and
weighed. Berries were crushed and the juice centrifuged briefly to remove solids.
94
°Brix of the juice was measured using a temperature compensated digital
refractometer (ATAGO Model PR101). Allometric analysis was conducted
according to Sadras and McCarthy (2007). The log of sugar (°Brix) per berry was
calculated using the mean berry weight for each replicate fifty berry sample.
Petiole and shoot analysis
Prior to spraying at 0 daa in the Nuriootpa 2005-2006 trial, fifty petioles from
leaves opposite proximal bunches were collected from each block of six vines,
twenty-five from the northern and the southern aspects of the vines. For the
shoots, fifty tips were cut below the second unfurled leaf from the shoot tip on
actively growing shoots, twenty-five from the northern and the southern aspects
of the vines at 0 daa. Samples were collected while wearing nitrile gloves and
placed into brown paper bags, then dried overnight at 80 ºC in an oven. Sampling
of petioles and shoot tips was repeated by the same method at 105 daa. Petiole
and shoot samples were not rinsed, but were exposed to seasonal rainfall for the
duration of the experiment. Samples were held overnight at 40 ºC prior to
grinding in a Fritsch Pulverisette 14 with titanium rotor and a 0.2 mm titanium
sieve ring at 20,000 rpm. Waite Analytical Services, University of Adelaide,
carried out a nitric/hydrochloric acid digest and ICPMS analysis on the samples.
Yield and pruning weights
The Nuriootpa 2005-2006 trial where the vines were sprayed at 0 daa was used
for this data. Fruit was harvested by hand and the number of bunches and fruit
weight was recorded for each vine of each treatment block. The fruit from the
control four blocks of six vines was picked at 112 daa and the fruit from the
molybdenum treated vines was picked 115 daa to ensure consistent sugar
content for wine making and sensory analysis. Vines were spur-pruned mid-
winter, 30 June 2006, and the fresh weight of canes from each vine was
measured.
95
Abscisic acid in berries
Fruit from the Nuriootpa 2005-2006 trial where the vines were sprayed at 0 daa
was used for ABA analysis. Samples of thirty berries from each experimental
block were collected by the same method as for weight and sugar analysis, put
into clip lock plastic bags, then into an esky containing dry ice and transported to
the laboratory where they were stored in a -80 ºC freezer. Ten berries were
randomly sub-sampled and analysis of ABA content of whole berries was
conducted (Soar et al. 2004; Soar et al. 2006). Briefly, whole berries were ground
to a powder in liquid nitrogen in an IKA Works A11 analytical mill. 100-250 mg of
powdered tissue was transferred into chilled pre-weighed centrifuge tubes and
sample weights determined. Extraction of ABA was carried out by adding 5 mL of
boiling water to the samples, boiling samples for ten minutes then chilling on ice.
100 ng of deuterated ABA was added to each sample as an internal standard
and mixed. Samples were centrifuged at 200 g for 5 minutes and the supernatant
collected. A Phenomonex extraction of the samples was completed using a
strata-X 33 !m polymeric Sorbent 500 mg / 6 mL cartridge with brief centrifuging
between each step to ensure the wash was complete. The cartridge was washed
with 5 mL of methanol then 5 mL of nanopure water. The samples were adjusted
to pH 2.5 using 1 M HCl, then loaded onto the cartridge. After a wash of 50%
methanol, each sample was eluted from the cartridge with a wash of 80%
methanol, the eluent collected in glass tubes, transferred to microfuge tubes and
dried using a Speedivac. The dried samples were dissolved in 30 µL of 0.2 %
acetic acid and 50% methanol, and ABA was quantified using Liquid
Chromatography-Mass Spectroscopy (Surveyor LC Pump Thermo Finnigan PDA
Detector) with a Phenomenex Synergi 4u Hydro RP-80A column.
96
Wine making, molybdenum and sensory analysis.
Wine was made from the fruit harvested from the replicates of the control and Mo
treated blocks of vines from the Nuriootpa trial sprayed at 0 daa (n=4). It was
prepared from the approximately 50 kg lots of fruit by Provisor Pty Ltd (Hartley
Grove, Urrbrae, South Australia), a commercial winemaking company. The Mo
content of the wine was assayed by Waite Analytical Services, University of
Adelaide, using a nitric/hydrochloric acid digest and ICPMS analysis. From the
four replicates of wine made from control and treated vines, three were chosen at
random for sensory analysis. Difference testing was carried out using the
triangle, or forced choice test according to Australian Standards (AS 2542.1
Sensory Analysis of Foods, General guide to methodology and AS 2542.2.2
Method 2.2: Triangle Test) initially to establish whether there were differences
between replicate wines and later between wine made from control and treated
fruit. Assessors were volunteers from staff and post-graduate students of the
University of Adelaide, and staff from South Australian Research and
Development Institute. A purpose built, multiple booth sensory analysis
laboratory with monochromatic sodium lighting was used for the testing. FIZZ
software (Biosystemes, France) was used to create and run each sensory
session. Samples were coded and labeled with Fizz generated random numbers
so that no Triangle test had repeated numbers, and there was a balanced,
random testing order for each triad (AAB, ABA, BAA, BBA, BAB, ABB). All
samples were measured (30 mL) and presented at room temperature (21.5-22.0
ºC) in a standard wine tasting glass on a white tray, with a glass of water to rinse
the mouth between tests. The assessors were asked to identify which wine in the
triad being offered was different to the other two. Samples were tasted in order
and expectorated. Six tests (a balanced set) were presented to an assessor in a
session.
97
The hypothesis tested: there was a difference between the wines. Binomial
distribution tests were used to calculate the Z statistic using the formula
where pobs is the proportion of tests answered correctly, pexp is the proportion of
tests correct by chance (eg 1/3) and q=1-pexp. The significance levels of p = 0.01,
0.05 and 0.1 correspond with Z values of 2.33, 1.65 and 1.28 respectively for a
one tailed Triangle test, and binomial distribution tables were consulted for critical
numbers of correct responses (Bi 2006; Lawless and Heymann 1999). To
consider type I and type II errors at the level p=0.05 or Z=1.65, with the allowable
proportion of discriminators at 50%, a minimum number of 23 tests was required
(Bi 2006). For difference testing between the replicates of control and Mo treated
wines, 43 tests were conducted for each replicate. For the testing between a
control and a Mo treated wine, 96 tests were conducted.
Results
Berry weight and sugar accumulation
The time courses of changes in berry weight during phase 2 and 3 are shown in
Fig 20 for treated and control vines for the two sites (Coombe vineyard, sprayed
45 and 68 daa, Fig 20 a) and different spraying times (Nuriootpa at anthesis Fig
20 b, and at veraison Fig 20 c). These data were well fitted by second order
polynomials. Phase 2 commenced with berry softening at 79 daa in the 2004-
2005 trial at the Coombe vineyard, and 66 daa for the 2005-2006 trials at
Nuriootpa. Significant differences between berry weight accumulation of control
and molybdenum treated vines were found in all trials (Fig 20). Each curve was
differentiated to establish the commencement of phase 3. For the Coombe
vineyard trial, control vines began phase 3 at 99 daa and treated vines at 106
98
daa. In the Nuriootpa vines treated at anthesis, control vines began phase 3 at
89 daa and treated vines at 91 daa. For vines treated at berry softening, control
vines began phase 3 at 89 daa and treated vines at 93 daa.
