Zinc finger transcription factor ZAT6 modulates pyroxsulam resistance by activating cytochrome CYP92C6 expression in Italian ryegrass ( Lolium perenne ssp. multiflorum )

Abstract Herbicide resistance threatens sustainable weed management. Cytochrome P450s (P450s) are well-established core enzymes that mediate metabolic resistance via herbicide detoxification. Nevertheless, the regulatory mechanisms of P450 overexpression in resistant weeds remain largely uncharacterized. Here, we aimed to characterize the functions of P450 genes in pyroxsulam-resistant Italian ryegrass (Lolium perenne ssp. multiflorum) and elucidate their transcriptional regulatory mechanisms. CYP92C6 was identified as a pivotal gene contributing to metabolic resistance in L. perenne ssp. multiflorum. Overexpression of CYP92C6 in Arabidopsis thaliana or rice significantly enhanced tolerances to pyroxsulam. Combined analysis of transcriptomic data and dual-luciferase reporter assays demonstrated the zinc finger transcription factor ZAT6 acts as a direct upstream regulator of CYP92C6, and ZAT6 specifically binds to the promoter region of CYP92C6. The critical cis-acting element responsible for ZAT6 binding within the CYP92C6 promoter was also defined. Overexpression of ZAT6 in Arabidopsis enhanced herbicide resistance, and nuclear localization of ZAT6 was indispensable for its regulatory activity. Collectively, we conclude that ZAT6 directly activates the expression of CYP92C6 to facilitate pyroxsulam detoxification, thereby conferring pyroxsulam resistance in L. perenne ssp. multiflorum. These findings deepen our mechanistic understanding of the transcriptional regulation underlying metabolic resistance to pyroxsulam in L. perenne ssp. multiflorum.