OsBZR1 turnover mediated by OsSK22-regulated U-box E3 ligase OsPUB24 in rice BR response

Oryza sativa BRASSINAZOLE RESISTANT 1 (OsBZR1) is the closest rice homolog of the Arabidopsis BZR1 and bri1-EMS-SUPPRESSOR 1 (BES1)/BZR2 transcription factors. OsBZR1 plays a central role in the rice brassinosteroid signaling pathway. Despite its functional importance, the control mechanism by which the cellular stability of OsBZR1 is regulated has not yet been fully elucidated. Here, we report that a rice U-box E3 ubiquitin (Ub) ligase OsPUB24 acts as a negative regulator in the BR signaling pathway via the 26S proteasome-dependent degradation of OsBZR1. The ospub24 T-DNA knock-out mutant and Ubi:RNAi-OsPUB24 knock-down rice plants displayed enhanced seedling growth, increased lamina joint bending, and hypersensitivity to brassinolide (BL). The expressions of the BR biosynthetic genes suppressed by BR in a negative feedback loop were lower in the mutant progeny than in the wild-type rice plants, which indicated increased BR responses in the mutant line. OsPUB24 ubiquitinated OsBZR1, resulting in the proteasomal degradation of OsBZR1. In addition, the stability of OsPUB24 was downregulated by BL and bikinin, an inhibitor of Oryza sativa Shaggy/GSK3-like kinase 22 (OsSK22). OsSK22, the homolog of Arabidopsis BRASSINOSTEROID INSENSITIVE 2 (BIN2) protein kinase, phosphorylated OsPUB24 and elevated the cellular stability of OsPUB24. Our findings suggest that OsPUB24 participates in OsBZR1 turnover, and that the regulatory networks of OsPUB24, OsSK22 and OsBZR1 are crucial for fine-tuning the BR response in rice.