Rice qGL3 negatively regulates brassinosteroid signaling by regulating the phosphorylation level and stability of OsGSK3, which modulates OsBZR1 phosphorylation

Brassinosteroids (BRs) play essential roles in regulating plant growth and development, however, gaps still remain in our understanding of the BR signaling network. We previously cloned a grain length quantitative trait locus qGL3, encoding a rice (Oryza sativa L.) protein phosphatase with Kelch-like repeat domain (OsPPKL1), that negatively regulates grain length and BR signaling. To further explore the BR signaling network, we performed phosphoproteomic analysis to screen qGL3-regulated downstream components. We selected a 14-3-3 protein OsGF14b from the phosphoproteomic data for further analysis. qGL3 promoted the phosphorylation of OsGF14b and induced the interaction intensity between OsGF14b and OsBZR1. In addition, phosphorylation of OsGF14b played an important role in regulating nucleocytoplasmic shuttling of OsBZR1. The serine acids (Ser²⁵⁸Ser²⁵⁹) residues of OsGF14b play an essential role in BR-mediated responses and plant development. Genetic and molecular analyses indicated that OsGF14b functions as a negative regulator in BR signaling and represses the transcriptional activation activity of OsBZR1. Collectively, these results demonstrate that qGL3 induces the phosphorylation of OsGF14b, which modulates nucleocytoplasmic shuttling and transcriptional activation activity of OsBZR1, to eventually negatively regulate BR signaling and grain length in rice.

Brassinosteroids (BRs) are a class of polyhydroxylated steroidal phytohormones and essential for plant growth and development. Rice BRASSINOSTEROID-INSENSITIVE1 (BRI1)-ASSOCIATED RECEPTOR KINASES (OsBAKs) are plasma membrane (PM)-localized receptor kinases, belonging to the subfamily of leucine-rich repeat (LRR) receptor kinases. In Arabidopsis, BRs induce the formation of BRI1-BAK1 heterodimer complex and transmit the signal cascade to the BRASSINAZOLE RESISTANT1/bri1-EMS-SUPPRESSOR1 (BZR1/BES1) to regulate BR signaling. In rice, we found that OsBZR1 binds directly to the promoter of OsBAK2, not OsBAK1, and represses the expression of OsBAK2 to form a BR feedback inhibition loop. Additionally, the phosphorylation of OsBZR1 by OsGSK3 reduced its binding activity to OsBAK2 promoter. The osbak2 displays a typical BR deficient phenotype and negatively modulates the accumulation of OsBZR1. Interestingly, the grain length of osbak2 mutant was increased whereas the cr-osbak2/cr-osbzr1 double mutant restored the reduced grain length of cr-osbzr1 mutant, implying that the increased grain length of osbak2 may be due to the rice SERKs-dependent pathway. Our study reveals a novel mechanism of OsBAK2 and OsBZR1 engaging in a negative feedback loop to maintain rice BR homeostasis, facilitating to a deeper understanding of BR signaling network and grain length regulation in rice.

Grain size and lodging resistance determine rice yield by affecting grain weight, and are also associated with secondary cell wall (SCW) thickness. In this study, we demonstrate the dual roles of OsCesAs, which encode cellulose synthases, in the regulation of grain size and culm mechanical strength through the brassinosteroid (BR) signaling pathway. RT-qPCR analysis revealed that the transcript levels of all rice CesA gene family members were induced by exogenous brassinolide (BL) treatment. Moreover, OsCesA4/7/9, which are involved in secondary cell wall synthesis, exhibited similar expression patterns during rice culm and grain development. Overexpression of OsCesA4/9 not only enhanced secondary cell wall thickness and cellulose content in culms, thereby improving mechanical strength, but also increased grain size. OsBZR1 directly binds to the promoters of OsCesA4/7/9 through a regulatory mechanism distinct from that in Arabidopsis, thus negatively regulating their expression. This ultimately results in increased culm cellulose content and mechanical strength in the cr-osbzr1 mutant. However, analysis of culm mechanical strength and grain size in mutants of qGL3, OsGSK3, and OsBZR1, which are key components of the BR signaling pathway, revealed a weak correlation between these two traits. This work helps breeders better understand the important functions of BR signaling in plant growth and development, enabling targeted molecular design breeding of rice for lodging tolerance and grain size.