Overexpression of CYP81A8 confers tolerance to flusulfinam in rice(Oryza sativa).

Flusulfinam is a novel 4-hydroxyphenylpyruvate dioxygenase (HPPD)-inhibiting herbicide independently developed in China for use in rice paddies. It exhibits characteristic advantages including high efficacy, broad-spectrum control, low residual persistence, and reduced environmental impact. Notably, flusulfinam displays exceptional selectivity for rice (Oryza sativa) under direct-seeding cultivation systems, emphasizing its practical significance. To elucidate the intrinsic mechanism underlying the significant differences in flusulfinam tolerance among diverse rice cultivars, we identified a high-tolerance cultivar (Jiahua 1) and a low-tolerance cultivar (Longyang 11). Biochemical analyses revealed that the tolerance difference was attributed to variations in metabolic detoxification and oxidative stress responses. The Jiahua 1 variety exhibited significantly enhanced metabolic detoxification of the flusulfinam, mediated by elevated Cytochrome P450 (CYP450) activity. Additionally, increased activities of antioxidant enzymes (peroxidase, superoxide dismutase, catalase) in Jiahua 1 effectively mitigated herbicide-induced oxidative damage. Importantly, these tolerance mechanisms were independent of differential herbicide uptake or translocation. Transcriptome sequencing coupled with quantitative real-time PCR (qRT-PCR) screening identified three candidate CYP genes with significantly increased expression levels. Functional validation using yeast expression systems, rice callus assays, and Arabidopsis overexpression assays, along with molecular docking, demonstrated that CYP81A8 effectively metabolizes flusulfinam. CRISPR/Cas9-mediated knockout of the CYP81A8 gene significantly increased rice sensitivity to flusulfinam. This study establishes that CYP81A8-mediated enhanced metabolic detoxification plays a key role in conferring flusulfinam tolerance in rice. These findings provide a theoretical foundation for the scientific application of this herbicide, and the CYP81A8 gene represents a valuable genetic resource for breeding herbicide-resistant crops.