Study On The Mechanism Of BIPP1 Protein Phosphatase Negatively Regulated Brassin Saponin Signal Pathway In Arabidopsis Thaliana

Brassinosteroids (BRs) are a class of plant steroid hormones and play important roles in a wide range of plant developmental and physiological processes. Stemelongation, vascular differentiation, male fertility, seed size and germination,flowering time, senescence, and resistance to biotic and abiotic stresses are under regulation of BRs.The BR synthesis pathway and signaling transduction pathway has been studied for tens of years and well understood. BR signaling was perceived by the extracellular domain of a transmembrane receptor kinase,BRASSINOSTEROID INSENSITIVE 1 (BRI1), leading to the auto-phosphorylation and trans-phosphorylation of BRI1, which gain the ability to deliver the signal to downstream components by phosphorylation. However, the molecular mechanism of BRI1 dephosphorylation and activation reductionwas still not clear. Therefore, we hope to have a further study on the molecular mechanism of BRI1 inactivation. In this paper, we constructed a library containing over 65% protein phosphatases in Arabidopsis, and conducted a yease two hybrid screening using BRI1 as the bait. BIPP1(BRI1 interacting protein phosphatase 1) was selected out in this screening as a direct BRI1-interacting protein phosphatase. Pull-down assay and BiFC assay confirmed the interaction between BRI1 and BIPP1. Western blotting assay by pSer and pThr antibody indicated that BIPP1 can dephosphate BRI1 rapidly. To study the function of BIPP1, we performed genetics analysis. BIPP1 knock out mutant showed a bigger and longer hypocotyl phenotype. RT-PCR analysis indicated that the expression level of marker genes CPD and DET2 dicreased in bippl mutant, moreover, western blotting assay showed the dephosphorylated BES1 accumulated in the mutant. All these data suggested that knocking out BIPP1 lead to a promoted BR signaling output. To address whether BIPP1 functions dependent on the kinase activity of BRI1, we obtained det2-1/bippl and bril-301/bippl double mutants by cross. The phenotype of the double mutants demonstrated that knocking out BIPP1 effected the phenotype of Arabidopsis on dependent of a normal wild type BRI1 protein. Finally, we found that the expression of BIPP1 was negatively regulated by BR signaling using molecular and cellular biology methods.This paper gives a new sight of the molecular mechanism of activity regulation of BRI1, and finds a novel protein phosphatase involed in BR signaling pathway.These results have a great significance in supplementing the known BR signaling transduction pathway and providing ideas for other kinases regulation through reversible phosphorylation.