A cell type-specific proteomics uncovers a guard cell-specific RAF15-SnRK2.6/OST1 kinase cascade
Ontology highlight
ABSTRACT:
ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)
SUBMITTER: Pengcheng Wang
PROVIDER: PXD034026 | JPOST Repository | Tue May 23 00:00:00 BST 2023
REPOSITORIES: jPOST
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Project description:Multicellular organisms such as plants contain different types of cells with specialized functions. Analyzing the characteristics of each type of cells will reveal specific cell functions and enhance understanding of how an organism organizes and works. Here we report a high-sensitive and efficient cell-type-specific multi-omics pipeline, combining simplified flow cytometry-based fluorescent cell-sorting for fluorescent protoplasts and optimized nano-scale proteomics and metabolomics methods, which allow us to in-depth analyze the proteomics and metabolomics of a particular type of cells. By this method, we quantitatively compared the proteomics and metabolomics between guard cells and mesophyll cells and revealed that the enrichment of signal transduction-related proteins enables guard cells to respond to various environmental stimuli quickly. We uncovered a guard-cell-specific kinase cascade consisting of RAF15 and OST1 mediates the ABA-induced stomatal closure in guard cells. This pipeline is applicable to various types of cells in plant or non-plant systems to acquire systemic knowledge of how cells work specifically and in highly organized multiple cell organisms.
2022-12-18 | PXD030511 | JPOST Repository
Project description:Multicellular organisms such as plants contain different types of cells with specialized functions. Analyzing the characteristics of each type of cells will reveal specific cell functions and enhance understanding of how an organism organizes and works. Here we report a high-sensitive and efficient cell-type-specific multi-omics pipeline, combining simplified flow cytometry-based fluorescent cell-sorting for fluorescent protoplasts and optimized nano-scale proteomics and metabolomics methods, which allow us to in-depth analyze the proteomics and metabolomics of a particular type of cells. By this method, we quantitatively compared the proteomics and metabolomics between guard cells and mesophyll cells and revealed that the enrichment of signal transduction-related proteins enables guard cells to respond to various environmental stimuli quickly. We uncovered a guard-cell-specific kinase cascade consisting of RAF15 and OST1 mediates the ABA-induced stomatal closure in guard cells. This pipeline is applicable to various types of cells in plant or non-plant systems to acquire systemic knowledge of how cells work specifically and in highly organized multiple cell organisms.
2022-12-17 | PXD030489 | JPOST Repository
Project description:Multicellular organisms such as plants contain different cell types with specialized functions. Analyzing the characteristics of each cell type reveals specific cell functions and enhances understanding of organization and function at the organismal level. Guard cells are specialized epidermal cells that play an important role in gaseous exchange in and out of plant leaves by regulating the opening and closing of the stomata, and provide a model genetic system for analyzing cell fate, signaling and function. Several proteomics analyses of guard cells are available, but these are limited in depth. Here we used enzymatic isolation and flow cytometry to enrich for guard cell and mesophyll cell protoplasts and acquire in-depth proteomics in these two major cell types in Arabidopsis leaves. We identified ~3,000 proteins not previously found in the guard cell proteome and more than 500 proteins that may be specific to guard cells. The depth of our proteomics enabled us to uncover a guard cell-specific kinase cascade whereby Raf-like kinase15(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 dephosphorylated inactive form of SnRK2.6/OST1. ABA-triggered SnRK2.6/OST1 activation and stomatal closure was impaired in raf15 mutants. Our study answers the long-standing question of how ABA activates SnRK2.6/OST1 in guard cells and represents a resource potentially providing many more insights into the molecular basis of guard cell and mesophyll cell development, structure, and function.
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Project description:Multicellular organisms such as plants contain different cell types with specialized functions. Analyzing the characteristics of each cell type reveals specific cell functions and enhances understanding of organization and function at the organismal level. Guard cells are specialized epidermal cells that play an important role in gaseous exchange in and out of plant leaves by regulating the opening and closing of the stomata, and provide a model genetic system for analyzing cell fate, signaling and function. Several proteomics analyses of guard cells are available, but these are limited in depth. Here we used enzymatic isolation and flow cytometry to enrich for guard cell and mesophyll cell protoplasts and acquire in-depth proteomics in these two major cell types in Arabidopsis leaves. We identified ~3,000 proteins not previously found in the guard cell proteome and more than 500 proteins that may be specific to guard cells. The depth of our proteomics enabled us to uncover a guard cell-specific kinase cascade whereby Raf-like kinase15(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 dephosphorylated inactive form of SnRK2.6/OST1. ABA-triggered SnRK2.6/OST1 activation and stomatal closure was impaired in raf15 mutants. Our study answers the long-standing question of how ABA activates SnRK2.6/OST1 in guard cells and represents a resource potentially providing many more insights into the molecular basis of guard cell and mesophyll cell development, structure, and function.
