Dissecting The Biochemical Mechanism Of One PP2C Gene Regulating ABA Signaling And Abiotic Stress In Arabidopsis Thaliana

The phytohormone abscisic acid(ABA)plays a crucial role in various plant growth processes such as seed maturation,germination,seedling growth,as well as in adaptation to abiotic stresses.Drought is one of the most important yield limiting stresses which occurs frequently in northwestern regions of China.During drought stress ABA acts as molecular messenger regulating various aspects of drought resistance and drought tolerance such as stomatal closure and reduced water loss.The core ABA signaling components consist of RCAR/PYR/PYL(regulatory component of ABA receptor/pyrabactin resistance/pyrabactin resistance 1-like)detecting extracellular ABA level.Sensing of ABA leads to a conformational change resulting in binding of the RCAR/PYR/PYLs to PP2 Cs which in turn releases Sn RK2 s from the Sn RK-PP2 C complex.Upon Release Sn RKs phosphorylate ABF/AREB-type b ZIP transcription factors which further modulate the expression of various downstream target genes.Previous work on ABA signaling pathways has been mostly focused on Clade A PP2 Cs in Arabidopsis thaliana.Here we reported a novel PP2 C that is involved in ABA signaling pathway and drought stress.On the basis of yeast two-hybrid screening several putative interacting partners were identified.We focused our work on MPK3(mitogen-activated protein kinase 3),CIPK11(CBL-interacting protein kinase11)and ABI5(ABA insensitive 5).We then confirmed the protein-protein interaction using bimolecular fluorescence complementation(Bi FC)and co-immunoprecipitation(Co-IP)assays in vivo and GST pull-down assay in vitro.The subcellular localization of PP2 Ca was determined to be in both cytoplasm and nucleus.We performed phenotypic analysis of PP2 Ca overexpression lines and mutant lines after crossed with abi5-1 mutant.In addition,stomatal aperture assay and quantification of proline contents were also performed with these lines.Collectively these results indicated that ABI5 acts downstream of PP2 Ca in the ABA signaling pathway.Dual Luciferase assay in Arabidopsis protoplasts further confirmed that PP2 Ca acted in a feed forward manner upstream of CIPK11 and ABI5.Interestingly it was also found that PP2 Ca could regulate activities of MPK3/6 in Western blots.The results altogether lay a solid foundation for depicting the role of PP2 Ca in ABA signaling network and drought tolerance.