Isoform-specific function underlies differential herbicide resistance of the W574L mutation in peanut AhALS isozymes

Peanut (Arachis hypogaea) is an important cash crop with high oil yield per unit area; it is considered an excellent source for producing premium edible oil and a variety of processed foods. Weed infestation poses a critical challenge to peanut cultivation. Although the acetolactate synthase (ALS)-inhibiting herbicide imazethapyr is extensively applied for weed control in peanut fields, it also causes significant phytotoxicity to the crop. The development of herbicide-resistant varieties through gene editing represents a promising sustainable solution; however, the identification of efficient gene-editing targets for conferring high herbicide resistance in peanut remains limited. Plant resistance to imazethapyr arises primarily from mutations in the target gene ALS.

In this study, a total of four AhALS genes (AhALS1a, AhALS1b, AhALS2a and AhALS2b) with high homology were identified in cultivated peanut. Importantly, we demonstrated for the first time that all four AhALS proteins localize to the chloroplast. Using site-directed mutagenesis, we introduced mutations at position 574 (W574L) in AhALS1a, AhALS2a, AhALS1b and AhALS2b. Both petri-dish and whole-plant bioassays revealed that transgenic Arabidopsis thaliana expressing AhALS2b-W574L remained susceptible to imazethapyr. By contrast, lines expressing AhALS1a-W574L, AhALS2a-W574L or AhALS1b-W574L exhibited high herbicide resistance. The reduced binding ability of resistance AhALS isoforms to imazethapyr was mainly responsible for transgenic lines resistance to imazethapyr. Furthermore, enzymatic and molecular interaction analyses critically showed that the W574L mutation in AhALS2b, unlike its paralog AhALS1b, did not reduce binding affinity to imazethapyr and thus conferred no resistance.

This demonstrates marked functional divergence among peanut ALS isozymes, where an identical mutation yields different phenotypic outcomes depending on the protein structure. Consequently, position 574 in AhALS2b is an ineffective editing target, underscoring the need for isozyme-specific screening to develop herbicide-resistant peanut varieties. © 2026 Society of Chemical Industry.