Functional Analyses Of Leucine-Rich Repeat Receptor-Like Kinases BIR3 And Its Homologues In Arabidopsis Thaliana

Brassinosteroids(BRs)are a class of steroidal hormones playing important roles in many developmental processes,from seed germination to flowering and senescence.BRI1,as a major receptor gene of BRs,encodes a leucine-rich repeat receptor-like kinase.It perceives BRs at the cell surface,causing BRI1 to dissociate from its kinase inhibitor BKI1 from the plasma membrane and subsequently associate with its co-receptor BAK1.The BR signaling cascade is thought to be activated via a sequential transphosphorylation mechanism of BRI1 and BAK1.BIRs(BAK1-interacting receptor-like kinases)belong to the same subfamily with BRI1 which contains four members,including BIR1,BIR2,BIR3 and BIR4,all of which encode small LRRs within their extracellular domain.Previous investigation demonstrated that BON1 interacts with BIR1 and BAK1 in modulating temperature-dependent plant growth and cell death in Arabidopsis.Whereas,BIR2 is a negative regulator of BAK1 in regulating plant innate immunity.Our research shows that overexpression of BIR3(BIR3OE)results in a number of defective phenotypes,similar to those of bak1-4,including round leaves,shorter petioles and smaller rosette leaves in comparison with those of wild-type Arabidopsis plants.Moreover,the roots and hypocotyls of BIR3 OE are much shorter than those of wild-type when grown either in light or dark.Interestingly,BIR3 OE plants show reduced sensitivity to exogenously applied BR compared to those of wild-type seedlings.Additional analyses such as BES1 phosphorylation status and BR biosynthetic genes expression in response to exogenous BR application all support that the BR signaling pathway in BIR3 OE is significantly attenuated.Overexpression of a kinase-dead mutant of BIR3,mBIR3,results in a phenotype similar to that of BIR3 OE,indicating that the role of BIR3 in negatively regulating BR signaling is independent of its kinase activity.Moreover,when BIR3 is overexpressed in bri1-301,the transgenic plants exhibit a more severe phenotype with reduced fertility.Co-immunoprecipitation assay shows that BIR3 can interact with BAK1 in vivo.Furthermore,both yeast two-hybrid and bimolecular fluorescence complementation analyses indicate that BIR2,BIR3 and BIR4 can interact with BAK1 and BRI1,respectively.Furthermore,overexpression of BIR3 caused hysteresis during seed germination.The expression of GA3ox1 is significantly down-regulated during the germination of BIR3 OE seeds.These results suggest that BIR3 may also play a role during seed germination.