Proteomic analysis of environmental stress responses in grapevine

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 daylength buds. We observed active growth as well as greater abundance of phenylpropanoid biosynthesis proteins in long daylength buds. Finally, we have introduced a new method for validation of quantitative shotgun proteomic data. We developed a protein quantitation false discovery rate and applied it as a noise-level threshold, to establish the significance of the proteomic results in this thesis. All the research studies in this thesis demonstrated the efficacy of label-free proteomics approaches in gaining information on grapevine responses to environmental stresses at the cellular level.