RESUMEN
A 2,4-dichlorophenoxyactic acid (2,4-D)-resistant population of Amaranthus tuberculatus (common waterhemp) from Nebraska, USA, was previously found to have rapid metabolic detoxification of the synthetic auxin herbicide 2,4-D. We purified the main 2,4-D metabolites from resistant and susceptible plants, solved their structures by nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HRMS), and synthesized the metabolites to determine their in planta toxicity. Susceptible plants conjugated 2,4-D to aspartate to form 2,4-D-aspartic acid (2,4-D-Asp), while resistant plants had a unique metabolic profile where 2,4-D was hydroxylated into 5-OH-2,4-D, followed by conjugation into a sugar metabolite (2,4-D-5-O-d-glucopyranoside) and subsequent malonylation into 2,4-D-(6'-O-malonyl)-5-O-d-glucopyranoside. Toxicological studies on waterhemp and Arabidopsis thaliana confirmed that the hydroxylated metabolite lost its auxinic action and toxicity. In contrast, the 2,4-D-Asp metabolite found in susceptible plants retained some auxinic action and toxicity. These results demonstrate that 2,4-D-resistant A. tuberculatus evolved novel detoxification reactions not present in susceptible plants to rapidly metabolize 2,4-D, potentially mediated by cytochrome P450 enzymes that perform the initial 5-hydroxylation reaction. This novel mechanism is more efficient to detoxify 2,4-D and produces metabolites with lower toxicity compared to the aspartic acid conjugation found in susceptible waterhemp.