Functional Analysis Of Two PP2C Genes In Regulating ABA And Drought Stress Responses In Arabidopsis

Drought stress is not only one of the most common stresses which plants suffer from but also the one that affects the crop yield and quality a lot.Because of immobility,plants have to mitigate the damage brought about by stresses through physiolygical and metabolic regulation.Abscisic acid(ABA)is the most important plant hormone that regulates various abiotic stresses including drought.Generally,the signaling pathways through which plants respond to drought stress can be divided into ABA-dependent and ABA-independent,and studies on ABA-dependent drought stress responses have been reported a lot.ABA,as a signaling molecule can induce the transmission of ABA signalling pathway and regulate the expression of relevant genes,which leads to stomatal closure and finally reduces the water loss under drought stress.The ABA signalling pathway contains a variety of regulatory factors,among which a few Clade A PP2Cs(protein phosphatases,type 2C)have been reported to act as negative regulators.PP2 Cs are protein serine/threonine phosphatases existing in both eukaryotes and prokaryotes and regulate the growth,development and adaptability to stresses of organisms by dephosphorylation.In the past years,the research of PP2 Cs has been conducted in many plant species.In Arabidopsis,researches show that Calde A PP2 Cs are involved in ABA and osmotic stress signalling pathway.However,little is known about the PP2 Cs of clades other than Clade A.On the basis of previous work,we took the newly identified two PP2 C genes as the subject which could interact with MAPK(mitogen-activated protein kinase).These two genes were named PP2 Ca and PP2 Cb respectively.By screening homozygous T-DNA insertion mutants of these non-Clade A PP2 C genes and further phenotypic analysis,we identified that mutants of these two PP2 C genes were responsive to ABA treatment at the germination level.Among the mutants,pp2ca-1 and pp2ca-2 were sensitive to ABA while pp2cb-1、pp2cb-2 and pp2cb-3 were insensitive,compared to wild-type(WT).In the meantime,by phenotypic analysis at the post-germination level and soil drought test,we detected the response of AtPP2 Ca and AtPP2 Cb mutant and overexpression(OE)lines to ABA and various abiotic stresses,and the result indicated that both AtPP2 Ca and AtPP2 Cb can regulate the response of plant to drought stress.Real-time fluorescence quantitative RT-PCR analysis of PP2 Ca and PP2 Cb response to various stresses and hormones showed that the expression of AtPP2 Ca was inhibited by ABA,PEG8000,cold and heat treatments while induced by NaCl.In comparison,AtPP2 Cb expression was also inhibited by PEG8000,cold and heat.The subcellular localization analysis showed that AtPP2 Ca localized in the cytomembrane,cytoplasm and nuclei.Expression analysis of GUS reporter gene driven by AtPP2 Ca promoter showed that AtPP2 Ca expressed highly in anther and stigma.Yeast two-hybrid(Y2H)and the bimolecular fluorescence complementation(BiFC)assays indicated that AtPP2 Ca can interact with AtCIPK10(CBL-interacting protein kinase 10)and AtCIPK18 and may also interact with AtCIPK7,AtCIPK11,AtABF1,-2,-3,-4(ABA responsive element binding factor 1,-2,-3,-4),AREB3(ABA responsive element binding protein 3),ABI5(ABA insensitive 5)and AtSnRK1.1、-1.2(SNF1-related protein kinase 1.1,-1.2).This research can help us to learn more about the function of non-Clade A PP2 C genes in ABA and abiotic stress responses,which will provides theoretical guidance in breeding drought-tolerant crops.