Brassinosteroid-associated transcriptional regulatory networks in rice.

Brassinosteroids (BRs) are crucial in regulating growth, development, and environmental adaptation in plants. To understand the complex regulatory network of BRs, we integrated RNA-Seq, sRNA-Seq, and degradome data from rice (Oryza sativa L.) seedlings subjected to brassinolide (BL) treatment and from BR core signaling gene mutants (qgl3, osgsk3, and osbzr1). Using guide-gene-directed network construction and machine learning-based transcription factor (TF) target prediction methods, we also constructed BR-associated gene co-expression and regulatory networks. This study revealed synergistic regulation at the transcriptional level between BR core signaling mutants and BL treatment, highlighting the central modules involving qGL3 and OsGSK3 in BR signaling. The constructed regulatory network was enriched for TFs centered on the qGL3/OsPPKL1-OsGSK3-OsBZR1 pathway. Potential BR-core transcription factor genes were identified using co-expression and regulatory network analyses, and their loss-of-function mutants were evaluated using BL sensitivity assays and immunoblot analyses. Additionally, a BR transcriptional regulatory network database was developed, providing a valuable resource for the rice BR research community.