Detoxification via rapid oxidative metabolism of 4-hydroxyphenylpyruvate dioxygenase (HPPD)-inhibiting herbicides, catalyzed by cytochrome P450s, primarily confers non-target-site resistance in waterhemp (Amaranthus tuberculatus). We investigated resistance mechanisms to a presumed metabolically blocked, non-selective HPPD-inhibiting herbicide, syncarpic acid-3 (SA3) (IUPAC name: 6-[2,4-bis(trifluoromethyl)benzoyl]-2,2,4,4-tetramethylcyclohexane-1,3,5-trione), in multiple-herbicide-resistant (MHR) waterhemp populations (SIR, NEB) compared with HPPD-inhibitor-sensitive populations (ACR, SEN). SA3 is a synthetic compound derived from the natural product, leptospermone (IUPAC name: 2,2,4,4-tetramethyl-6-(3-methylbutanoyl)cyclohexane-1,3,5-trione). SA3 and two other non-selective triketones (SA1 and SA2) that possess a 2-benzoyl or 2-heteroaroyl cyclohexane-1,3,5-trione moiety were investigated. Dose-response experiments with SA3 determined fold-resistance levels and metabolism experiments using excised leaves quantified parent SA3 during a 16-h time course. SA3 metabolites were identified and quantified in two untargeted metabolomics experiments. The first experiment used excised leaf extracts of SIR and ACR at 4 and 12 h and consisted of three biological replicates. The second experiment used whole plant extracts of SIR, NEB, SEN and ACR at 12 and 24 h and consisted of five biological replicates. Resistant-to-sensitive ratios based on plant survival were at least 18-fold. MHR populations metabolized SA3 faster than HPPD-inhibitor-sensitive populations. Multivariate statistics displayed clear separation among waterhemp populations and time points after treatment. Several putative SA3 metabolites were characterizing for MHR waterhemp, and their mass spectra revealed lack of metabolism on the CF3-substituted aryl ring of SA3. A Phase I metabolite consistent with ketone reduction followed by water elimination was detected, creating a putative α,ß-unsaturated carbonyl resembling a Michael acceptor, a potential glutathione S-transferase substrate. A Phase II glutathione-SA3 conjugate, minus an oxygen, and its catabolites were characterizing for resistance. Our results revealed a novel reduction-dehydration-GSH conjugation detoxification mechanism. SA3 metabolism in MHR waterhemp is thus atypical compared to commercial HPPD inhibitors and presents a unique opportunity for biorational herbicide design and investigating new inhibitors of plant detoxification enzymes.

Detoxification via reduction-dehydration-GSH conjugation of a non-selective HPPD-inhibiting herbicide in waterhemp (Amaranthus tuberculatus)

ACS Fall 2021

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