Novel amino acid substitutions in Protoporphyrinogen oxidase 1 endow resistance to PPO-inhibiting herbicides in Bassia scoparia

Abstract PPO-inhibiting herbicides are widely used to manage weeds in different cropping systems, yet resistance evolution threatens their long-term efficacy. Here, we investigated the molecular basis of resistance to PPO-inhibiting herbicides in Bassia scoparia biotypes collected from four locations in North Dakota, USA. Greenhouse dose–response assays revealed high levels of resistance to saflufenacil and carfentrazone-ethyl, while fomesafen retained full efficacy across all biotypes. Resistant plants did not show increased copy number or elevated expression of PPO1 or PPO2 . Sequencing of survivor plants revealed conserved PPO2 sequences, but consistent target-site substitutions at position F454 in PPO1, including F454I, F454L, and F454V. In vitro enzyme assays demonstrated that these substitutions impair PPO1 sensitivity to saflufenacil and carfentrazone-ethyl, but not to fomesafen. Ectopic expression of B. scoparia PPO1 F454 mutant variants in Arabidopsis thaliana conferred tolerance to saflufenacil and carfentrazone-ethyl, but not to fomesafen, supporting greenhouse and in vitro results. Molecular modeling indicated that the conformational flexibility and interaction profile of fomesafen enables it to maintain binding to mutated PPO1 variants, in contrast to the more rigid structures of saflufenacil and carfentrazone-ethyl. A yeast-based complementation system further confirmed that F454 substitutions decrease herbicide sensitivity. In addition, developmental profiling showed distinct expression patterns of PPO1 and PPO2 during early growth stages in B. scoparia and Amaranthus spp., highlighting isoform-specific roles. Together, these findings represent the first reported PPO1 target-site mutations in a broadleaf weed species as a key mechanism of resistance and highlight that fomesafen is effective to control resistant B. scoparia populations.