The influence of viticultural treatments on the accumulation of … · 2010. 10. 4. · The influence of viticultural treatments on the accumulation of flavonoid compounds in grapes

The influence of viticultural treatments on the accumulation of flavonoid compounds in grapes and their

contribution to wine quality

Nicole Cordon

B. Biotechnology (Hons), The Flinders University of South Australia

A thesis submitted for the degree of

Doctor of Philosophy

The University of Adelaide

School of Agriculture, Food and Wine

Discipline of Wine and Horticulture

in collaboration with

CSIRO Plant Industry,

Horticulture Unit

Urrbrae, Adelaide

May 2008

I

TABLE OF CONTENTS

ABSTRACT ...................................................................................................... VII

DECLARATION ................................................................................................. X

ACKNOWLEDGEMENTS ............................................................................... XI

ABBREVIATIONS ........................................................................................ XIII

LIST OF FIGURES ......................................................................................... XV

LIST OF TABLES ........................................................................................ XVII

CHAPTER 1 General introduction 1.1 Introduction ................................................................................................................................... 1 1.2 Wine production .............................................................................................................................. 1

1.2.1 Winemaking ............................................................................................................................... 1 1.2.2 The wine industry ...................................................................................................................... 1 1.2.3 The health benefits of wine ........................................................................................................ 2

1.3 Grape development .......................................................................................................................... 2 1.4 Flavonoids ....................................................................................................................................... 3 1.5 Flavonoids in grapes & wine ........................................................................................................... 4

1.5.1 Anthocyanins ............................................................................................................................. 4 1.5.2 Flavonols.................................................................................................................................... 5 1.5.3 Tannins ...................................................................................................................................... 5

1.6 Biosynthesis of flavonoids .............................................................................................................. 7 1.6.1 Anthocyanins ............................................................................................................................. 7 1.6.2 Flavonols.................................................................................................................................... 8 1.6.3 Tannins ...................................................................................................................................... 9

1.7 Transcriptional regulation of flavonoids ....................................................................................... 10 1.7.1 Activation by transcription factors .......................................................................................... 10

1.8 Factors affecting the flavonoid composition of grapes ................................................................. 11 1.8.1 Light exposure ......................................................................................................................... 12 1.8.2 Temperature ............................................................................................................................. 13

1.9 The definition of grape & wine quality ......................................................................................... 14 2.0 Summary & project aims ............................................................................................................ 14

CHAPTER 2 General methods 2.1 Sample processing & preparation .............................................................................................. 17

2.1.1 Grape samples .......................................................................................................................... 17 2.1.2 Wine samples ........................................................................................................................... 17

II

2.2 Analytical methods ...................................................................................................................... 18 2.2.1 Determination of anthocyanin & flavonol content .................................................................. 18

2.2.1.1 Extraction: grape skins & wine .......................................................................................... 18 2.2.1.2 High Performance Liquid Chromatography (HPLC) analysis ........................................... 18 2.2.1.3 Liquid Chromatography-Mass Spectrometry (LC-MS) analysis ....................................... 21 2.2.1.4 UV-VIS Spectrophotometric analysis ................................................................................ 23

2.2.2 Determination of tannin content ............................................................................................. 23 2.2.2.1 HPLC analysis of tannin composition following acid-catalysis in the presence of excess

phloroglucinol (i.e. Phloroglucinol Analysis (PGA)) ........................................................ 23 2.2.2.1.1 Extraction .................................................................................................................... 23 2.2.2.1.2 Acid-cleavage in the presence of phloroglucinol ........................................................ 24 2.2.2.1.3 HPLC separation ......................................................................................................... 24

2.2.2.2 Total tannin determination following protein precipitation & spectrophotometric detection (i.e. Protein Precipitation Analysis (PPA)) ....................................................... 25

2.2.2.2.1 Extraction .................................................................................................................... 25 2.2.2.2.2 Protein precipitation & spectrophotometric detection ................................................ 25

2.3 Detailed molecular methods ........................................................................................................ 26 2.3.1 Primer design ........................................................................................................................... 26 2.3.2 Polymerase Chain Reaction (PCR) .......................................................................................... 26 2.3.3 Agarose gel electrophoresis of DNA ....................................................................................... 26 2.3.4 Purification & quantifying DNA ............................................................................................. 27 2.3.5 DNA ligation ............................................................................................................................ 27 2.3.6 Transformation of bacteria & overnight cultures..................................................................... 27 2.3.7 Restriction enzyme (RE) digestion of DNA ............................................................................ 27 2.3.8 Sequencing & sequence analysis ............................................................................................. 28 2.3.9 Preparation of total grape RNA ............................................................................................... 28 2.3.10 First-strand cDNA synthesis .................................................................................................. 29 2.3.11 Real Time PCR (RT-PCR) analysis of gene expression ........................................................ 29

CHAPTER 3 The flavonoid composition of Shiraz grapes in a warm & cool

climate region & their role in grape & wine quality 3.1 Introduction .................................................................................................................................. 31

3.1.1 Background information .......................................................................................................... 33 3.1.1.1 Regional descriptions ......................................................................................................... 33

3.1.1.1.1 Riverland ...................................................................................................................... 33 3.1.1.1.2 McLaren Vale ............................................................................................................... 34