Figure 20 Comparative analysis of second order polynomial fits to phase 2 and 3 berry weight accumulation data shows significant differences in berry weight accumulation of control and treated vines in all trials, n=4 x 50 berry samples. (a) Trial conducted in 2004-2005 season at the Coombe vineyard. Vines sprayed at 45 and 68 daa, p=0.005. (b) Trial at Nuriootpa in 2005-2006. Vines sprayed at flowering, 0 daa, p=0.016. (c) Trial at Nuriootpa in 2005-2006. Vines sprayed at
berry softening, 62 daa, p=0.015.
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Given the significant difference in the time of onset for peak berry weight
between treatments and controls, sugar accumulation was analysed
allometrically relative to berry weight during development (Fig 21).
Figure 21 Allometric analysis of the log of sugar accumulation in berries relative to the log of berry
weight shows distinctly different patterns in phase 2 and 3 of development, n=4 x 50 berry samples.(a) Trial conducted in 2004-2005 season at the Coombe vineyard, vines sprayed at 45 and 68 daa. (b) Nuriootpa trial 2005-2006, vines sprayed at flowering. (c) Nuriootpa trial 2005-2006, vines sprayed at berry softening, 59 daa.
Phase 2 and 3 of berry development show clearly different patterns, with the
allometric coefficients (slopes) in phase 2 all being greater than 1, and less than
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1 in phase 3 (Fig 21, Table 3). The allometric coefficients for sugar accumulation
in fruit from molybdenum treated vines were all lower than control vines with a
single exception; phase 3 where the vines were sprayed at anthesis. In testing for
differences, the only significant difference in allometric coefficients was between
phase 2 from control and untreated vines sprayed at anthesis in the Nuriootpa
trial (p=0.042) (Table 3).
Table 3 Allometric analysis of sugar accumulation of berries in phase 2 and 3 of development show that, with a single exception, the allometric coefficients of molybdenum treated fruit were less than
control fruit in three separate trials. All the coefficients were > 1 in phase 2 and < 1 in phase 3 of berry development. There was a significant difference in the coefficient of control and molybdenum treated fruit in phase 2 of the Nuriootpa trial where molybdenum was sprayed on the vines at anthesis.
Petiole and shoot analysis
Analysis of petioles from vines that were sampled at anthesis before spraying,
and later sampled at 105 daa, indicated a significant difference in molybdenum
content (2 way ANOVA, p<0.0001). Petiole molybdenum content was similar at
anthesis in all experimental blocks, immediately prior to the vines being sprayed.
At 105 daa, petioles from the control vines maintained similar molybdenum
content as at anthesis, whereas the petioles from the treated vines had a nine
fold increase (Fig 22 a). For untreated vines the petiole concentrations of Mo
were low and in the range (0.05 – 0.09 mg/kg) that was associated with a yield
response in Merlot to Mo application (Williams et al. 2004).
Phase 2 Phase 3
Molybdenum treatment
Control + Mo Pvalue Control + Mo Pvalue
45 daa and veraison, 04-05
2.14 +/-0.45 1.92 +/-0.16 0.621 0.31 +/-0.19 -0.13 +/-0.16 0.344
Anthesis, 05-06 2.92 ± 0.17 2.53 +/-0.09 0.042* -0.32 +/-0.17 -0.21 +/-0.23 0.774
Veraison, 05-06 2.87 ± 0.26 2.62 +/-0.12 0.343 -0.41 ± 0.28 -0.59 +/-0.92 0.853
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Figure 22 Petioles and shoot tips from vines of the Nuriootpa trial sprayed at anthesis were analysed for molybdenum content prior to treatment and at 105 daa, before harvest; different letters indicate significant difference between groups in post tests. Note that error bars are present for all
data sets, but not all are visible due to minimal variation.
(a) Molybdenum content was stable in control petioles at the measured time points, and 9 times higher at 105 daa in response to molybdenum treatment of vines at flowering (n=4 x 50 petioles, 2 way ANOVA p<0.0001). The grey shaded area indicates the petiolar concentration 0.05-0.09 mg/kg that was associated with a yield response in Merlot (Williams et al. 2004).
(b) In shoot tips, molybdenum content of control and treated vines were significantly different in response to the molybdenum treatment (n=4 x 50 shoot tips, 2 way ANOVA p=0.003). The shoot tips of treated vines maintained higher concentrations of molybdenum compared to untreated vines.
For vines sprayed at anthesis, there was significantly less reduction in shoot tip
molybdenum concentration of treated vines compared to control vines at 105 daa
(Fig 22 b). At 0 daa, shoot tips contained similar molybdenum concentrations in
all blocks as expected. At 105 daa, shoot tips showed less molybdenum content
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than at anthesis in all vines. This suggests that less molybdenum is transported
to shoot tips late in the season compared to anthesis.
Abscisic acid in berries
There was no significant difference in berry ABA concentration in fruit from
control and treated vines during berry development (Fig 23 a). Applying the
allometric analysis to ABA content per berry (Fig 23 b) showed a clear difference
between phase 2 and phase 3 allometric coefficients. The log ABA content per
berry appears relatively static relative to the log of berry weight during phase 2,
then a distinct reduction in phase 3. There were no significant differences in the
allometric coefficients between fruit of control and treated vines. During phase 3
the berries of the treated vines had a higher concentration of ABA, but it was not
significantly different. In control and treated berries, ABA concentration declined
at the same relative rate (Fig 23 b).
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Figure 23 Analysis of abscisic acid (ABA) in berries from the trial at Nuriootpa 2005-2006, where molybdenum was applied at anthesis, n=4 x 10 berry samples. Berry weight, n=4 x 50 berry samples.
(a) Berry ABA, initially low in phase 1, has risen sharply by 66 daa (after berry softening at 59 daa) and is maintained with high variability in phase 2. It drops to pre-veraison levels in phase 3 as
berries lose weight.
(b) Log of ABA concentration in berries relative to log of berry weight increases in phase 1, is maintained to the transition from phase 2 into phase 3of berry development at 90 and 92 daa (by differentiation) for berries of control and treated vines respectively. By 98 daa, abscisic acid content per berry reduced to pre-veraison levels.
Yield and pruning weights
Fruit was harvested from the vines of the Nuriootpa trial with the vines sprayed at
anthesis. The mean yield of fruit per vine from control vines was 8.67 kg
compared to 7.83 kg for the treated vines. The fruit from the control vines was
harvested at 112 daa, and from the molybdenum treated vines at 115 daa to
104
reduce the likelihood of confounding data from planned sensory analysis of wine
made from the fruit. As a result, the yields were not significantly different (2 way
ANOVA, p=0.21) (Fig 24 a). If the fruit had been harvested on the same day, it is
likely that a significant difference in yield would have been measured, as berry
weights were 5% heavier on treated vines at 112 daa.