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Project description:Plant organs are comprised of distinct cell types with unique assemblages of mRNAs. This is a collection of CEL files of mRNA profiles of the total steady-state mRNAs and polysomal mRNAs of distinct cell types of the whole root and shoot of 7-d-old Arabidopsis thaliana seedlings. The cell type specific mRNA populations are those present in ribosome-mRNA complexes. This sub-population of mRNAs was obtained by first establishing a collection of Arabidopsis lines that express a FLAG-epitope tagged ribosomal protein L18 (RPL18) directed by promoters expressed in specific cell types and regions. Thirteen different promoter:FLAG-RPL18 lines were used. The targeted cell types and promoters included root atrichoblast (non-hair) epidermal cells (pGL2), root endodermis (pSCR), root stelar xylem and pericycle (pWOL, pSHR), root phloem companion cells (phloem CC) (pSUC2, pSultr2;2), root proliferating cells (pRPL11C), root cortex meristematic cells (pCO2), root cortex elongation/maturation cells (pPEP), shoot mesophyll (pRBCS), shoot epidermis (pCER5), shoot guard cells (pKAT1), shoot bundle sheath (pSultr2;2), shoot phloem CC (pSUC2) and shoot trichomes (pGL2). A CaMV 35S promoter:FLAG-RPL18 line was used to obtain the polysomal mRNA of multiple cell types. The immunopurification of ribosome-mRNA complexes of specific cell types/regions was accomplished by the method described in Zanetti et al. (Plant Physiology, 138, 624-635; 2005). Hybridization of the immunopurified mRNAs to the Affymetrix ATH1 DNA microarray platform and subsequent data analysis permitted the identification of transcripts that are enriched or depleted in specific cell types/regions of roots and shoots. The dataset includes samples from cell types/regions from seedlings grown under control conditions and cell types/regions of seedlings exposed to low oxygen stress (hypoxia) for 2 h. Keywords: cell-type specific expression, hypoxic stress, polysomal mRNA, abiotic stress, atrichoblasts, epidermis, cortex, endodermis, stele, phloem companion cells, guard cells, mesophyll
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Project description:Stomata are highly specialized organs which consist of pairs of guard cells and regulate gas and water vapor exchange in plants. While early stages of guard cell differentiation have been described and were interpreted in analogy to processes of cell type differentiation in animals, the downstream development of functional stomatal guard cells remains poorly understood. We have isolated an Arabidopsis mutant, scap1 (stomatal carpenter 1), that develops irregularly shaped guard cells and lacks the ability to control stomatal aperture, including CO2-induced stomatal closing and light-induced stomatal opening. SCAP1 was identified as a plant-specific Dof-type transcription factor expressed in maturing guard cells but not in guard mother cells. SCAP1 regulates the expression of genes encoding key elements of stomatal functioning and morphogenesis, such as a K+ channel protein, MYB60 transcription factor, and pectin methyl esterase. Consequently, ion homeostasis was disturbed in scap1 guard cells, and esterification of extracellular pectins was impaired so that the cell walls lining the pores did not mature normally. We conclude that SCAP1 regulates essential processes of stomatal guard cell maturation and functions as a key transcription factor regulating the final stages of guard cell differentiation. We isolated guard cell protoplasts from 4-week-old WT(Col-0) and scap1 mutant plants and extracted RNA independently. Four biological replicates were performed for each experiment.
2013-02-28 | E-GEOD-43964 | biostudies-arrayexpress
Project description:Stomata are highly specialized organs which consist of pairs of guard cells and regulate gas and water vapor exchange in plants. While early stages of guard cell differentiation have been described and were interpreted in analogy to processes of cell type differentiation in animals, the downstream development of functional stomatal guard cells remains poorly understood. We have isolated an Arabidopsis mutant, scap1 (stomatal carpenter 1), that develops irregularly shaped guard cells and lacks the ability to control stomatal aperture, including CO2-induced stomatal closing and light-induced stomatal opening. SCAP1 was identified as a plant-specific Dof-type transcription factor expressed in maturing guard cells but not in guard mother cells. SCAP1 regulates the expression of genes encoding key elements of stomatal functioning and morphogenesis, such as a K+ channel protein, MYB60 transcription factor, and pectin methyl esterase. Consequently, ion homeostasis was disturbed in scap1 guard cells, and esterification of extracellular pectins was impaired so that the cell walls lining the pores did not mature normally. We conclude that SCAP1 regulates essential processes of stomatal guard cell maturation and functions as a key transcription factor regulating the final stages of guard cell differentiation.
2013-02-28 | GSE43964 | GEO