Table Of ContentProteomic Analysis of Environmental
Stress Responses in Grapevine
by
Iniga Seraphina George
This thesis is presented for the award of the degree of
Doctor of Philosophy
Department of Chemistry and Biomolecular Sciences
Faculty of Science and Engineering
Macquarie University, Sydney, New South Wales, Australia
April 2015
“I am the vine, you are the branches. He that abides in me, and I
in him, he bears much fruit: for without me you can do nothing.”
- Jesus Christ
Dedicated to my husband Martin William,
and my parents.
Declaration
I certify that this thesis titled ‘Proteomic analysis of environmental stress responses in
grapevine’ is my original research. Some parts of this research was achieved in
collaboration with other researchers; any form of assistance received from others has
been duly acknowledged and their contribution recognised. All sources of information
and cited material have been referenced in this thesis. No part of this thesis has been
submitted to any other institution as a component of a degree or award. I also certify
that this thesis contains no information that has been formerly written or published by
anyone else except where due reference is cited in the text.
Iniga Seraphina George
April 2015
Table of Contents
Acknowledgements .......................................................................................................... 1
Abstract ............................................................................................................................ 4
List of Manuscripts ......................................................................................................... 6
Conference Presentations and Awards ......................................................................... 8
Abbreviations ................................................................................................................. 10
Chapter 1 ........................................................................................................................ 13
1. Introduction ........................................................................................................... 13
1.1 ‘Change’ is constant in this world ..................................................................... 13
1.2 The importance of plants ................................................................................... 14
1.3 Abiotic environmental stresses in plants ............................................................ 15
1.4 Grapevine - genetics, history and diversity ........................................................ 16
1.5 Wine regions and wine production .................................................................... 18
1.6 Proteomics and mass spectrometry .................................................................... 20
1.7 Proteomic technologies for studying abiotic stress in plants ............................. 21
1.8 Grapevine proteomics ........................................................................................ 23
1.8.1 Grape genomic resources ............................................................................ 23
1.8.2 The era of 2D gels ....................................................................................... 24
1.8.3 The renaissance in grapevine proteomics: Emergence of various qualitative
and quantitative approaches ................................................................................. 32
1.8.4 Abiotic and biotic stress response studies in grapevine .............................. 34
1.8.5 Photoperiod associated molecular investigations in grapevine ................... 36
1.8.6 Berry development and fruit proteomics ..................................................... 37
1.8.7 Wine proteomics ......................................................................................... 39
1.9 Concluding remarks and research objectives ..................................................... 40
Chapter 2 ........................................................................................................................ 46
2. Materials and Methods ......................................................................................... 46
2.1 Methodology for in-solution digestion and shotgun proteomic analysis of
grapevine samples .................................................................................................... 47
2.1.1 Protein extraction ........................................................................................ 47
2.1.2 Filter aided sample preparation ................................................................... 47
2.1.3 Gas phase fractionation ............................................................................... 48
2.1.4 Tandem mass spectrometry ......................................................................... 48
2.1.5 Protein identification ................................................................................... 49
2.1.6 Data processing and statistical analysis ...................................................... 50
2.1.7 Functional annotations ................................................................................ 51
2.2 Methodology for in-gel digestion and shotgun proteomic analysis of plant
tissues ...................................................................................................................... 52
2.2.1 Synopsis of Publication I: Analysis of rice proteins using SDS-PAGE
shotgun proteomics .............................................................................................. 52
2.2.2 Publication I ................................................................................................ 53
Chapter 3 ....................................................................................................................... 69
3. Protein identification and quantification from riverbank grape, Vitis riparia:
Comparing SDS-PAGE and FASP-GPF techniques for shotgun proteomic
analysis ....................................................................................................................... 69
3.1 Synopsis of Publication II ................................................................................. 69
3.2 Key Findings ..................................................................................................... 70
3.3 Contributions ..................................................................................................... 70
3.4 Publication II ..................................................................................................... 71