Figure 24 Field data for the Nuriootpa trial vines sprayed at flowering. Note that the fruit from the control vines was harvested at 112 daa whereas the molybdenum treated vines were harvested at 115 daa, therefore reducing the yield difference, n=4 blocks of 6 vines. (a) Mean yields were higher from molybdenum treated vines but not significant, 2 way ANOVA, p=0.21. (b) Mean number of bunches was higher on molybdenum treated vines but not significant, 2 way ANOVA, p=0.26. (c) The weight of canes pruned from the vines was significantly higher from the molybdenum treated vines, 2 way ANOVA, p=0.04. (d) Yield to pruning weight ratio showed no significant difference, 2 way ANOVA, p=0.63.
The mean number of bunches per vine was higher on vines treated with
molybdenum (82.3 compared to 76.8), but not significantly different (2 way
ANOVA, p=0.26) (Fig 24 b). It was noted that not all inflorescences were
maintained on the shoot in the two weeks after anthesis. Some did not set fruit,
105
then shrivelled and abscised from the shoot in that period. As bunches were
counted at harvest and not at anthesis, this was not quantified.
Pruning weights measured in winter of 2006 for the Nuriootpa trial were
significantly higher for Mo treated vines than control vines (2 way ANOVA,
p=0.042) (Fig 24 c). The yield to pruning weight ratios were not significantly
different (Fig 24 d).
Wine making and sensory analysis
Fruit was picked from control blocks of vines at 112 daa and at 115 daa for fruit
of molybdenum treated vines to ensure consistent ºBrix values for winemaking
(Fig 25 a &b). Fruit receival reports from Provisor indicated that the juice of each
lot of fruit showed no significant differences in ºBrix, pH and titratable acidity.
Juice ºBrix ranged from 26.2 -27, pH from 3.82-3.96 and titratable acidity from
4.2-4.7 g/L of tartaric acid. To reduce the likelihood of “stuck” ferments due to
excessive alcohol being produced by yeasts as a result of high sugar levels in the
juice, the winemaker removed 5.3% of the juice from each lot and replaced that
volume with condensate. This ensured that all lots of juice were no more than
26.1 ºBrix or 14.5 Baumé.
18-20 bottles of wine were bottled from each lot of fruit approximately four
months after fruit delivery. The bottling report indicated no significant differences
between wine made from fruit of control and molybdenum treated vines (t-test,
p>0.05), with ranges of pH (3.45-3.49), titratable acidity (6.4-6.7 g/L tartaric acid),
total sulfur dioxide (80 ppm), percent alcohol (15.1-15.4 %), and volatile acidity
(0.21-0.28 g/L acetic acid). Malic acid was 0.04-0.16 g/L several days before
bottling, below the tasting threshold, and malo-lactic acid fermentation was
complete when wine was bottled.
106
Molybdenum content in wine made from fruit from treated blocks of vines was
five times higher than that made from control blocks of vines (n=4 lots of wine, t-
test p=0.0001) (Fig 25 c). The limited spread of data for the wine made from the
molybdenum treated vines confirms an even application of sodium molybdate
across the blocks of vines.
Figure 25 Fruit from Nuriootpa trial with molybdenum treatment applied at flowering. The control vines were harvested three days earlier than the vines sprayed with molybdenum to ensure sugar content of the berries was consistent for winemaking and subsequent sensory analysis. Fruit from control vines was harvested 112 daa and treated vines on 115 daa. (a) Sugar accumulation (ºBrix) of berries was tracked during phase 3 of development (black symbols) to predict harvest dates (grey symbols), n=4 x 50 berry samples. Diamond shaped symbols indicate the ºBrix of the juice from the harvested fruit when crushed for winemaking, n=4. (b) Berry weight loss in phase 3 of development (black symbols) with data predicted (grey symbols) for post 105 daa period, n=4 x 50
berry samples. (c) Wine made from control and untreated fruit shows five fold higher molybdenum content in fruit from molybdenum treated vines, n=4 lots of wine, t-test p=0.0001.
107
Sensory analysis of the wines found the hypothesis of a difference between the
wines was rejected for all the control replicates of wine (n=43, 11-17/43 correct
responses, Z=-1.08 - 1.51, p>0.05), and was rejected for two replicates of the
molybdenum treated wine lots (n=43, 19/43 correct responses, Z=1.51, p>0.05).
The third replicate was found to have a difference (n=43, 20/43 correct
responses, Z=1.83, p=0.034).
We considered that an exploratory approach would be useful and continued to
the next phase of sensory analysis. One of the replicate wines made from control
vines was tested against one of the pair of replicate wines from treated vines that
were found to be indistinguishable. In sensory testing of these wines, the
hypothesis that there was a difference between the wine made from fruit of the
control vines versus wine from fruit of the Mo treated vines was accepted (n=96,
45/96 correct responses, Z=1.92, p=0.027).
Discussion
At both experimental sites the application of Mo to the vines changed the time
course of berry weight accumulation, regardless of the timing of the application.
In all cases, the transition from phase 2 to phase 3 of berry development was
delayed by between 2 and 7 days by Mo treatment. Allometric analysis of berry
sugar accumulation relative to the change in berry weight showed distinct
differences between phase 2 and phase 3. The allometric coefficients were
greater than 1 in phase 2, and less than 1 in phase 3, which is consistent with
other published data for Shiraz (Sadras and McCarthy 2007). The negative
slopes in phase 3, although not significantly different from zero, may indicate a
net loss in berry material, supporting the hypothesis of backflow from the berries
(Tilbrook and Tyerman 2009). With one exception, the allometric coefficients,
were lower for the Mo treated fruit compared to controls. Thus there are two
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effects of Mo treatment, a delay in the onset of phase 3 (ontogentic shift) and a
slowing in sugar accumulation relative to berry weight accumulation in phase 2.
This translates to more weight increase per unit sugar increase, presumably due
to a greater uptake of water into the berry. It is possible that the delay in the start
of phase 3 and the lower allometric coefficient could explain the visual
differences in extent of shrivel observed in fruit of Mo treated and untreated vines
at harvest in Merlot (Longbottom 2007). It is also possible that these changes
contribute to differences in yields seen as a result of Mo treatment on Merlot
vines.
A concentration of 0.1 mg/kg of Mo in dried shoot tips of plants is an indication
that sufficient Mo is available in the soil for plant growth (Marschner 1995). This
correlates with data showing that when the shoots of Merlot vines have 0.1 mg/kg
Mo at anthesis, there is optimal fruit set which increases yield and overcomes the
issue of poor fruit set that frequently occurs in that variety (Longbottom 2007).
The shoot tips of the Shiraz vines used in our study had a Mo content of 0.3-0.4
mg/kg at anthesis and no visible symptoms of Mo deficiency. The Mo content of
petioles in this Shiraz trial were similar to the range where a yield response to an
application of Mo was seen in Merlot (Williams et al. 2004). In own rooted Merlot
grown in McLaren Vale, South Australia, a linear relationship has been found
between petiolar and shoot Mo content; at anthesis, a Merlot vine with a petiole
Mo concentration of 0.1 mg Mo would be expected to have shoots with a
concentration of around 3 mg/kg of Mo (Longbottom 2007). In these experiments
on Shiraz, the relationship between petioles and shoots is ten-fold smaller but
this is not indicative of any trend as data from a variety of plants shows that the
location and concentration of Mo in plant organs varies between plant species
and between varieties within species (Marschner 1995).Soil analysis from a site
approximately 150 m from the trial vines indicated a soil pH ranging from 7.9-9.2
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over a depth of 0-1.6 m (DWLBC 2007), which suggests that sufficient soil Mo
was available for vine growth at the Nurioootpa site.
