Biochemical Characterization of Fatty Acid Thioesterase Target Site Mutants and their Implication on Herbicide Resistance

Herbicide resistance threatens effective weed control in modern agriculture, particularly in grass weeds such as Alopecurus myosuroides and Lolium multiflorum . Cinmethylin is a pre-emergence herbicide with a novel mode of action that inhibits plastidial fatty acid thioesterases (FATs), enzymes essential for fatty acid biosynthesis. Although no cases of field resistance to cinmethylin have been reported, its resistance risk has not been fully assessed. In this study, we biochemically characterized defined amino acid substitutions in FAT A and FAT B to evaluate their effects on cinmethylin inhibition profile. Some substitutions in FAT A reduced inhibition in vitro , with mutations at residue R171 causing the largest shifts in sensitivity. However, these highly resistant variants required multiple specific nucleotide polymorphisms and are therefore predicted to be unlikely to arise in weed populations. In FAT B, sensitivity shifts were generally moderate. Importantly, most substitutions that reduced cinmethylin sensitivity also impaired enzymatic activity, suggesting limited viability in planta . Overall, these results indicate that while theoretical target-site resistance mechanisms exist, the practical risk of rapid resistance evolution to cinmethylin is low, supporting its value for integrated grass weed management