Chapter 4 ....................................................................................................................... 78
4. Quantitative proteomic analysis of Cabernet Sauvignon grape cells exposed to
thermal stresses reveals alterations in sugar and phenylpropanoid metabolism 78
4.1 Synopsis of Publication III ................................................................................ 78
4.2 Key Findings ..................................................................................................... 78
4.3 Contributions ..................................................................................................... 79
4.4 Publication III .................................................................................................... 80
Chapter 5 ....................................................................................................................... 95
5. Shotgun proteomic analysis of photoperiod regulated dormancy induction in
grapevine .................................................................................................................... 95
5.1 Synopsis of Publication VII (manuscript prepared for submission) ................. 95
5.2 Key Findings ..................................................................................................... 95
5.3 Contributions ..................................................................................................... 96
5.4 Publication VII (manuscript prepared for submission) ..................................... 97
Chapter 6 ..................................................................................................................... 158
6. A new approach for validation: measurement of protein-fold changes by
spectral counting to determine protein quantitation false discovery rates ........ 158
6.1 Synopsis ........................................................................................................... 158
6.2 Introduction ..................................................................................................... 159
6.3 Materials and Methods .................................................................................... 161
6.3.1 Cell cultures .............................................................................................. 161
6.3.2 Protein extraction, digestion and peptide extraction ................................. 161
6.3.3 Gas phase fractionation, chromatography and tandem MS/MS ............... 162
6.3.4 Protein identification and data processing ................................................ 163
6.3.5 Control - control experiment and statistical analysis ................................ 164
6.3.6 Minimum spectral count analysis .............................................................. 165
6.3.7 Comparison of false discovery rates of protein quantitation of control
versus control and control versus temperature stresses ...................................... 166
6.4 Results and Discussion .................................................................................... 166
6.4.1 Protein identification and t-test comparisons ............................................ 166
6.4.2 Determination of protein quantitation FDR .............................................. 169
6.4.3 Comparison of protein quantitation FDRs calculated from different
minimum spectral cut-off values ........................................................................ 171
6.4.4 Control versus temperature stresses .......................................................... 173
6.4.5 Discussion of results .................................................................................. 174
6.5 Concluding Remarks ........................................................................................ 177
Chapter 7 ...................................................................................................................... 179
7. Conclusions and future directions...................................................................... 179
7.1 Conclusions ...................................................................................................... 179
7.2 Future directions .............................................................................................. 181
7.3 Synopsis of Publication IV: Current perspectives in proteomic analysis of
abiotic stress in grapevines .................................................................................... 185
7.4 Publication IV .................................................................................................. 186
Appendix ...................................................................................................................... 192
References .................................................................................................................... 217
Acknowledgements
The success and final outcome of this thesis required a lot of guidance and support from
many people. I am greatly indebted in gratitude to many and will try to thank as many
as I can here.
Firstly, I thank my principal supervisor, Professor Paul Haynes for the opportunity to
work on this project. His leadership, excellent supervision, knowledgeable guidance in
directing my project, constant encouragement and his trust and confidence in me
motivated me to accomplish my tasks in time. I am grateful to my co-supervisor,
Associate Professor Robert Willows for his instructions and comments on my project
whenever I needed it. I gratefully acknowledge both my supervisors for their time,
availability, instructions, and insights and also for the freedom to pursue my own
thoughts in this project.
Thanks to Professor Brian Atwell for his advice and Muhammad Masood for the help
and friendly assistance while working in the greenhouse and plant growth facility.
Special and sincere thanks to Dr. Dana Pascovici, for her prompt assistance and great
help in data analysis. I also thank Professor Anne Fennell, South Dakota State
University, Brookings, USA, for the opportunity to work on collaborative projects in
grapevine and Professor Grant Cramer, University of Nevada, Reno, USA, for allowing
me to visit his laboratory. Thanks to Dr. Chris Davies, CSIRO Plant Industry, South
Australia for starter grape calli. I thank all those whom I have co-authored with, for the
opportunity to collaborate and also for their intellectual contribution. I thank Ms. Jane
Yang for her kind assistance in regard to higher degree research matters. I thank
Catherine Wong, Michelle Kang and Jinxia Ke for administrative assistance.
