title: Genetics and phosphoproteomics reveal a protein phosphorylation network in the abscisic acid signaling pathway in Arabidopsis thaliana.
abstract: Abscisic acid (ABA) is a phytohormone that regulates diverse plant processes, including seed germination and the response to dehydration. In Arabidopsis thaliana, protein kinases of the SNF1-related protein kinase 2 (SnRK2) family are believed to transmit ABA- or dehydration-induced signals through phosphorylation of downstream substrates. By mass spectrometry, we identified proteins that were phosphorylated in Arabidopsis wild-type plants, but not in mutants lacking all three members of the SnRK2 family (srk2dei), treated with ABA or subjected to dehydration stress. The number of differentially phosphorylated peptides was greater in srk2dei plants treated with ABA than in the ones subjected to dehydration, suggesting that SnRK2 was mainly involved in ABA signaling rather than dehydration. We identified 35 peptides that were differentially phosphorylated in wild-type but not in srk2dei plants treated with ABA. Biochemical and genetic studies of candidate SnRK2-regulated phosphoproteins showed that SnRK2 promoted the ABA-induced activation of the mitogen-activated protein kinases AtMPK1 and AtMPK2; that SnRK2 mediated phosphorylation of Ser(45) in a bZIP transcription factor, AREB1 (ABA-responsive element binding protein 1), and stimulated ABA-responsive gene expression; and that a previously unknown protein, SnRK2-substrate 1 (SNS1), was phosphorylated in vivo by ABA-activated SnRK2s. Reverse genetic analysis revealed that SNS1 inhibited ABA responses in Arabidopsis. Thus, by integrating genetics with phosphoproteomics, we identified multiple components of the ABA-responsive protein phosphorylation network.
authors.affiliation: Faculty of Agriculture and Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan. taishi@cc.tuat.ac.jp
authors.forename: Taishi
authors.initials: T
authors.lastname: Umezawa
authors.forename: Naoyuki
authors.initials: N
authors.lastname: Sugiyama
authors.forename: Fuminori
authors.initials: F
authors.lastname: Takahashi
authors.forename: Jeffrey C
authors.initials: JC
authors.lastname: Anderson
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Ishihama
authors.forename: Scott C
authors.initials: SC
authors.lastname: Peck
authors.forename: Kazuo
authors.initials: K
authors.lastname: Shinozaki
mesh_terms: D000040:Abscisic Acid / Q000378:metabolism*; D017360:Arabidopsis / Q000235:genetics*; D029681:Arabidopsis Proteins / Q000378:metabolism*; D001483:Base Sequence; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics* / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D040901:Proteomics / Q000379:methods; D036226:Seedlings / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology*
chemical_list: D029681:Arabidopsis Proteins; D010455:Peptides; D010750:Phosphoproteins; C583907:SNS1 protein, Arabidopsis; C492978:SnRK2 protein, Arabidopsis; D000040:Abscisic Acid; D017346:Protein Serine-Threonine Kinases