At berry softening, the ABA content per berry increased markedly as expected
(Davies et al. 1997) and was maintained, with high variation, until about 90 daa
after which levels returned to pre-veraison levels. When ABA per berry was
allometrically examined relative to berry weight, phase2 and 3 of berry
development show a clear difference and a change in coefficients but there were
no significant differences between control and treated berries. In phase 2, the
allometric coefficient was around zero as the log of ABA per berry did not
increase relative to the log of berry weight. From maximum berry weight and the
commencement of phase 3 there was a sharp reduction in ABA content per
berry. More data points would be useful, however this data indicates that ABA
may be associated with the transition between phase 2 and phase 3.
ABA content per berry had no clear peak as berry weight increased in phase 2.
This may be a reflection of the high variability in the data. Data plotted as ABA
per gram fresh berry weight followed essentially the same pattern as Fig 23 a
(data not shown). Another issue to consider is varietal difference in ABA
accumulation in berries through development. In cv Doradillo, Coombe and Hale
(1973) found that ABA per berry peaked at 14 days after veraison, then dropped
sharply by 21 days after veraison as berries approached maximum weight, No
replicate measurements were presented. Davies et al. (1997) presented data
showing ABA per gram in Cabernet Sauvignon berry tissue peaked at 84 daa as
weight accumulation slowed, and ABA content in tissue fell in what appeared to
be phase 3 of berry development. Again no replicate measurements were
presented to indicate what degree of variability was present during development.
Also in Cabernet Sauvignon, free ABA in berries increased from veraison and
peaked around 70 daa (Susan Wheeler, pers. com.). In cvs Cavus, Italia and
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Perlette berries, Goktürk Baydar and Harmankaya (2005) found varietal
differences with ABA content in Perlette berry tissue peaking at veraison,
maintained during phase 2, then falling in phase 3 of development, similar to the
Shiraz data in this work. ABA accumulation in Cavus and Italia berries appeared
to peak during phase 2 of development. In all the mentioned studies and this
paper, berry ABA falls back to pre-veraison levels in phase 3. The variability in
the ABA content in Shiraz berries during phase 2 makes it difficult to interpret the
data except to say that it is not inconsistent, but in phases 1 and 3 it is consistent
with other published work.
If all the fruit had been harvested on the same day it is likely that the Mo treated
vines would have had significantly higher yield than that of the control vines. It is
estimated that the fruit from the Mo treated vines lost 5% in weight in the three
days between harvest dates. The weight loss at this point in Shiraz berry
development is probably due to water loss via xylem backflow into the vine
(Tilbrook and Tyerman 2009).
The pruning weight of canes from the Mo treated vines was significantly higher
than that of the control vines. This suggests that the changes seen in berry
development might be related to increased vigour of the vine, potentially caused
by greater capacity to reduce nitrate for assimilation. The molybdoenzyme nitrate
reductase may be limited by the availability of Mo that can be incorporated into it,
especially when Mo concentrations are low (Srivastava 1997). This may have
altered the relative strengths of the source (leaves) and sinks (fruit) of the vine as
the photo-assimilates were partitioned. The increase in Shiraz pruning weights is
different to what was observed in Merlot where vines sprayed with the same
concentration of Mo before anthesis when shoots were 10 cm in length. The
Merlot had a reduction in pruning weights over two seasons at sites in the
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Adelaide Hills and McLaren Vale and no differences in vine vigour were recorded
at veraison (Longbottom 2007).
When the fruit was crushed for winemaking, the basic juice parameters of ˚Brix,
titratable acidity and pH were not significantly different for all the harvested lots of
fruit from treated and untreated vines. Application of metallic elements such as
Mo, chromium and tungsten to grape vines have been implicated in increased
berry sugars (Fernández Pereira 1988) but this was not evident in this study.
The ˚Brix of the crush juice of untreated and Mo treated lots of fruit was lower
than that obtained by the vineyard experimental method. This suggests that the
sampling method, while producing consistent and reliable data, could be modified
to better account for bunches that are shaded within the canopy and likely to
have lower ˚Brix.
The Mo content of the lots of wine from the treated vines was five times that of
the control wines, clearly indicating that Mo was carried into the wine. A reduction
in total acidity and an increase in total alcohol of wine when it has high levels of
Mo, manganese and zinc have been reported, but again this was not evident in
our data (reviewed in Fernández Pereira 1988).
Although Mo is an essential trace element needed by people as well as plants,
human deficiency is rarely documented (Turnland et al. 1995). The
recommended daily intake of Mo for adults is 45 µg/day (NHMRC 2005). It has
been found that Mo is conserved in the body when intake is low, but is turned
over more rapidly and excreted mainly via the urinary system as consumption
increases. An intake of 1,500 µg/day by men has shown no adverse symptoms or
effects over a 120 day period (Turnland et al. 1995). Wine Mo content in fifty-one
Italian red and rosé wines has been assayed in the range 10 – 330 µg/L
(Interesse et al. 1984). The 2 µg/L Mo content of the wine made from fruit of
112
control vines in this study is low in comparison, and the wine made from Mo
treated vines has a Mo content similar to the lowest of the Italian wines. It is not
likely that the Mo treatment used in these experiments would cause any
problems with human health, but wine made from Mo treated vines should be
followed over some years as the long term effects are not known. The carry over
of Mo into wine is also likely to occur when applied to Merlot to improve yields.
Sensory analysis of three lots of wine from treated and untreated vines showed
that the lots made from the control vines could not be distinguished from each
other (p > 0.05). Two lots of the wine from the Mo treated vines could not be
separated from each other, but one was found to be different (p=0.034). Despite
this, an exploratory approach was decided on, and triangle testing of one control
wine and one of the undistinguishable treated wines was conducted. This
resulted in the hypothesis that there was a difference being accepted (p=0.027).
In summary, the application of Mo to Shiraz vines at flowering and prior to berry
softening can delay the transition to phase 3 and the onset of berry weight loss. It
also decreases the relative rate of sugar accumulation to weight accumulation in
phase 2. There was an increase in pruning weights of Mo treated vines. We did
not observe a significant difference in ABA concentration in berries as a result of
Mo treatments. A significant amount of Mo applied to vines will be carried into
wine made from the fruit and this wine could be sensorially differentiated from
control wines.
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Chapter 6
General Discussion
A figure in this chapter has been published in peer reviewed Proceedings
and a modified version is presented:
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 job-optimal ripening of Cabernet Sauvignon and Shiraz
pp 4-12 Openbook Australia.
114
Summary of findings
Interesting findings emerged from each research strand and provided significant
new information about similarities and differences between the wine grape
varieties Shiraz and Chardonnay and the table grape Thompson Seedless. The
principle findings are summarised.
Varietal differences in berry xylem hydraulics
• Highly negative pressures are generated in the berry pedicel xylem in
pre-veraison Shiraz, Chardonnay and Thompson Seedless grapes.
