Unraveling the Role of P450 Reductase in Herbicide Metabolic Resistance Mechanism

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 <i>CYP81A12</i> from <i>Echinochloa phyllopogon</i>, which metabolizes a broad spectrum of herbicides, were crossed with CPR knockout <i>atr1</i> or <i>atr2</i> mutant lines. Homozygous gene knockout was confirmed using PCR, and gene copy number of <i>CYP81A12</i> was determined using ddPCR. Arabidopsis lines expressing <i>CYP81A12</i> in combination with <i>atr1</i> or <i>atr2</i> knockout were used for herbicide dose-response and metabolism studies. Knocking out <i>ATR1</i> in transgenic Arabidopsis <i>CYP81A12</i> significantly reduced herbicide resistance. Transgenic mutant plants (<i>CYP81A12 atr1-b</i>) 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 <i>ATR2</i> also decreased herbicide resistance but to a lower magnitude than <i>ATR1</i>. These results corroborate ½ MS medium assay, and herbicide resistance reduction was observed for additional herbicides including bensulfuron-methyl, propoxycarbazone-sodium, 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 resistance. 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.