Identification of tonoplast membrane proteins involved in cellular response to environmental stress by quantitative/comparative- and phospho-proteomics

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Proteom analyses represent a formidable tool to identify vacuolar proteins and to enlighten their relevance in stress response. During the past period of the research grant we have mainly focused on establishing a platform to isolate highly pure vacuoles applicable for mass spectrometry based proteomics. We isolated vacuoles from cold incubated and control plants to investigate changes in the tonoplast proteome during the process of cold acclimation. Thereby several changes in the abundance of tonoplast proteins and alterations in protein phosphorylation were identified providing first insights into the physiological relevance of the vacuole on cold acclimation. By these proteome analyses we identified several unknown proteins residing in the tonoplast and characterized one of these in detail. The tonoplast copper transporter COPT5 belongs to the eukaryotic Ctr family, catalyses the export of copper ions from the vacuole and is responsible for the inter organ allocation of copper in Arabidopsis. In the proposed second funding period we will expand the comparative analysis of changes in the proteome of the tonoplast induced by other abiotic factors like drought, salt, heat and osmotic stress. In parallel we will investigate how such stress factors alter the phosphorylation of tonoplast proteins to understand the relevance of this post-translational protein modification on the regulation of tonoplast transport mechanisms. Finally, we will investigate the relevance of individual tonoplast proteins for the plants stress response and we will effort to clarify the biochemical and physiological function of so far unknown tonoplast proteins.