• Pedicel xylem pressures increase to around 0 MPa around veraison
and are maintained until harvest in the wine grape varieties despite
berry contents reaching an osmotic potential of -3 to -4 MPa.
• In Thompson Seedless berries, highly negative pedicel xylem
pressures are maintained while the berry contents have an osmotic
potential of -3 to -4 MPa.
• The differences in xylem pressures during development of the wine
varieties and the table variety are significant. In Shiraz and
Chardonnay, it suggests that the cell membranes of the cells
surrounding the xylem have a reflection coefficient close to 1 pre-
veraison, decreasing to about 0.1-0.2 at veraison and reducing again
to around 0 at harvest. In contrast, the Thompson Seedless cell
membranes appear to maintain a high reflection coefficient throughout
berry development.
• Hydraulic conductance via the xylem into Shiraz and Chardonnay
berries reduces gradually from veraison or berry softening until
harvest. It does not cease suddenly at veraison. Hydraulic
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conductance into Thompson Seedless berries remains high
throughout development.
• Conductance into Shiraz berry pedicel xylem is 2 to 5 fold greater than
into Chardonnay throughout berry development.
• Late in berry development, Shiraz do not hydraulically isolate from the
vine as do Chardonnay.
o Hydraulic conductance for water flow in to Chardonnay berries
was 14% of the conductance for water flow in to Shiraz
berries.
o Hydraulic conductance for water flow out of Chardonnay
berries was 4% of the conductance for water flow out of
Shiraz.
• Xylem function dye studies confirmed that hydraulic backflow to the
vine is likely in Shiraz but not Chardonnay.
• Calculations based on hydraulic conductance out of the berry xylem
and the vine stem water potential estimate that outflow via pedicel
xylem could be 7% of berry weight per day, which is consistent with
the berry weight loss measured in Shiraz berries from 90-100 days
after anthesis.
Varietal differences in berry cell vitality and cell membrane
competence
• The mesocarp and endocarp tissue of berries of wine varieties Shiraz
and Chardonnay maintained vital cells past veraison. Cells lost
membrane competence at or near the time maximum berry weight
was reached and cell death commenced around 100 days after
anthesis. Little or no loss of membrane competence or cell death
occurred in Thompson Seedless berries. The data is consistent with
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the membrane reflection coefficients indicated by xylem hydraulic
measurements.
• Cell death across the mesocarp and endocarp tissue corresponded
with the beginning of weight loss in Shiraz but not Chardonnay
berries. It also corresponded with a rate change or slowing in solute
accumulation.
• Relative cell vitality continuously declined in Chardonnay and Shiraz
from at or near berry maximum weight until harvest, reaching between
25 and 40 % loss in vitality respectively. Thompson Seedless berries
maintained 100% vitality.
• To prevent backflow to the vine, berry xylem hydraulic conductance
should be restricted. This occurs in Chardonnay but not Shiraz.
Thompson Seedless berries prevent backflow by maintaining living
tissue across the mesocarp and endocarp which have competent cell
membranes. Foliar molybdenum application alters the kinetics of berry
weight accumulation late in development
Molybdenum effect on berry development
• 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.
• Berry sugar accumulation was slowed relative to berry weight
accumulation in phase 2.
• Allometric analysis of ABA content of berries relative to weight
accumulation showed no significant differences as a result of
molybdenum treatment, suggesting molybdenum availability was not a
limiting factor during development.
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• Pruning weights from molybdenum treated vines were significantly
higher than control vines. This indicates increased vigour which
probably implicates improved nitrate assimilation.
• Molybdenum applied to vines at anthesis is reflected in the
molybdenum content of wine made from the fruit.
• Sensory analysis suggests that increased molybdenum content in the
wine is likely to affect the organoleptic qualities of the wine.
Conclusions
The viticultural experiments with molybdenum did not prevent Shiraz berry weight
loss late in development, but did alter the kinetics of berry sugar and weight
accumulation. The time course of berry development was shifted as a result of
molybdenum application. Also, molybdenum was carried into the wine and is
likely to affect organoleptic qualities.
Xylem hydraulic inflow into Shiraz and Chardonnay berries does not cease at
veraison, instead, it tapers down gradually. In Chardonnay, it continues to
reduce until it is close to zero at harvest. In Shiraz, hydraulic inflow stays
constant from at or around berry maximum weight until harvest maturity.
Hydraulic conductance out of Shiraz pedicel xylem remains relatively high
compared to Chardonnay. Together with the qualitative evidence of the dye
studies, this shows that Shiraz berries do not isolate as effectively from the vine
as Chardonnay, and backflow is likely which results in berry weight loss.
At the end of phase 2 of berry development, there is a relatively sudden and
significant loss of cell membrane competence across the pericarp of Shiraz and
Chardonnay berries. It indicates the commencement of a third phase of
development where programmed cell death or senescence occurs in the berry
tissue, which continues until harvest. At the same time, Thompson Seedless
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berries show no loss of cell vitality and maintain highly negative xylem pressures
at 105 daa, an indication of perfect, functioning cell membranes. The crisp,
turgidity of this variety of table grape late in development is indicative of living
cells and an osmotic gradient maintained across competent cell membranes. The
continuing negative pressures measured in berry xylem and the maintenance of
cell vitality support this.
As cell death occurs in Chardonnay, there is a concomitant reduction in xylem
hydraulic connection with the vine, preventing backflow. Berry weight is
maintained with a balance between phloem hydraulic inflow and transpiration. In
Shiraz, a xylem hydraulic connection with the vine continues in phase 3 of berry
development as cell vitality and osmotic competence is lost, allowing backflow
and berry weight loss to occur. A summary of these events is shown in the
context of berry weight accumulation, transpiration, phloem inflow and cell turgor
(Fig 26).
119
Figure 26 Berry size through the phases of development in wine grapes is shown relative to changes in xylem (Tilbrook and Tyerman 2006) and phloem function (Greenspan et al. 1994), transpiration (Rogiers et al. 2004), cell turgor (Thomas et al. 2006), apoplast tension (negative
pedicel xylem pressure), and cell viability (Tilbrook and Tyerman 2008) in the mesocarp and exocarp. Berry softening is indicated by the vertical line at 60 daa and is the transition point for phase 1 to phase 2 of berry development. Phase 3 reflects the berry weight loss in Shiraz (Sadras and McCarthy, 2007). Note that accumulation of aroma and flavour compounds commences from around maximum berry weight (Coombe and McCarthy 1997) when cell death has commenced in Shiraz and Chardonnay. Diagram redrawn from Coombe and Iland (2004).
Future research
Little is known about how molybdenum application changes the quality of wine
made from fruit of treated vines. This needs to be investigated further in Shiraz
and Merlot, as molybdenum is commonly applied to Merlot vines to improve fruit
set at anthesis.
The 25-40% cell death of the wine grape berry endocarp and mesocarp has
interesting implications for flavour development in berries for winemaking. The
120
more structured or organized cell death in the premium Shiraz fruit is tantalizing.
Does cell death have a role in flavour development in berries that carries into
wine? Is it connected with the formation or release of flavour or aroma
compounds in the berries, perhaps making their extraction easier in winemaking?
