Resistance to a nonselective 4‐hydroxyphenylpyruvate dioxygenase‐inhibiting herbicide via novel reduction–dehydration–glutathione conjugation in Amaranthus tuberculatus

Metabolic resistance to 4‐hydroxyphenylpyruvate dioxygenase (HPPD)‐inhibiting herbicides is a threat in controlling waterhemp (Amaranthus tuberculatus) in the USA. We investigated resistance mechanisms to syncarpic acid‐3 (SA3), a nonselective, noncommercial HPPD‐inhibiting herbicide metabolically robust to Phase I oxidation, in multiple‐herbicide‐resistant (MHR) waterhemp populations (SIR and NEB) and HPPD inhibitor‐sensitive populations (ACR and SEN). Dose–response experiments with SA3 provided ED(50)‐based resistant : sensitive ratios of at least 18‐fold. Metabolism experiments quantifying parent SA3 remaining in excised leaves during a time course indicated MHR populations displayed faster rates of SA3 metabolism compared to HPPD inhibitor‐sensitive populations. SA3 metabolites were identified via LC‐MS‐based untargeted metabolomics in whole plants. A Phase I metabolite, likely generated by cytochrome P450‐mediated alkyl hydroxylation, was detected but was not associated with resistance. A Phase I metabolite consistent with ketone reduction followed by water elimination was detected, creating a putative α,β‐unsaturated carbonyl resembling a Michael acceptor site. A Phase II glutathione–SA3 conjugate was associated with resistance. Our results revealed a novel reduction–dehydration–GSH conjugation detoxification mechanism. SA3 metabolism in MHR waterhemp is thus atypical compared to commercial HPPD‐inhibiting herbicides. This previously uncharacterized detoxification mechanism presents a unique opportunity for future biorational design by blocking known sites of herbicide metabolism in weeds.