3.1.1.2 Differences in grape sampling ........................................................................................... 35 3.2 Methods ........................................................................................................................................ 38

3.2.1 Sample selection, collection & processing .............................................................................. 38 3.2.2 Sample measurements .............................................................................................................. 38 3.2.3 Data details .............................................................................................................................. 39 3.2.4 Statistical analysis .................................................................................................................... 40

3.3 Results ........................................................................................................................................... 41 3.3.1 Grape & wine sampling ........................................................................................................... 41 3.3.2 Analysis of tannins ................................................................................................................... 43

3.3.3.1 Comparisons of extraction solvents in the PPA ................................................................. 43 3.3.3.2 Comparison of PPA & PGA .............................................................................................. 44

III

3.3.3 Grape & wine phenolic measurements .................................................................................... 45 3.3.4 Grape berry composition in both regions & seasons ............................................................... 48 3.3.5 Relationships with berry weight .............................................................................................. 51 3.3.6 Relationships between the flavonoid compounds (anthocyanins, flavonols & tannins) in

grapes ...................................................................................................................................... 51 3.3.7 Relationships between the flavonoids (anthocyanins & flavonols) in wine ............................ 55 3.3.8 Indicators of grape quality ....................................................................................................... 56 3.3.9 Indicators of wine quality ........................................................................................................ 61 3.3.10 Miscellaneous relationships ................................................................................................... 67

3.4 Discussion ..................................................................................................................................... 68 3.4.1 The flavonoid content & composition of grapes from a range of warm & cool climate

vineyards .................................................................................................................................... 69 3.4.1.1 Grape berry characteristics ................................................................................................ 70 3.4.1.2 Anthocyanin content & composition ................................................................................. 71 3.4.1.3 Flavonol content & composition ........................................................................................ 73 3.4.1.4 Tannin content ................................................................................................................... 73 3.4.1.5 Summary ............................................................................................................................ 74

3.4.2 The relationship between the flavonoid compounds in grapes ................................................ 75 3.4.2.1 Anthocyanins & flavonols ................................................................................................. 75 3.4.2.2 Anthocyanins & tannins ..................................................................................................... 76 3.4.2.3 Regulation of the flavonoid biosynthetic pathway ............................................................ 78

3.4.2.3.1 Transcriptional regulation ............................................................................................ 78 3.4.2.3.2 Post-transcriptional regulation ..................................................................................... 79

3.4.2.3.2.1. Enzymatic regulation ............................................................................................ 79 3.4.2.3.2.2. Organisation in the cell ......................................................................................... 80

3.4.2.4 Summary ............................................................................................................................ 82 3.4.3 Indicators of grape & wine quality .......................................................................................... 83

3.4.3.1 Berry weight ...................................................................................................................... 83 3.4.3.2 Juice composition (oBrix) .................................................................................................. 85 3.4.3.3 Grape flavonoid content & composition (anthocyanins, flavonols & tannins) ................. 86 3.4.3.4 Vineyard Characteristics (vine age, yield & canopy measurements) ................................ 89 3.4.3.5 Wine anthocyanins & wine grade ...................................................................................... 91 3.4.3.6 Determining grape & wine quality: a complex subject ..................................................... 94 3.4.3.7 Social, financial & future impacts on the Australian wine industry .................................. 97 3.4.3.8 Summary ............................................................................................................................ 98

3.5 Conclusion .................................................................................................................................... 99

CHAPTER 4 The influence of bunch light exposure on flavonol synthesis in

Shiraz & Chardonnay grapes during berry development 4.1 Introduction ............................................................................................................................... 101 4.2 Methods ...................................................................................................................................... 103

4.2.1 Experimental approach .......................................................................................................... 103 4.2.1.1 The influence of bunch light exposure on VvFLS1 expression & flavonol accumulation103

4.2.1.1.1 Plant material ............................................................................................................. 103 4.2.1.1.2 Shading treatments ..................................................................................................... 103 4.2.1.1.3 Sampling & analysis of fruit ...................................................................................... 104

4.2.1.1.3.1 RT-PCR expression analysis of the flavonoid pathway genes ............................. 105

IV

4.2.1.1.3.2 HPLC analysis of anthocyanin & flavonol composition & concentration ........... 105 4.2.1.2 Experiment 1 Chardonnay bunch light exposure, pre-veraison (2002-2003)

(Preliminary experiment) ................................................................................................. 106 4.2.1.3 Experiment 2 Shiraz & Chardonnay bunch light exposure during berry development

(2003-2004) ..................................................................................................................... 107 4.2.1.4 Experiment 3 Hourly light induction of Shiraz berries, pre-veraison (2004-2005) ......... 108 4.2.1.5 The diurnal pattern of VvFLS1 gene expression in Shiraz bunches at different stages

during development ......................................................................................................... 108 4.3 Results ......................................................................................................................................... 110

4.3.1 Experiment 1- The influence of bunch light exposure on flavonol synthesis in pre-veraison Chardonnay berries (Preliminary experiment) ...................................................................... 110

4.3.2 Experiment 2- The influence of bunch light exposure on flavonol synthesis in Shiraz & Chardonnay bunches during berry development ................................................................... 111