It is a novel view of berry development that has not been considered in the
literature and should be investigated further.
The shift from symplastic to apoplastic unloading of sugars at veraison may have
varietal differences. It is clearly separated in time from the loss of membrane
competence that commences around maximum berry weight in the wine
varieties, and the work of Zhang et al. (2006) and Wada et al. (2008) on table
and wine grapes imply that varietal differences could be quantified.
The loss of osmotically competent cells in Shiraz and Chardonnay berries means
that hydraulic conductance must be reduced to prevent pre- harvest weight loss
due to hydraulic backflow to the vine. The obvious question is: what is the
mechanism that prevents backflow in Chardonnay that is not in action in Shiraz?
In Thompson Seedless it is the maintenance of osmotically competent plasma
membranes of cells across the endocarp and mesocarp throughout berry
development. This has the potential problem of berries splitting at high water
potentials, something that is seen in this variety with rain events in vineyards
close to harvest maturity. In Shiraz and Chardonnay, despite significant cell
death, it is noticeable that cell vitality is maintained around the central and
peripheral vasculature of berries late in development. This implies that there is
the potential for control of water movement into and out of the xylem at the later
stages. It could be that the cells adjacent to the xylem vessels act as a barrier
with the apoplast, perhaps with variability in hydraulic conductance controlled by
aquaporins. This is possible as the dye studies indicate that the berry xylem is
121
functional. Is there a mistiming in the structures or mechanisms that reduce
hydraulic conductance in Chardonnay berries compared to Shiraz?
The research presented provides quantitative evidence that in addition to
transpiration, weight loss late in Shiraz berry development is caused by backflow
from the berries as they do not effectively hydraulically isolate from the vine.
While characterising the Shiraz berry weight loss, a significant body of new
information about varietal differences in grape berry development has emerged.
Acknowledgements
Chapter 3
Thankyou to Professor Ken Shackel and Dr Mark Krasnow for sharing the FDA
method developed in Professor Shackel’s laboratory at Davis, University of
California.
Chapter 5
Thankyou to Dr Mike McCarthy and SARDI Nuriootpa Research Centre for the
use of experimental vines for the 2005-2006 trials.
Thankyou to Wendy Sullivan (University of Adelaide), Treva Hebberman and
Tony Gerlach (SARDI) for assistance with fruit harvesting and pruning.
Thankyou to Professor Brian Loveys and Sue Maffei (Plant Industry, Adelaide,
CSIRO) for assistance with ABA analysis.
122
123
References
Adams DO (2006) Phenolics and ripening in grape berries. American Journal of
Enology and Viticulture 57, 249-256.
Bernas T, Zarebski M, Cook RR, Dobrucki JW (2004) Minimising photobleaching during confocal microscopy of fluorescent probes bound to chromatin:
role of anoxia and photon flux. Journal of Microscopy 215, 281-296.
Bi J (2006) Standard discrimination tests. In Sensory discrimination tests and measurements. Statistical principles, procedures and tables, pp 6-20.
Blackwell Publishing, Iowa, USA.
Blanke MM, Leyhe A (1987) Stomatal activity of the grape berry cv. Riesling,
Muller-Thurgau and Ehrenfelser. Journal of Plant Physiology 127, 451-460.
Bondada BR, Matthews MA, Shackel KA (2005) Functional xylem in the post-
veraison grape berry. Journal of Experimental Botany 56: 2949-2957.
Brummell DA (2006) Cell wall disassembly in ripening fruit. Functional Plant
Biology 33, 103-119.
Brummell DA, Dal Cin V, Crisosto C, Labavitch J (2004) Cell wall metabolism
during maturation, ripening and senescence of peach fruit. Journal of Experimental Botany 55, 2029-2039.
Chatelet D, Rost T, Matthews M, Shackel K (2008a) The peripheral xylem of
grapevine (Vitis vinifera) berries. 2. Anatomy and development. Journal of Experimental Botany 59, 1997-2007.
Chatelet D, Rost T, Shackel K,Matthews M (2008b) The peripheral xylem of
grapevine (Vitis vinifera). 1. Structural integrity in post-veraison berries. Journal of Experimental Botany 59, 1987-1996.
Chen G, Hackett R, Walker D, Taylor A, Lin Z, Grierson D (2004) Identification of
a specific isoform of tomato lipoxygenase (TomloxC) involved in the
generation of fatty acid-derived flavor compounds. Plant Physiology 136, 2641-2651.
Cochard H, Bodet C, Améglio T, Cruiziat P (2000) Cryo-scanning electron
microscopy observations of vessel content during transpiration in walnut petioles. Facts or artifacts? Plant Physiology 124, 1191-1202.
Coombe, BG, Iland PG (2004) Grape berry development and winegrape quality.
In Viticulture Volume 1 – Resources pp 210-248. Eds PR Dry and BG Coombe. Winetitles, Adelaide, South Australia.
Coombe BG, McCarthy MG. (2000) Dynamics of grape berry growth and
physiology of ripening. Australian Journal of Grape and Wine Research 6,
131-135.
Coombe BG (1992) Research on development and ripening of the grape berry.
American Journal of Enology and Viticulture 43, 101-110.
124
Coombe BG, Bishop GR (1980) Development of the grape berry. II. Changes in
diameter and deformability during veraison. Australian Journal of
Agricultural Research 31, 499-509.
Coombe BG, Hale CR (1973) The hormone content of ripening grape berries and the effects of growth substance treatments. Plant Physiology 51, 629-634.
Coombe BG, McCarthy MG (1997) Identification and naming of the inception of
aroma development in ripening grape berries. Australian Journal of Grape and Wine Research 3, 18-20.
Cosa G, Focsaneanu KS, McLean JRN, McNamee JP, Scalano JC (2001)
Photophysical properties of fluorescent DNA-dyes bound to single-and double-stranded DNA in aqueous buffered solution. Photochemistry and
Photobiology 73, 585-599.
Creasy G, Price SF & Lombard PB (1993) Evidence for xylem discontinuity in
Pinot noir and Merlot grapes: Dye uptake and mineral composition during berry maturation. American Journal of Enology and Viticulture 44, 187-
192.
Davies C, Boss P, Robinson SP (1997) Treatment of grape berries, a nonclimacteric fruit with a synthetic auxin, retards ripening and alters the
expression of developmentally regulated genes. Plant Physiology 115,
1155-1161.
Davies C, Robinson SP (1996) Sugar accumulation in grape berries. Plant
Physiology 111, 275-283.
Delrot S, Picaud S, Gaudillere, J-P (2001) Water transport and aquaporins in
grapevine. In Molecular Biology & Biotechnology of the Grapevine, pp 241-262. Ed K.A. Roubelakis-Angelakis, Kluwer Academic Publishers,
Dordrecht, Netherlands.
Dreier LP, Hunter JJ, Ruffner HP (1998) Invertase activity, grape berry development and cell compartmentation. Plant Physiology and
Biochemistry 36, 865-872.
Dreier LP, Stoll GS, Ruffner HP (2000) Berry ripening and evapotranspiration in
Vitis vinifera L. American Journal of Enology and Viticulture 51, 340-346.
