OsbHLH069通过对LAX1-LAX2复合体的竞争性干扰负调控水稻穗发育OA

[Objective]The LAX1-LAX2 complex is a key module regulating axillary meristem development in the rice panicle.The bHLH(basic helix-loop-helix)transcription factor OsbHLH069 interacts with LAX1,and its gain-of-function mutant nsp1-D(no spikelet 1-dominant)exhibits a sparse panicle phenotype.This study aimed to elucidate the molecular interaction model among these three proteins and clarify the mechanism by which they regulate panicle branching development.[Methods]The nuclear localization signal and transcriptional activation domain of OsbHLH069 were analyzed using rice protoplasts and yeast systems.The interaction between OsbHLH069 and LAX2 was investigated through yeast two-hybrid assays,bimolecular fluorescence complementation,and in vitro pull-down assays.The lax2-4 nsp1-D double mutant was generated by field crossing,and genetic interactions were analyzed by quantitative real-time PCR.The interaction regions among the three proteins were mapped using truncation mutants and yeast two-hybrid assays.The interaction model was further explored via luciferase complementation assays.[Results]OsbHLH069 is a typical nuclear-localized transcriptional activator,with its nuclear localization signal located at amino acids 266‒285 and its transcriptional activation domain at the N-terminal region(amino acids 1‒120).OsbHLH069 directly interacted with LAX2 both in vivo and in vitro.OsbHLH069 interacted with LAX1 through its bHLH domain and with LAX2 through its C-terminal region;these interaction regions overlapped with the LAX1-LAX2 interaction interface.OsbHLH069 competitively inhibited the stability of the LAX1-LAX2 complex.The lax2-4 nsp1-D double mutant showed more severe defects in branch number and spikelets per panicle than either single mutant,resembling the previously reported phenotype of the lax1 nsp1-D double mutant.[Conclusion]A phased regulatory model is proposed:during early panicle axillary meristem development,the LAX1-LAX2 complex promotes axillary meristem initiation;as development progresses,OsbHLH069 achieves fine-tuned negative regulation of the panicle axillary meristem by competitively disassembling the LAX1-LAX2 complex.