4.3.2.1 Pre-flowering sampling .................................................................................................... 111 4.3.2.2 Pre-veraison sampling ...................................................................................................... 116 4.3.2.3 Post-veraison sampling .................................................................................................... 116 4.3.2.4 Pre-harvest sampling ........................................................................................................ 120 4.3.2.5 The influence of bunch light exposure on expression of other flavonoid genes

(VvLDOX & VvCHI) during development ....................................................................... 124 4.3.3 Experiment 3- Hourly light induction in Shiraz berries, pre-veraison ................................... 126 4.3.4 The diurnal pattern of VvFLS1 gene expression in Shiraz fruit at different stages during

development .......................................................................................................................... 127 4.4 Discussion ................................................................................................................................... 131

4.4.1 Expression of VvFLS1 & accumulation of flavonols during grape berry development ........ 131 4.4.2 The effect of shading on berry development, VvFLS1 expression & flavonol accumulation 134 4.4.3 The effect of light induction on VvFLS1 expression & flavonol accumulation .................... 137

4.4.3.1 Summary .......................................................................................................................... 141 4.4.4 The diurnal pattern of VvFLS1, VvUFGT & VvLDOX gene expression during development142

4.4.4.1 Summary .......................................................................................................................... 144 4.5 Conclusion .................................................................................................................................. 145

CHAPTER 5 The molecular mechanism of flavonol gene regulation in grapevines 5.1 Introduction ................................................................................................................................ 147 5.2 Methods ...................................................................................................................................... 149

5.2.1 Isolating & cloning VvFLS1 promoter fragment(s) into the luciferase vector (pLUC) ......... 149 5.2.2 Chardonnay suspension cell cultures ..................................................................................... 149

5.2.2.1 Growth conditions & preparation for assays ................................................................... 149 5.2.2.2 Light induction experiments ........................................................................................... 149

5.2.3 Transient assays testing the VvFLS1 promoter(s) .................................................................. 150 5.2.4 Identifying transcriptional regulators of VvFLS1 .................................................................. 151

5.2.4.1 Database searching for AtMYB12 homologs & RT-PCR expression analysis of potential transcription factor candidate genes ............................................................... 151

5.2.4.2 Microarray analysis ......................................................................................................... 151 5.2.4.2.1 Plant material ............................................................................................................ 151 5.2.4.2.2 Data analysis ............................................................................................................ 151 5.2.4.2.3 RT-PCR expression analysis of potential transcription factor candidate genes ....... 152

5.3 Results ......................................................................................................................................... 153 5.3.1 Cloning & sequence analysis of VvFLS1 promoter fragment(s) ........................................... 153

V

5.3.2 Functional analysis of VvFLS1 promoter using a transient assay .......................................... 156 5.3.3 Light induction of Chardonnay cell cultures ......................................................................... 158 5.3.4 Identifying transcriptional regulators of VvFLS1 .................................................................. 160

5.3.4.1 Database searching for AtMYB12 homologs & RT-PCR expression of potential candidate genes ............................................................................................................... 160

5.3.4.2 Microarray analysis. ........................................................................................................ 163 5.3.4.2.1 Plant material ........................................................................................................... 163 5.3.4.2.2 Data analysis ............................................................................................................ 163 5.3.4.2.3 RT-PCR expression of potential transcription factor candidate genes .................... 166

5.4 Discussion ................................................................................................................................... 169 5.4.1 The light induction of flavonoid gene expression & accumulation in Chardonnay cell

cultures .................................................................................................................................. 169 5.4.2 Cloning & sequence analysis of VvFLS1promoter fragment(s) ............................................ 171 5.4.3 Development of a transient reporter assay for VvFLS1 promoter(s) ..................................... 174 5.4.4 Identifying transcriptional regulators of VvFLS1 & RT-PCR expression of transcription

factor candidate genes ........................................................................................................... 175 5.4.4.1 Database searching for AtMYB12 homologs in grapevine ............................................. 175 5.4.4.2 Microarray analysis for candidate transcription factors .................................................. 176

5.4.5 Transcriptional regulation of VvFLS1 ................................................................................... 178 5.4.6 Summary ................................................................................................................................ 180

5.5 Future directions ....................................................................................................................... 181 5.5.1 Isolating additional VvFLS1 promoter sequence .................................................................. 181 5.5.2 Isolating transcriptional factor candidate genes ................................................................... 182

CHAPTER 6 Conclusions & future directions 6.1 General introduction ................................................................................................................. 185 6.2 The flavonoid composition of Shiraz grapes from a warm & cool climate region & their role

in grape & wine quality .............................................................................................................. 186 6.3 The influence of bunch light exposure on flavonol synthesis in Shiraz & Chardonnay grapes

during berry development .......................................................................................................... 187 6.4 The molecular mechanisms of flavonol gene regulation in grapevines ...................................... 189 6.5 Relevance of current findings to the Australian grape & wine industry ............................. 190 6.6 Suggestions for future research ................................................................................................ 192

6.6.1 The influence of temperature, independent of light, on the flavonoid content & composition of grapes .......................................................................................................... 192

