Unraveling the Role of P450 Reductase in Herbicide Metabolic Resistance Mechanism

Abstract

SUMMARYPlants require cytochrome P450 reductase (CPR) to supply two electrons for cytochrome P450 monooxygenase enzymes (P450) to react with an organic substrate. The transfer of electrons to the P450 active site in the P450 catalytic site relies on a robust and intricate CPR:P450 complex in the endoplasmic reticulum membrane.Transgenic Arabidopsis plants carryingCYP81A12fromEchinochloa phyllopogon, which metabolize a broad spectrum of herbicides, were crossed with CPR knockoutatr1oratr2mutant lines. Homozygous gene knockout was confirmed using PCR, and gene copy number ofCYP81A12was determined using ddPCR. Arabidopsis lines expressingCYP81A12in combination withatr1oratr2knockout were used for herbicide dose-response and metabolism studies.Knocking outATR1in transgenic ArabidopsisCYP81A12significantly reduced herbicide resistance. Transgenic mutant plants (CYP81A12 atr1-b) had a 3.6-, 5.6-, 6.8- and at least 26- fold reduction in resistance to mesotrione, 2,4-D, penoxsulam and chlorsulfuron, respectively, in the dose-response assay. Knockouts of theATR2also decreased herbicide resistance, but to a lower magnitude thanATR1. These results were confirmed through the ½ MS medium assay, and herbicide resistance reduction was observed for additional tested herbicides, bensulfuron, propoxycarbazone and bentazon.Our findings highlight the importance of CPRs in metabolic herbicide resistance in plants, specifically identifying thatATR1is the most important in the CPR:P450 complex in Arabidopsis for herbicide metabolism. The different CPRs found in weeds have potential as target genes to manage herbicide resistance evolution. We further provide an in-depth exploration of the evolutionary implications in weed management arising from the results.