The brassinosteroid signaling-related ILI–OsAIF–OsbHLH92 transcription factor module antagonistically controls leaf angle and grain size in rice

Atypical basic helix-loop-helix (bHLH) transcription factors, which lack the basic region for DNA binding, are important elements of brassinosteroid (BR) signaling. Recently, our systematic characterization of the rice (Oryza sativa) INCREASED LEAF INCLINATION (ILI) subfamily of atypical bHLHs revealed their indispensable roles in BR-mediated growth and development. Here, we reported the isolation of two additional rice ILI-interacting atypical bHLHs, ATBS1-INTERACTING FACTOR 1 (OsAIF1)/OsbHLH176 and OsAIF2/OsbHLH178. Genetic and cytological analyses of the OsAIFs knockout mutants and overexpression lines revealed that OsAIF1 and OsAIF2 negatively regulate rice leaf inclination and grain size in a synergistic and redundant manner. Compared to the wild type, osaif knockout mutants exhibited hypersensitivity to BR, while OsAIF1 and OsAIF2 overexpression lines showed greatly reduced sensitivity or complete insensitivity to BR, indicating that these two OsAIFs act as major negative regulators of rice BR signaling. As ILI-interacting negative atypical HLHs, OsAIF1 and OsAIF2 genetically counteracted the positive ILI subfamily of atypical HLHs. Moreover, OsAIF1 and OsAIF2 physically interacted with and antagonized OsbHLH92, a positive regulator of BR signaling, thereby modulating rice development and gene transcription. These findings suggested that the atypical HLHs (ILIs and OsAIF1/OsAIF2) and the bHLH (OsbHLH92) transcription factors form a triantagonistic cascade in rice, counteracting each other to fine-tune leaf angle and grain size through BR signaling. Our results provide insights into the mechanisms balancing BR signaling and growth in rice.