6.6.2 Post-transcriptional regulation of the flavonoid compounds in grapes ................................. 193 6.6.3 Improve vineyard sampling techniques & methods used to analyse grape composition ...... 194 6.6.4 Better understanding of the transfer of flavonoid compounds from fruit to wine during

fermentation ......................................................................................................................... 196 6.6.5 Closer examination of the activity of the VvF3’H & VvF3’5’H genes in relation to

flavonoid composition & in response to different environmental cues ............................... 197 6.6.6 The influence of light quantity & quality on flavonol biosynthesis ...................................... 197 6.6.7 Isolation of the transcriptional regulator of flavonol biosynthesis ........................................ 198

6.7 Summary .................................................................................................................................... 200 REFERENCES ............................................................................................................................ 201

VI

APPENDICES APPENDIX 2A Buffers, solutions, reagents & media .................................................................... 237

APPENDIX 2B HPLC chromatogram ............................................................................................. 239

APPENDIX 2C Table of primers ..................................................................................................... 242

APPENDIX 3A South Australian grape growing regions: The Riverland & McLaren Vale ......... 243

APPENDIX 3B Supplementary results for Chapter 3 (Flavonoid composition of Shiraz grapes).

McLaren Vale & Riverland sample details and data sets (On CD) ....................... 245

APPENDIX 4A Maps of experimental plots for Chapter 4 (Light induction of flavonol

biosynthesis) ........................................................................................................... 256

APPENDIX 4B Supplementary results for Chapter 4 (Light induction of flavonol biosynthesis).

The influence of bunch light exposure on expression of VvLDOX & VvCHI in

Shiraz & Chardonnay berries during development ................................................ 258

APPENDIX 5A Supplementary results for Chapter 5 (Molecular mechanisms of flavonol

biosynthesis). Microarray data (On CD) .............................................................. 263

APPENDIX 5B Supplementary results for Chapter 5 (Molecular mechanisms of flavonol

biosynthesis). Testing potential transcription factor candidate genes by RT-PCR

expression analysis ................................................................................................. 264

APPENDIX 5C Supplementary results for Chapter 5 (Molecular mechanisms of flavonol

biosynthesis). Nucleotide sequence alignment of grapevine VvFLS1 genomic

sequences (On CD) ................................................................................................ 267

FILES ON CD APPENDIX 3B (MV03, MV04, RL03, RL04). McLaren Vale & Riverland data sets. Provided as

Microsoft Excel documents.

APPENDIX 5A Microarray data. Provided as a Microsoft Excel document.

APPENDIX 5C FLS nucleotide alignment. Provided as a Microsoft Word document.

VII

ABSTRACT

The grape flavonoids include anthocyanins, tannins and flavonols, all of which contribute to grape

and wine quality by influencing the colour and mouthfeel of red wine. These compounds are

synthesized in different parts of the berry and during different stages of berry development. In

addition, environmental and viticultural factors such as light exposure can also alter the flavonoid

composition of grapes. An understanding of how synthesis of these compounds is coordinated, their

relationship to wine quality and the influence of bunch light exposure on the flavonoid composition

of grapes, could be used to improve fruit quality by enhanced viticultural management.

The first part of this study sought to investigate the relationship between the different products of

the flavonoid biosynthetic pathway (anthocyanins, flavonols and tannins), from two climatic regions

(warm and cool) and determine their role in grape and wine quality. In collaboration with a major

winery, whole Shiraz grapes were sampled at the weighbridge from a range of different vineyards

from two climatic regions; warm (Riverland) and cool (McLaren Vale) in 2003 and 2004. A total of

80 grape samples were collected in each season and processed (i.e. 100 berries, separated into skin,

seeds and juice, weighed and frozen). Anthocyanins and flavonols were measured, in triplicate, in

skins by HPLC. Tannins were determined in the skins and seeds by two methods; phloroglucinol

hydrolysis (HPLC) and protein precipitation (UV-VIS spectrophotometer). A comprehensive

comparison of the two methods is discussed.

In both years, the grapes from warm and cool climates formed two distinct data sets based on

flavonoid composition. There was a correlation between anthocyanins and flavonols for both the

warm and cool climate samples in both years, however those from the warm region had lower

anthocyanin for a given level of flavonol. As expected, the level of tannin in the seeds was greater

than in skin for all samples. In both years, there was a weak correlation between anthocyanin levels

in the skin and skin tannins, but no relationship with seed tannins. These results suggest there is

some co-ordination in the synthesis of anthocyanins, flavonols and skin tannins. Also, the two

regions clearly separated based on yield and despite the weak correlations in both regions, the levels

of total anthocyanins were inversely related to yield. In addition, there was no relationship with any

of the flavonoids and grape quality, indicating the need for improvement in streaming fruit for

quality using these flavonoid compounds.

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The second part of the study was to investigate the effect of bunch light exposure on flavonol

synthesis and accumulation in Shiraz and Chardonnay grapes during development. Light-excluding

boxes were applied to bunches at budburst. Boxes were removed at four sampling times; flowering,

pre-veraison, veraison and harvest. At each sampling time, berry skins were sampled when the

boxes were removed and then every second day (light induced), along with exposed controls for one

week. Flavonol accumulation and flavonol synthase (VvFLS1) gene expression was determined by

HPLC and Real Time-PCR (RT-PCR) respectively.

