Target-site and metabolic mechanisms confer broad resistance to PPO-inhibiting herbicides in Amaranthus retroflexus

Amaranthus retroflexus is among the most problematic and widely distributed weed species in soybean production systems across China. Fomesafen (FSA), a herbicide that blocks protoporphyrinogen oxidase (PPO), has been extensively used for its control. However, repeated applications of FSA have promoted the evolution of resistance in A. retroflexus. In this study, two resistant genotypes of A. retroflexus (R1 and R2) were identified, both exhibiting resistance indices (GR₅₀ R/S) exceeding 20-fold relative to the wild type. The results of PPO enzyme activity assays were consistent with those obtained from whole-plant bioassays. Sequence analysis of the PPX2 gene revealed that genotype R2 harbored an Arg-128-Gly mutation. Inhibitors of CYP450 and GST significantly reversed resistance to FSA; however, CYP450 inhibitors produced a stronger reversal effect, whereas GST inhibitors were less effective. The reduced uptake and enhanced metabolic degradation of FSA observed in R2 (R¹²⁸G) likely account for its high level of resistance. High-resolution LC-Q-TOF-MS/MS analysis identified four primary FSA metabolites in A. retroflexus (C₁₃H₇ClF₃NO₃, C₁₄H₇ClF₃NO₅, C₁₅H₁₁ClF₃NO₅S, C₇H₄ClF₃O). To identify candidate resistance genes, transcriptome sequencing combined with RT-qPCR validation revealed two common CYP450 genes (ArCYP76AD1, ArCYP77A2) and one glucosyltransferase gene (ArUGT88B1) shared by both R1 and R2. Molecular docking analysis further supported ArCYP77A2 as candidate genes involved in resistance. Heterologous expression of ArCYP77A2 in Arabidopsis thaliana conferred resistance to FSA. These findings indicate the importance of CYP450 genes in non-target-site resistance to FSA in A. retroflexus through enhanced metabolic detoxification, although further functional characterization is still required.