Scientist 2 Pacific Northwest National Laboratory Richland, Washington
Body of Abstract: Plant responses to abiotic stresses were predominantly studied at the whole tissue level and mostly under single stress conditions. We took an alternative approach to fill the knowledge gap of how poplar’s two major leaf cell-types (palisade and vascular) respond to single stresses (drought, salinity, heat) and combination stress conditions. We isolated the leaf cell-types using cryo-sectioning and laser-capture microdissection (LCM) for low-input transcriptomics and proteomics analyses, where the cell-type proteomics was enabled by a nanodroplet-based proteomic processing system (nanoPOTS). The omics data showed cell-type unique differentially expressed genes (DEGs) (5899 DEGs, of which 56% were unique to palisade and 26% unique to vascular) and differentially abundant proteins (DAPs) (6172 DAPs, of which 14% were unique to palisade and 33% unique to vascular) in response to single and combined stress conditions. From the proteomics data, we identified an isoform of Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein that showed nearly 12-fold upregulation uniquely in palisade cells during combination stress while showing no change under single stress conditions. Using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), we found the metabolites associated with GAPDH, i.e. glyceraldehyde-3-phosphate and 3-phosphoglycerate, showing higher abundances under combined stress conditions in palisades, which was correlated to an increase in hexoses and sugar alcohols in palisade cells. Complementary GCMS-based metabolomics confirmed the increase in sugar alcohols in palisade cells. Currently, through enzyme assays and stable isotope labeling procedures, we try to reveal a cell-type specific role of GAPDH under combination stress. Further, we have developed synthetic promoters based on the cell-type-specific transcriptomics data, which showed stress inducibility exclusively in palisade cell-type. These promoters will be used to functionally validate the role of GAPDH. Our findings allow future attempts to map molecular machineries to cellular domain and contribute to the design of plants with enhanced tolerance to abiotic stress combinations.