As expected, for all four sampling times, flavonol accumulation and VvFLS1 expression in the

boxed fruit was significantly less than bunches exposed to light. On removal of boxes at flowering,

pre-veraison and veraison, flavonols accumulated to levels similar to that of the exposed control

fruit over a period of 4-6 days. There was a significant increase in VvFLS1 expression 2 days after

exposure to light in parallel with the accumulation of flavonols. At harvest, in Chardonnay, VvFLS1

expression peaked by day 4, while in Shiraz VvFLS1 expression increased linearly and was highest

at day 6. In contrast to the results for the earlier sampling times, the total amount of flavonols

accumulated at harvest was less than 50% of exposed controls in Chardonnay and Shiraz grapes.

These results show that flavonols are able to be induced by bunch light exposure at different times

during berry development, including times when flavonols are not normally being synthesised. This

suggests bunch light exposure can override the developmental control of flavonol accumulation.

To further investigate the light induced expression of VvFLS1 in grapevines the molecular

mechanism of transcriptional control was explored. Using genomic walking PCR techniques, two

Shiraz VvFLS1 promoter sequences were cloned and their sequences were analysed. These

promoter sequences were ~800bp in length and were 99% identical. A putative MYB responsive

element (MRE) and several light responsive elements (LRE) were identified in the promoter region

of these genes.

To functionally test the VvFLS1 promoter(s), a transient assay was developed in Chardonnay

suspension cells. Cells were bombarded with constructs containing potential transcription factors

and the VvFLS1 promoter(s), fused to a luciferase reporter vector. After 48hrs incubation in the

dark, cells were harvested and luciferase activity measured as an indicator of VvFLS1 promoter

activity. Of the different transcription factors tested with the VvFLS1 promoter(s) the highest

luciferase activity was observed using AtMYB12 (a flavonol-specific regulator of AtFLS1 in

IX

Arabidopsis (Mehrtens et al. 2005). While this result shows activation of the VvFLS1 promoters by

AtMYB12 and the development of a transient reporter assay for testing the VvFLS1 promoter(s) a

grapevine transcription factor specific for VvFLS1 was sought.

Two techniques were employed to identify potential transcription factor regulators of the VvFLS1

promoter(s). The first involved BLAST sequence search analysis in a grapevine expression (EST)

database with AtMYB12 and the second involved using DNA microarray technology to identify

candidate transcription factors that were up-regulated in light exposed Chardonnay cell suspension

cultures. Thirteen potential transcription factors were identified and after correlative RT-PCR

analysis (with VvFLS1 expression patterns) two candidates were selected for further isolation and

characterisation. These results have made significant progress in unravelling the molecular

mechanisms of regulation of the flavonol biosynthetic, however additional experiments are required

to unravel the transcriptional control of flavonol biosynthesis.

This investigation contributes to our knowledge of flavonoid synthesis in grapes; how it is

coordinated, the relationship with wine quality, and the influence of light particularly on synthesis of

flavonols. It also explores the molecular mechanisms of VvFLS1 control, through isolation of the

VvFLS1 promoter and identification of potential transcription factors, which may regulate it. An

understanding of the synthesis of flavonoids and how they may be coordinated, particularly in

response to light, could be used to improve fruit quality by enhanced viticultural management.

Keywords: Grapevine, Vitis Vinifera L. cv. Shiraz, Chardonnay, flavonoid, anthocyanin, tannin, flavonol, phloroglucinol analysis, protein precipitation assay, quality, berry development, seeds, skin, light, temperature, shading, transcription factors, promoter

X

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 and belief,

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 available

for loan and photocopying.

SIGNED………………………………. DATE……………………………….

Nicole Cordon

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ACKNOWLEDGEMENTS

I am most grateful to the following people for their valuable contribution to the production of this

PhD thesis:

• To Dr Simon Robinson, my sincere thanks for giving me the opportunity of undertaking such a

fascinating and stimulating project, and for your guidance, support and encouragement

throughout the year.

• To everyone in the CSIRO laboratory;

The “doctors”: Dr Mandy Walker, Dr Jim Spiers, Dr Jochen Bogs, Dr Adam Takos, Dr Felix

Jaffe, Dr Chris Soar, Dr Aggie Cox

The technical staff: Debra McDavid, Karin Sefton, Liz Lee, Lorraine Caruthers, Sue Maffei,

Ange Jermaklow, Karina Swann, Lauren Hooper

The help, support, encouragement and friendships developed during the course of the PhD project

have been greatly appreciated.

• To staff and students at Adelaide University, including my co-supervisor Dr Graham Jones as

well as course coordinators, Dr Peter Dry and Dr Chris Ford.

• To those whom I have collaborated with including:

Mark Thomas & Pat Iocco (CSIRO) for microarray work.

Yogi Hayasaka, Gale Baldock, Marcus Herdenrich, Paul Smith, Daniel Cozzilino, Heather

Smyth (AWRI) for assistance with LC-MS, tannin and statistical analysis.

Alex Sas, Audrey Lim, Chris Bevin, Nicole Inwood, Catherine Anderson (HWC) for

providing grape and wine samples.

The Harvey family (Chalk Hill Wines) for allowing me to conduct experiments in their

Willunga vineyard.

