title: Rapid Phosphoproteomic Effects of Abscisic Acid (ABA) on Wild-Type and ABA Receptor-Deficient A. thaliana Mutants.
abstract: Abscisic acid (ABA)¹ is a plant hormone that controls many aspects of plant growth, including seed germination, stomatal aperture size, and cellular drought response. ABA interacts with a unique family of 14 receptor proteins. This interaction leads to the activation of a family of protein kinases, SnRK2s, which in turn phosphorylate substrates involved in many cellular processes. The family of receptors appears functionally redundant. To observe a measurable phenotype, four of the fourteen receptors have to be mutated to create a multilocus loss-of-function quadruple receptor (QR) mutant, which is much less sensitive to ABA than wild-type (WT) plants. Given these phenotypes, we asked whether or not a difference in ABA response between the WT and QR backgrounds would manifest on a phosphorylation level as well. We tested WT and QR mutant ABA response using isotope-assisted quantitative phosphoproteomics to determine what ABA-induced phosphorylation changes occur in WT plants within 5 min of ABA treatment and how that phosphorylation pattern is altered in the QR mutant. We found multiple ABA-induced phosphorylation changes that occur within 5 min of treatment, including three SnRK2 autophosphorylation events and phosphorylation on SnRK2 substrates. The majority of robust ABA-dependent phosphorylation changes observed were partially diminished in the QR mutant, whereas many smaller ABA-dependent phosphorylation changes observed in the WT were not responsive to ABA in the mutant. A single phosphorylation event was increased in response to ABA treatment in both the WT and QR mutant. A portion of the discovery data was validated using selected reaction monitoring-based targeted measurements on a triple quadrupole mass spectrometer. These data suggest that different subsets of phosphorylation events depend upon different subsets of the ABA receptor family to occur. Altogether, these data expand our understanding of the model by which the family of ABA receptors directs rapid phosphoproteomic changes.
authors.affiliation: Department of Biochemistry and Biotechnology Center, University of Wisconsin, Madison, Wisconsin, 53706.
authors.forename: Benjamin B
authors.initials: BB
authors.lastname: Minkoff
authors.affiliation: Department of Biochemistry and Biotechnology Center, University of Wisconsin, Madison, Wisconsin, 53706.
authors.forename: Kelly E
authors.initials: KE
authors.lastname: Stecker
authors.affiliation: Department of Biochemistry and Biotechnology Center, University of Wisconsin, Madison, Wisconsin, 53706.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Sussman
mesh_terms: D000040:Abscisic Acid / Q000378:metabolism / Q000494:pharmacology*; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000235:genetics* / Q000378:metabolism; D017353:Gene Deletion; D018506:Gene Expression Regulation, Plant; D005838:Genotype; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods; D058977:Molecular Sequence Annotation; D008969:Molecular Sequence Data; D010641:Phenotype; D010750:Phosphoproteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D010937:Plant Growth Regulators / Q000378:metabolism / Q000494:pharmacology*; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism; D040901:Proteomics / Q000379:methods; D015398:Signal Transduction
chemical_list: D029681:Arabidopsis Proteins; D007527:Isoenzymes; C569327:PYL2 protein, Arabidopsis; D010750:Phosphoproteins; D010937:Plant Growth Regulators; C492978:SnRK2 protein, Arabidopsis; D000040:Abscisic Acid; D017346:Protein Serine-Threonine Kinases