Proteomic Analysis of Environmental Stress Responses in Grapevine
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Proteomic 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