Metabolic resistance to acetolactate synthase inhibitors in Beckmannia syzigachne: identification of CYP81Q32 and its transcription regulation

SUMMARY Frequent herbicide use selects for herbicide resistance in weeds. Cytochrome P450s are important detoxification enzymes responsible for herbicide resistance in plants. We identified and characterized a candidate P450 gene (BsCYP81Q32) from the problematic weed Beckmannia syzigachne to test whether it conferred metabolic resistance to the acetolactate synthase‐inhibiting herbicides mesosulfuron‐methyl, bispyribac‐sodium, and pyriminobac‐methyl. Transgenic rice overexpressing BsCYP81Q32 was resistant to the three herbicides. Equally, rice overexpressing the rice ortholog gene OsCYP81Q32 was more resistant to mesosulfuron‐methyl. Conversely, an OsCYP81Q32 gene knockout generated using CRISPR/Cas9 enhanced mesosulfuron‐methyl sensitivity in rice. Overexpression of the BsCYP81Q32 gene resulted in enhanced mesosulfuron‐methyl metabolism in transgenic rice seedlings via O‐demethylation. The major metabolite, demethylated mesosulfuron‐methyl, was chemically synthesized and displayed reduced herbicidal effect in plants. Moreover, a transcription factor (BsTGAL6) was identified and shown to bind a key region in the BsCYP81Q32 promoter for gene activation. Inhibition of BsTGAL6 expression by salicylic acid treatment in B. syzigachne plants reduced BsCYP81Q32 expression and consequently changed the whole plant response to mesosulfuron‐methyl. Sequence polymorphisms in an important region of the BsTGAL6 promoter may explain the higher expression of BsTGAL6 in resistant versus susceptible B. syzigachne plants. Collectively, the present study reveals the evolution of an herbicide‐metabolizing and resistance‐endowing P450 and its transcription regulation in an economically important weedy plant species. Significance Statement Schematic illustration of the molecular basis of metabolic resistance to commonly used acetolactate synthase‐inhibiting herbicides in Beckmannia syzigachne with CYP81Q32 being the key player in the resistance evolution under regulation of a transcription factor (TGAL6). This knowledge provides insights into the evolutionary mechanisms underlying the dominance of B. syzigachne as a hard‐to‐control weed in agriculture system. Beckmannia syzigachne is a problematic weed in wheat and rapeseed cropping especially in China. Our study establishes the molecular basis of CYP81Q32‐mediated metabolic resistance to commonly used ALS‐inhibiting herbicides in B. syzigachne. Resistance evolution via CYP81Q32 was regulated by a transcription factor (TGAL6).