Mark Downey (DPI Victoria) for enthusiastic shared discussion.

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• To mum and dad (and the rest of the family), who have always supported and encouraged me

from day one.

• Finally, I want to thank my wonderful partner Corey Hancox, whose patience, understanding

and support has kept me going through the high and lows of undertaking the project, particularly

in the last 12 months. I could not have done it without his love and support and I am humbled by

his devotion to me.

This PhD project is a joint venture of the following participants:

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ABBREVIATIONS

Units Units % Percent M Molar °C Degrees Celsius min Minute aa Amino acid nt Nucleotide bp Base pairs rpm Revolutions per minute g Gram s Second g Relative centrifugal force T Tonnes Ha Hectare U Unit hr Hour V Volt km Kilometers w/v Weight per volume L Litre v/v Volume per volume m Milli

Genes/enzymes PAL Phenylalanine ammonia lyase CHS Chalcone synthase CHI Chalcone isomerase F3H Flavanone-3ß-hydroxylase F3’H Flavonoid-3’-hydroxylase F3’5’H Flavonoid-3’5’-hydroxylase FLS Flavonol synthase DFR Dihydroflavonol-4- reductase LAR Leucoanthocyanidin reductase LDOX Leucoanthocyanidin dioxygenase UFGT UDP-glucose flavonoid –3-O-glucosyltransferase ANR Anthocyanidin reductase FGT Flavonol glucosyl-transferase UBIQ Ubuiquitin

Plant species Md Malus domestica Apple At Arabidopsis thaliana Arabidopsis Dc Daucus carota Carrot Vv Vitis vinifera Grapevine Zm Zea mays Maize Am Anthurium andraeanum Lilly Ps Pisum sativum Pea Pc Petroselinum crispum Parsley Ph Petunia hybrida Petunia Le Lycopersicon esculentum Tomato

XIV

General %CV Coefficient of Variation (%) cv. Cultivar +SEM Standard Error of the Mean AVI(s) Anthocyanic Vacuolar Inclusion(s) BLAST Basic Local Alignment Search Tool BSA Bovine Serum Albumin cDNA Complementary DNA CSIRO Commonwealth Scientific Industry Research Organisation DEA Differential Expression Analysis (Microarray analysis) DNA Deoxyribonucleic acid dNTP Deoxynucleotide triphosphate dsDNA Double-stranded DNA EC Exposed Control E-L Stage Eichhorn-Lorenz grapevine growth stages (Coombe 1995) EST Expressed Sequence Tag HLH Helix-Loop-Helix HPLC High-Performance Liquid Chromatography HWC Hardy Wine Company LC-MS Liquid Chromatography Mass Spectrometry LI Light Induced LSD Least Significant Difference MJT Mean January Temperature mRNA Messenger RNA MV McLaren Vale n Number of samples N2 Liquid nitrogen NIR Near Infrared Spectroscopy ORF Open Reading Frame (coding region) p Probability PAR Photosynthetically Active Radiation PCR Polymerase Chain Reaction PCV Packed Cell Volume PGA Phloroglucinol Analysis PPA Protein Precipitation Assay RE Restriction Enzyme RL Riverland RNA Ribonucleic acid RT-PCR Real-Time PCR SC Shaded Control TA Total acidity TC Tentative Consensus TF Transcription Factor Tm Temperature of DNA dissociation (melt) TSS Total Soluble Solids (°Brix) UV Ultraviolet VIS Visible Vv Vitis vinifera L cv (cultivar) ZIP Zinc Finger Protein

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LIST OF FIGURES

CHAPTER 1

Figure 1.1 Schematic representation of grape berry development showing the bimodal pattern of

berry growth & the pattern of secondary metabolite accumulation. .............................. 2

Figure 1.2 The flavan skeleton of the plant secondary metabolites, flavonoids, showing the

numbering of carbon atoms & ring labels. .................................................................... 3

Figure 1.3 Stylised phenylpropanoid pathway showing major products, intermediates &

biosynthetic enzymes. .................................................................................................... 8

Figure 1.4 Schematic diagram showing transcriptional regulation of flavonoid pathway genes. ...... 11

CHAPTER 2

Figure 2.1 Typical HPLC separation of anthocyanins & flavonols in Shiraz skin at commercial

harvest. ......................................................................................................................... 20

Figure 2B.1 (APPENDIX 2B) Typical Shiraz skin HPLC chromatogram. .................................... 240

Figure 2B.2 (APPENDIX 2B) Typical Shiraz wine HPLC chromatogram. ................................... 241

CHAPTER 3

Figure 3.1 Schematic representation showing the relationship between wine quality &

anthocyanins. ............................................................................................................... 32

Figure 3.2 Schematic representation of the fruit streaming process in warm & cool regions. ........... 37

Figure 3.3 Maselli & YUBA sampling at the weighbridge in warm & cool regions ......................... 37

Figure 3.4 Comparison of tannin assays & extraction solvents.......................................................... 45

Figure 3.5 The relationship between anthocyanins & flavonols in Shiraz grape skins from

McLaren Vale & Riverland. ........................................................................................ 52

Figure 3.6 The relationship between anthocyanins & tannins in Shiraz grape skins & seeds from


Figure 3.7 The relationship between flavonols & tannins in Shiraz grape skins & seeds from


