Unraveling the Role of P450 Reductase in Herbicide Metabolic Resistance Mechanism

Abstract

Plants 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 carrying CYP81A12 from Echinochloa phyllopogon, which metabolizes a broad spectrum of herbicides, were crossed with CPR knockout atr1 or atr2 mutant lines. Homozygous gene knockout was confirmed using PCR, and gene copy number of CYP81A12 was determined using ddPCR. Arabidopsis lines expressing CYP81A12 in combination with atr1 or atr2 knockout were used for herbicide dose-response and metabolism studies. Knocking out ATR1 in transgenic Arabidopsis CYP81A12 significantly 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 the ATR2 also decreased herbicide resistance, but to a lower magnitude than ATR1. These results corroborate [1/2] 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, by identifying that a single CPR knockout can reverse herbicide sensitivity . The different CPRs found in weeds have potential as target genes to manage metabolic herbicide resistance evolution. We further provide an in-depth exploration of the evolutionary implications in weed management arising from the results. HIGHLIGHTSKnocking out cytochrome P450 reductase 1 in herbicide tolerant Arabidopsis reduces herbicide resistance, highlighting CPRs as targets for managing herbicide resistance evolution in weeds.