Düring H, Lang A, Oggioni F (1987) Patterns of water flow in Riesling berries in
relation to developmental changes in their xylem morphology. Vitis 26,
123-131.
DWLBC (2007) Regional Land Resource Information for Southern South
Australia [DVD]. Soil and Land Program. Department of Water, Land and
Biodiversity Conservation, Government of South Australia
Fernández Pereira C (1988) The importance of metallic elements in wine. A
literature survey. Zeitschrift fur Lebensmittel-Untersuchung und –
Forschung 186, 295-300.
Findlay N, Oliver KJ, Nii N, Coombe BG (1987) Solute accumulation by grape pericarp cells. Journal of Experimental Botany 38, 668-679.
Fitzpatrick K, Tyerman S, and Kaiser B (2008) Molybdate transport through the
plant sulfate transporter SHST 1. FEBS Letters 582, 1508-1513.
Goktürk Baydar N, Harmankaya N (2005) Changes in endogenous hormone
levels during the ripening of grape cultivars having different berry set
mechanisms. Turkish Journal of Agriculture and Forestry 29, 205-210.
125
Greenspan MD, Schultz HR, Matthews MA (1996) Field evaluation of water
transport in grape berries during water deficits. Physiologia Plantarum 97,
55-62.
Greenspan MD, Shackel KD, Matthews MA (1994) Developmental changes in the diurnal water budget of the grape berry exposed to water deficits.
Plant, Cell and Environment 17, 811-820.
Gupta UC (1997a) Deficient, sufficient, and toxic concentrations of molybdenum in crops. In Molybdenum in agriculture, pp 150-159. Ed. UC Gupta.
Cambridge University Press: Cambridge, U.K.
Gupta UC (1997b) Soil and plant factors affecting molybdenum uptake by plants. In Molybdenum in agriculture, pp 71-91. Ed. UC Gupta, Cambridge
University Press: Cambridge, U.K.
Hardie WJ, O'Brien TP (1988) Considerations of the biological significance of
some volatile constituents of grape (Vitis spp.) Australian Journal of Botany 36, 107-117.
Hardie WJ, O'Brien TP, Jaudzems VG (1996) Morphology, anatomy and
development of the pericarp after anthesis in grape, Vitis vinifera L. Australian Journal of Grape and Wine Research 2, 97-142.
Hrazdina G, Parsons GF, Mattick LR (1984) Physiological and biochemical
events during development and maturation of grape berries. American Journal of Enology and Viticulture, 35, 220-227.
Iland P, Gago P (1997) Australian wine from the vine to the glass. Patrick Iland
Wine Promotions, Adelaide, South Australia.
Interesse FS, Lamparelli F, Alloggio V (1984) Mineral contents of some southern Italian wines. Zeitschrift fur Lebensmittel-Untersuchung und –Forschung
178, 272-278.
Jones KH, Senft JA (1985) An improved method to determine cell viability by simultaneous staining with fluorescein diacetate-propidium iodide. Journal
of Histochemistry & Cytochemistry 33, 77-79.
Jones ML (2004) Changes in gene expression during senescence. In Plant cell
death processes, pp 51-71. Ed. L Noodén. Elsevier Science, California, USA.
Kaiser B, Gridley K, Ngaire Brady J, Phillips T, Tyerman S (2005) The role of
molybdenum in agricultural plant production. Annals of Botany 96, 745-754.
Katsuhara M, Hanba Y, Shiratake K, Maeshima M (2008) Expanding roles of
plant aquaporins in plasma membranes and cell organelles. Functional Plant Biology 35, 1-14.
Keller M, Smith JP, Bondada BR (2006) Ripening grape berries remain
hydraulically connected to the shoot. Journal of Experimental Botany 57,
2577-2587.
Kral T, Widerak K, Langner M, Hof M (2005) Propidium iodide and PicoGreen as
dyes for the DNA fluorescence correlation spectroscopy measurements.
Journal of Fluorescence 15, 179-183.
Krasnow M, Matthews M, Shackel K (2008) Evidence for substantial
maintenance of membrane integrity and cell viability in normally
developing grape (Vitis vinifera L.) berries throughout development. Journal of Experimental Botany 59, 849-859.
126
Lang A, Düring H (1991) Partitioning control by water potential gradient: evidence
for compartmentation breakdown in grape berries. Journal of
Experimental Botany 42, 1117-1122.
Lang A, Thorpe MR (1989) Xylem, phloem and transpiration flows in a grape: application of a technique for measuring the volume of attached fruits to
high resolution using Archimedes' Principle. Journal of Experimental
Botany 219, 1069-1078.
Lang A, Thorpe MR (1986) Water potential, translocation and assimilate
partitioning. Journal of Experimental Botany 37, 495-503.
Lang A, Thorpe MR (1989) Xylem, phloem and transpiration flows in a grape: application of a technique for measuring the volume of attached fruits to
high resolution using Archimedes' Principle. Journal of Experimental
Botany 219, 1069-1078.
Lawless H, Heymann H (1999) Sensory evaluation of food. Principles and practices. Aspen Publishers, Maryland USA.
Longbottom ML (2007) The reproductive biology of grapevines - factors that
affect flowering and fruitset. PhD Thesis, University of Adelaide.
Loureiro J, Rodriguez E, Dolezel J, Santos C (2006) Flow cytometric and
microscopic analysis of the effect of tannic acid on plant nuclei and
estimation of DNA content. Annals of Botany 98, 515-527.
Marschner H (1995) Mineral nutrition of higher plants. Academic Press, London.
McCarthy MG (1997) The effect of transient water deficit on berry development of
cv. Shiraz (Vitis vinifera L.). Australian Journal of Grape and Wine
Research 3, 102-108.
McCarthy MG (1999) Weight loss from ripening berries of Shiraz grapevines
(Vitis vinifera L. cv Shiraz). Australian Journal of Grape and Wine
Research 5, 10-16.
McCarthy MG (2000) Developmental variation in sensitivity of Vitis vinifera L.
(Shiraz) berries to soil water deficit. Australian Journal of Grape and Wine
Research 6, 136-140.
McCarthy MG, Coombe BG (1999) Is weight loss in ripening grape berries cv. Shiraz caused by impeded phloem transport? Australian Journal of Grape
and Wine Research 5, 17-21.
Mendel R, Hänsch R (2002) Molybdoenzymes and molybdenum cofactor in plants. Journal of Experimental Botany 375, 1689-1698.
NHMRC (2005) Nutrient reference values for Australia and New Zealand
including recommended dietary intakes. National Health and Medical Research Council, Department of Health and Aging, Australia and
Ministry of Health, New Zealand.
Noirot M, Barre P, Duperray C, Louarn J, Hamon S (2003) Effects of caffeine and
chlorogenic acid on propidium iodide accessibility to DNA: consequences on genome size evaluation in coffee tree. Annals of Botany 92, 259-264.
Noodén L (2004) Plant cell death processes. Academic Press, U.S.A.
Nunan KJ, Sims IM, Bacic A, Robinson SP, Fincher GB (1998) Changes in cell wall composition during ripening of grape berries. Plant Physiology 118,
783-792.