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Figure 3.8 The relationship between anthocyanins in the grape & anthocyanins in the wine in

Shiraz samples from McLaren Vale in 2003 & 2004. ................................................. 55

Figure 3.9 The relationship between anthocyanins & flavonols in Shiraz wine samples from

McLaren Vale in 2003 & 2004. ................................................................................... 56

Figure 3.10 The relationship between anthocyanins & grape quality stream in Shiraz grape skins

from McLaren Vale & Riverland. ................................................................................ 59

Figure 3.11 The relationship between vine age & grape quality stream in Shiraz samples from

McLaren Vale. ............................................................................................................. 60

Figure 3.12 The relationship between anthocyanins & yield (T/Ha) in Shiraz grape skins from


Figure 3.13 The relationship between anthocyanins (ratio of grape/wine) & grape quality stream

in Shiraz samples from McLaren Vale. ....................................................................... 61

Figure 3.14 The relationship between anthocyanins (in the grape & wine) & wine grade in Shiraz

samples from McLaren Vale. ....................................................................................... 64

Figure 3.15 The relationship between wine anthocyanins & $/bottle (wholesale) in Shiraz

samples from McLaren Vale in 2003. .......................................................................... 65

Figure 3.16 The relationship between anthocyanins & wine grade in Shiraz wine samples from

the Riverland. ............................................................................................................... 65

Figure 3.17 The relationship between grape quality stream & wine grade in Shiraz samples from


Figure 3A.1 (APPENDIX 3A) Map South Australia grape growing regions Riverland &

McLaren Vale. ........................................................................................................... 244

CHAPTER 4

Figure 4.1 Polypropylene boxes applied to Shiraz & Chardonnay vines. ........................................ 104

Figure 4.2 The influence of bunch light exposure on VvFLS1 expression & flavonol

accumulation in pre-veraison Chardonnay berries. ................................................... 110


accumulation in Shiraz & Chardonnay inflorescences. ............................................. 113

Figure 4.4 Phenotypic & developmental differences between exposed & shaded Shiraz (A) &

Chardonnay (B) bunches at different stages in development. ................................... 114


accumulation in Shiraz & Chardonnay leaves. .......................................................... 115

XVII

Figure 4.6 The influence of bunch light exposure, pre-veraison, on VvFLS1 expression &

flavonol accumulation in Shiraz berries. ................................................................... 116

Figure 4.7 The influence of bunch light exposure, post-veraison, on VvFLS1 expression &

flavonol accumulation in Shiraz & Chardonnay berries. ........................................... 118

Figure 4.8 The influence of bunch light exposure, post-veraison, on VvUFGT expression &

anthocyanin accumulation in Shiraz berries. ............................................................. 120

Figure 4.9 The influence of bunch light exposure, pre-harvest, on VvFLS1 expression & flavonol

accumulation in Shiraz & Chardonnay berries. ......................................................... 122

Figure 4.10 The influence of bunch light exposure, pre-harvest, on VvUFGT expression &

anthocyanin accumulation in Shiraz berries. ............................................................. 123

Figure 4.11 The (hourly) influence of bunch light exposure, pre-veraison, on VvFLS1 expression

& flavonol accumulation in Shiraz berries. ............................................................... 127

Figure 4.12 The diurnal pattern of VvFLS1 expression in Shiraz, post-flowering. .......................... 128

Figure 4.13 The diurnal pattern of VvFLS1 expression in Shiraz, post-veraison ............................. 128

Figure 4.14 The diurnal pattern of VvFLS1 expression in Shiraz, pre-harvest. ................................ 129

Figure 4.15 The diurnal pattern of VvLDOX & VvUFGT expression in Shiraz, during

development. .............................................................................................................. 130

Figure 4.16 Schematic representation of the flavonoid biosynthetic pathway ................................. 133

Figure 4A.1 (APPENDIX 4A) Map of Slate Creek Vineyard, Willunga. ....................................... 256

Figure 4A.2 (APPENDIX 4A) Map of Coombe Vineyard, Adelaide University Waite Campus ... 257

Figure 4B.1 (APPENDIX 4B) RT-PCR expression of VvCHI & VvLDOX in Shiraz berries

during development. .................................................................................................. 259

Figure 4B.2 (APPENDIX 4B) RT-PCR expression of VvCHI & VvLDOX in Chardonnay berries

during development. .................................................................................................. 261

CHAPTER 5

Figure 5.1 Chardonnay cells, in suspension culture & on disks contained on GC agar. .................. 150

Figure 5.2 Nucleotide sequence alignment of the VvFLS1 promoter fragments identified by PCR 154

Figure 5.3 Transient assays on the VvFLS1 promoter sequence(s) to compare the activation

capacities of transcription factors AtMYB12, VvMYBF, VvMYBA, VvMYBPA. . 158

Figure 5.4 Light induction in Chardonnay cell suspension culture; VvFLS1 expression &

flavonol accumulation................................................................................................ 159

XVIII

Figure 5.5 Light induction in Chardonnay cell suspension culture; VvFLS1 expression &

flavonol accumulation. ............................................................................................... 160

Figure 5.6 RT-PCR expression patterns for candidate (AtMYB12 homolog) genes. ...................... 162

