Promiscuous cytochrome P450s confer metabolic resistance to synthetic auxin herbicides in the weedy grass Echinochloa phyllopogon

Abstract

Auxinic herbicides are critically important tools for weed control in modern agriculture. Although resistant weed populations have been increasing in recent years, the underlying mechanisms remain largely unknown. In a multiple-herbicide resistant line of the paddy weed Echinochloa phyllopogon, overexpression of catalytically promiscuous CYP81A P450 enzymes has been shown to confer resistance to diverse herbicides. Here, we investigated the role of these P450s in quinclorac resistance in E. phyllopogon. Arabidopsis thaliana plants expressing CYP81A12 and CYP81A21 genes, previously demonstrated to metabolize other herbicides, showed low but detectable levels of quinclorac resistance. A similar activity was also observed for CYP81A24, another promiscuous enzyme present in E. phyllopogon. Moreover, Escherichia coli strains expressing these genes produced putative hydroxylated metabolites of quinclorac. In silico analyses revealed that quinclorac binds stably near the catalytic heme center of CYP81A12, CYP81A21, and CYP81A24, but not CYP81A18, the importance of spatial alignment between the substrate recognition site and the heme center in determining metabolic activity and resistance potential. Consistent with the phenotype of resistant E. phyllopogon, A. thaliana expressing CYP81A12 and CYP81A21 produced significantly less ethylene following quinclorac treatment, linking P450-mediated metabolism to the suppression of quinclorac-induced phytotoxic responses. Finally, we demonstrated that CYP81A enzymes also confer resistance to other auxinic herbicides, florpyrauxifen-benzyl and 2,4-D. These findings provide new insights into the molecular basis of auxinic herbicide resistance and underscore the role of CYP81A P450s in metabolic detoxification and the prevention of herbicide-induced phytotoxic responses.