I extend my gratitude to all my colleagues and friends in the Department of Chemistry
and Biomolecular Sciences (CBMS) and the Australian Proteome Analysis Facility
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(APAF). Special thanks to Karlie, Mehdi, Gayani, Steve, Sridevi and Samantha from
whom I have learnt many things in experimentation. I appreciate Shabnam, Sri,
Vignesh, Manveen, Karthik, Katherine, Tony and other past and present PhD students
for their motivation and support during my study. Special thanks to Shabnam, Sammi
and Manveen for their friendship, which I value very much. I thank all the members of
the Haynes’ group, Mehdi, David, Sammi, Vineet and Yunqi for being great team
mates, and the others in the Biomarker Discovery Lab for the pleasant research
environment. I also gratefully acknowledge the help and motivation from my friends
outside university, Kanika, Dorji, Ronita, Jude, Rukshana and Cherag and also from
church, for their prayers and concern.
My heartfelt thanks to my family; my husband - Martin William who is an epitome of
love and patience, my father - Selvam George who is my role model and inspiration,
mother - Vasanthi for her care, concern and prayers, sister - Monica, for her long night
vigils to give me company me as I wrote this thesis and also for her cheerfulness,
parents-in-law, Joseph William and Cecilia, for their constant concern, sacrifice,
support and encouragement. I will ever be indebted in gratitude to my immediate family
for their unconditional love, sacrifice and help.
I also thank my loving extended family in Sydney, Aunty Regina, Uncle John, Kevin,
Aunty Pramila, Uncle Paul, Jemima and Joseph for their love, prayers, support and
words of encouragement. I admit my gratefulness to Premi, Mother Mercy and (Late)
Uncle Thivian who have been my family when I was away from home, especially
during the initial stages of my PhD. Words cannot explain the gratitude I feel towards
my family, my backbone of support, for their prayers and patience during my doctoral
study.
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Above all and most importantly, I would like to thank Jesus Christ, the God Almighty
and also my best friend, who gives me the strength to do all things. For with God, all
things are possible.
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Abstract
Understanding environmental stress responses is essential to improve abiotic stress
tolerance in commercially important crops such as grapevine. Erratic abiotic stresses
can pose a threat to viticulture practices. Changes in temperature and light exposure,
either suddenly or seasonally, are crucial environmental factors that can influence
grapevine productivity. The grape genome sequence was released in 2007 and the
availability of this genomic information facilitated proteomic investigations to be
executed in the Vitis species with ease. In this thesis, we aimed to enhance our
understanding on biological pathways activated by the impact of abiotic stresses in
grapevine, by characterising proteins associated with stress responses.
Firstly, we optimised a sample preparation and fractionation methodology for label-free
quantitative shotgun proteomic analysis of grapevine. We then implemented this
optimised protocol for all the studies included in this thesis.
Subsequently, we examined the influence of thermal stresses on Vitis vinifera and
compared protein expression patterns between the control temperature and two different
heat and cold stresses. This is the first label-free shotgun proteomic study on grape
exposed sudden temperature changes. We demonstrated that proteins involved in
phenylpropanoid biosynthesis were more abundant at extreme cold stress and could be
cold-responsive proteins. We also observed that sugar metabolism switched between the
alternative and classical pathways at thermal stresses.
Next, two hybrid grapevine species - Vitis riparia and Seyval, were used to investigate
cellular mechanisms associated with photoperiod regulated biological processes of
growth cessation and dormancy induction. Several enzymes involved in glycolysis, and
dormancy induction were up-regulated in short daylength buds compared to long
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Description:1.7 Proteomic technologies for studying abiotic stress in plants .. 1.8.4 Abiotic and biotic stress response studies in grapevine . the hybrid and phylloxera resistant Vitis cultivars which are commercially valued for.