Figure 5.7 RT-PCR expression patterns for candidate (microarray) genes. ..................................... 168

Figure 5B.1 (APPENDIX 5B) RT-PCR expression pattern for candidate genes (TC10 & TC12)

in Shiraz & Chardonnay fruit. .................................................................................... 265

Figure 5B.2 (APPENDIX 5B) RT-PCR expression pattern for candidate genes (TC10 & TC12)

in Shiraz (hourly induction). ...................................................................................... 266

XIX

LIST OF TABLES

CHAPTER 2 Table 2.1 LC-MS analysis of flavonols in Shiraz grape skins. ........................................................... 22

Table 2A.1 (APPENDIX 2A) Suppliers of chemicals, enzymes, growth media & other

miscellaneous consumables. ...................................................................................... 237

Table 2A.2 (APPENDIX 2A) Downloadable sequence management programs & online

databases. ................................................................................................................... 238

Table 2A.3 (APPENDIX 2A) Solutions commonly used in this research. ...................................... 238

Table 2C.1 (APPENDIX 2C) RT-PCR primers used for analysis of flavonoid gene expression. .. 242

Table 2C.2 (APPENDIX 2C) RT-PCR primers used in testing potential AtMYB12 homolog

candidate genes. ......................................................................................................... 242

Table 2C.3 (APPENDIX 2C) RT-PCR primers used in testing potential microarray candidate

genes. ......................................................................................................................... 242

Table 2C.4 (APPENDIX 2C) General primers used in PCR cloning. ............................................ 242

CHAPTER 3 Table 3.1 Mean tannin content of McLaren Vale (seed & skin) & Riverland (skin) samples in

2004 extracted with 70% acetone & model wine. ....................................................... 44

Table 3.2 Relationships between different measurements of anthocyanins & phenolics. .................. 47

Table 3.3 Mean values for grape berry factors & flavonoid composition in McLaren Vale &

Riverland in 2003 & 2004. .......................................................................................... 50

Table 3.4 R2 table of relationships between BERRY WEIGHT & berry factors & flavonoid content.51

Table 3.5 R2 table of relationships between GRAPE QUALITY STREAM & various factors as

measured by HWC & CSIRO. ..................................................................................... 58

Table 3.6 R2 table of relationships between WINE GRADE & various factors as measured by

HWC & CSIRO. .......................................................................................................... 63

Table 3.7 R2 table of miscellaneous relationships between different factors in McLaren Vale &

Riverland in 2003 & 2004. .......................................................................................... 67

Table 3A.1 (APPENDIX 3A) Riverland and McLaren Vale regional characteristics. ................... 243

Table 3B.1 (APPENDIX 3B) Vineyard assessment measurements recorded by Hardy Wine

Company (HWC). ..................................................................................................... 251

XX

CHAPTER 4

Table 4.1 Key grapevine growth stages & sampling dates for Chardonnay in the 2002-2003

season (preliminary light induction experiment). ...................................................... 106

Table 4.2 Key grapevine growth stages & sampling dates for Shiraz in the 2003-2004 season

(light induction experiment). ..................................................................................... 107

Table 4.3 Key grapevine growth stages & sampling dates for Chardonnay in the 2003-2004

season (light induction experiment). .......................................................................... 108


(hourly light induction experiment). .......................................................................... 108


(diurnal pattern of VvFLS1 expression experiment). ................................................. 109

Table 4.6 Berry weight & oBrix of Shiraz & Chardonnay exposed & shaded fruit. ......................... 114

Table 4.7 Summarised light induction data for VvFLS1, VvLDOX & VvCHI gene expression, in

Shiraz & Chardonnay berries at different stages in development. ............................. 125

Table 4.8 Summarized light induction data (VvFLS1 expression & flavonol concentration) in

Shiraz & Chardonnay fruit, at different stages during development. ........................ 135

CHAPTER 5

Table 5.1 Key motifs identified within the VvFLS1 promoter1 sequence (811bp) after analysis in

the PLACE database. ................................................................................................. 156

Table 5.2 Flavonoid composition of dark-grown Chardonnay cells & after three days exposure

to light. ....................................................................................................................... 158

Table 5.3 Putative AtMYB12 homologs identified by BLAST analysis in the grapevine TIGR

EST database. ............................................................................................................. 161

Table 5.4 Microarray results: The total number of genes changed in response to light ................... 163

Table 5.5 Unigenes identified by microarray analysis showing significant differences in gene

expression after Chardonnay cells were exposed to light for 24 hrs. ........................ 164

Table 5.6 Potential transcriptional regulator candidate genes identified by microarray analysis ..... 165

Table 5.7 Flavonoid gene expression in Chardonnay cells after exposed to light for 24 hrs as

determined by microarray analysis. ........................................................................... 166

TITLE PAGE: The influence of viticultural treatments on the accumulation of flavonoid compounds in grapes and their contribution to wine qualityTABLE OF CONTENTSABSTRACTDECLARATIONACKNOWLEDGEMENTSABBREVIATIONSLIST OF FIGURESLIST OF TABLES

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The influence of viticultural treatments on the accumulation of … · 2010. 10. 4. · The influence of viticultural treatments on the accumulation of flavonoid compounds in grapes