摘要Multicellular organisms such as plants contain various cell types with specialized functions.An-alyzing the characteristics of each cell type re-veals specific cell functions and enhances our understanding of organization and function at the organismal level.Guard cells(GCs)are specialized epidermal cells that regulate the movement of the stomata and gaseous exchange,and provide a model genetic system for analyzing cell fate,sig-naling,and function.Several proteomics analyses of GC are available,but these are limited in depth.Here we used enzymatic isolation and flow cy-tometry to enrich GC and mesophyll cell proto-plasts and perform in-depth proteomics in these two major cell types in Arabidopsis leaves.We identified approximately 3,000 proteins not pre-viously found in the GC proteome and more than 600 proteins that may be specific to GC.The depth of our proteomics enabled us to uncover a guard cell-specific kinase cascade whereby Raf15 and Snf1-related kinase2.6(SnRK2.6)/OST1(open stomata 1)mediate abscisic acid(ABA)-induced stomatal closure.RAF15 directly phosphorylated SnRK2.6/OST1 at the conserved Ser175 residue in its activation loop and was sufficient to reactivate the inactive form of SnRK2.6/OST1.ABA-triggered SnRK2.6/OST1 activation and stomatal closure was impaired in raf15 mutants.We also showed enrichment of enzymes and flavone me-tabolism in GC,and consistent,dramatic accu-mulation of flavone metabolites.Our study an-swers the long-standing question of how ABA activates SnRK2.6/OST1 in GCs and represents a resource potentially providing further insights into the molecular basis of GC and mesophyll cell development,metabolism,structure,and function.