The BELL-type homeobox transcription factor RLC3/OsBLH4 controls leaf rolling and drought tolerance via KNOX-BELL-lignin regulatory network in rice

Abstract Moderate leaf rolling in rice is crucial for plant architecture and stress adaptation, but its molecular regulation remains unclear. We investigated the role of RLC3/OsBLH4, a BELL-type homeobox transcription factor, in controlling leaf rolling and drought tolerance, addressing gaps in lignin biosynthesis and cell wall development mechanisms. We used gene map-based cloning ( rlc3-1 , rlc3-2 ), CRISPR/Cas9 knockout lines ( rlc3-ko#11 , rlc3-ko#12 ), and allelic complementation to validate RLC3 ’s function. Additionally, we employed biochemical assays, gene expression analysis, and protein interaction studies to explore its regulatory network. RLC3 mutations impaired lignin biosynthesis and secondary cell wall formation, reducing bulliform cells area and causing midrib defects. These structural abnormalities accelerated water loss, leading to excessive leaf rolling and compromised drought tolerance. Mechanistically, RLC3 directly activates lignin synthesis genes ( OsPAL5 , OsCOMT5 , OsCCR4, OsCAld5H1 ) and interacts with KNOX transcription factors (OSH1, OSH45, OSH71) to form a KNOX-BELL complex, further regulating lignin content and cell wall development. RLC3 orchestrates lignin deposition and secondary cell wall development to control leaf rolling, water transport, and drought tolerance. This study reveals a novel KNOX-BELL-lignin regulatory module governing leaf morphology and stress adaptation, offering targets for crop improvement under drought conditions.