127
Ojeda H, Deloire A, Carbonneau A, Ageorges A, Romieu C (1999) Berry
development of grapevines: relations between the growth of berries and
their DNA content indicate cell multiplication and enlargement. Vitis 38,
145-150.
Oparka KJ, Read ND (1994) The use of fluorescent probes for studies of living
plant cells. In, Plant cell biology, a practical approach. pp 27-50. Eds. N
Harris, KJ Oparka. Oxford University Press, United States.
Paliyath G, Droillard MJ (1992) The mechanisms of membrane deterioration and
disassembly during senescence. Plant Physiology and Biochemistry 30,
789-812.
Patrick JW (1997) Phloem unloading: sieve element unloading and post-sieve
element transport. Annual Review of Plant Physiology and Plant
Molecular Biology 48, 191-222.
Picaud S, Becq F, Dédaldéchamp F (2003) Cloning and expression of two plasma membrane aquaporins expressed during the ripening of grape
berry. Functional Plant Biology 30, 621-630.
Pratt C (1971) Reproductive anatomy in cultivated grapes-a review. American Journal of Enology and Viticulture 23, 93-109.
Price HJ, Hodnett G, Johnston JS (2000) Sunflower (Helianthus annus) leaves
contain compounds that reduce nuclear propidium iodide fluorescence. Annals of Botany 86, 929-934.
Robinson B (2000) Another look at the Merlot problem - could it be molybdenum
deficiency? Australian Grapegrower and Winemaker 438a, 21-23.
Rogiers S, Greer D, Hatfield J, Orchard B, Keller M (2006) Solute transport into Shiraz berries during development and late-ripening shrinkage. American
Journal of Enology and Viticulture 57, 73-80.
Rogiers S, Hatfield J, Gunta Jaudzems V, White R, Keller M (2004) Grape berry cv. Shiraz epicuticular wax and transpiration during ripening and
preharvest weight loss. American Journal of Enology and Viticulture 55,
121-127.
Rogiers S, Keller M, Holzapfel BP, Virgona JM (2000) Accumulation of potassium and calcium by ripening berries on field vines of Vitis vinifera (L) cv.
Shiraz. Australian Journal of Grape and Wine Research 6, 240-243.
Rogiers S, Smith JA, White R, Keller M, Holzapfel BP, Virgona JM (2001) Vascular function in berries of Vitis vinifera (L) cv. Shiraz. Australian
Journal of Grape and Wine Research 7, 46-51.
Rogiers S, Mohan Kumar GN, Knowles NR (1998) Maturation and ripening of fruit of Amelanchier alnifolia Nutt. are accompanied by increasing
oxidative stress. Annals of Botany 81, 203-211.
Sadras V, McCarthy MG (2007) Quantifying the dynamics of sugar concentration
in berries of Vitis vinifera cv. Shiraz: a novel approach based on allometric analysis. Australian Journal of Grape and Wine Research 13, 66-71.
Schwarz G, Mendel R (2006) Molybdenum cofactor biosynthesis and
molybdenum enzymes. Annual Review of Plant Biology 57, 623-647.
Shackel KA, Greve C, Labavitch JM, Hahmadi H (1991) Cell turgor changes
associated with ripening in tomato pericarp tissue. Plant Physiology 97,
814-816.
128
Smart RE, Turkington CR, Evans JC (1974) Grapevine response to furrow and
trickle irrigation. American Journal of Enology and Viticulture 25, 62-66.
Soar C, Speirs J, Maffei S, Loveys B (2004) Gradients in stomatal conductance,
xylem sap ABA and bulk leaf ABA along canes of Vitis vinifera cv. Shiraz: molecular and physiological studies investigating their source. Functional
Plant Biology 31, 659-669.
Soar C, Speirs J, Maffei S, Penrose A, McCarthy M, Loveys B (2006) Grape vine varieties Shiraz and Grenache differ in their stomatal response to VPD:
apparent links with ABA physiology and gene expression in leaf tissue.
Australian Journal of Grape and Wine Research 12, 2-12.
Srivastava PC (1997) Biochemical significance of molybdenum in crop plants. In
Molybdenum in agriculture, pp 47-70. Ed. UC Gupta. Cambridge
University Press, Cambridge, U.K.
Steudle E, Murrmann M, Peterson CA (1993) Transport of water and solutes across maize roots modified by puncturing the endodermis. Plant
Physiology 103, 335-349.
Steudle E & Jeschke WD (1983) Water transport in barley roots. Measurements of root pressure and hydraulic conductivity of roots in parallel with turgor
and hydraulic conductivity of root cells. Planta 158, 237-248.
Symons G, Davies C, Shavrukov Y, Dry I, Reid J, Thomas M (2006) Grapes on Steroids. Brassinosteroids are involved in grape berry ripening. Plant
Physiology 140, 150-158.
Thomas H, Ougham HJ, Wagstaff C, Stead AD (2003) Defining senescence and
death. Journal of Experimental Botany 54, 1127-1132.
Thomas TR, Matthews MA, Shackel KA (2006) Direct in situ measurement of cell
turgor in grape (Vitis vinifera L.) berries during development and in
response to plant water deficits. Plant, Cell and Environment 29, 993-1001.
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 job-optimal 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, 173-184.
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, 541-550.
Turnland J, Keyes W, Peiffer G (1995) Molybdenum absorption, excretion, and
retention studied with stable isotopes in young men at five intakes of
dietary molybdenum. American Journal of Clinical Nutrition 62, 790-796.
Tyerman SD, Niemietz CM, Bramley H (2002) Plant aquaporins: multifunctional
water and solute channels with expanding roles. Plant Cell and
Environment 25, 173-194.
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, 170-181.
129
van Bel A (2003) The phloem, a miracle of ingenuity. Plant, Cell and Environment
26, 125-149.
Wada H, Shackel K, Matthews M (2008) Fruit ripening in Vitis vinifera: apoplastic
solute accumulation accounts for pre-veraison turgor loss in berries. Planta 227, 1351-1361.
WFA (2009) WFA vintage report. Winemakers' Federation of Australia, Kent
Town, South Australia.
Williams C, Maier N, Bartlett L (2004) Effect of molybdenum foliar sprays on
yield, berry size, seed formation, and petiolar nutrient composition of
"Merlot" grapevines. Journal of Plant Nutrition 27, 1-26.
Yu XC, Li MJ, Gao GF, Feng HZ, Geng XQ, Peng CC, Zhu SY, Wang XJ, Shen
YY, Zhang DP (2006) Abscisic acid stimulates a calcium-dependent
protein kinase in grape berry. Plant Physiology 140, 558-579.
Zhang B, Chen K, Bowen J, Allan A, Espley R, Karunairetnam S (2006)
Differential expression within the LOX gene family in ripening kiwifruit.
Journal of Experimental Botany 57, 3825-3836.
Zhang DP, Li M, Wang Y (1997) Ultrastructural changes in the mesocarp cells of
grape berry during its development. Acta Botanica Sinica 39, 389-396.
Zhang XY, Wang XL, Wang X, Xia GH, Pan QH, Fan RC, Wu FQ, Yu XC, Zhang DP (2006) A shift of phloem unloading from symplasmic to apoplasmic
pathway is involved in developmental onset of ripening in grape berry.
Plant Physiology 142, 220-232.
