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Weed infestation poses a critical challenge to peanut cultivation. Although the acetolactate synthase (ALS)\u2010inhibiting herbicide imazethapyr is extensively applied for weed control in peanut fields, it also causes significant phytotoxicity to the crop. The development of herbicide\u2010resistant varieties through gene editing represents a promising sustainable solution; however, the identification of efficient gene\u2010editing targets for conferring high herbicide resistance in peanut remains limited. Plant resistance to imazethapyr arises primarily from mutations in the target gene ALS.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>RESULTS<\/jats:title>\n                    <jats:p>\n                      In this study, a total of four\n                      <jats:italic>AhALS<\/jats:italic>\n                      genes (\n                      <jats:italic>AhALS1a<\/jats:italic>\n                      ,\n                      <jats:italic>AhALS1b<\/jats:italic>\n                      ,\n                      <jats:italic>AhALS2a<\/jats:italic>\n                      and\n                      <jats:italic>AhALS2b<\/jats:italic>\n                      ) 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\u2010directed mutagenesis, we introduced mutations at position 574 (W574L) in\n                      <jats:italic>AhALS1a<\/jats:italic>\n                      ,\n                      <jats:italic>AhALS2a<\/jats:italic>\n                      ,\n                      <jats:italic>AhALS1b<\/jats:italic>\n                      and\n                      <jats:italic>AhALS2b<\/jats:italic>\n                      . Both petri\u2010dish and whole\u2010plant bioassays revealed that transgenic\n                      <jats:italic>Arabidopsis thaliana<\/jats:italic>\n                      expressing\n                      <jats:italic>AhALS2b<\/jats:italic>\n                      \u2010W574L remained susceptible to imazethapyr. By contrast, lines expressing\n                      <jats:italic>AhALS1a<\/jats:italic>\n                      \u2010W574L,\n                      <jats:italic>AhALS2a<\/jats:italic>\n                      \u2010W574L or\n                      <jats:italic>AhALS1b<\/jats:italic>\n                      \u2010W574L 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.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>CONCLUSIONS<\/jats:title>\n                    <jats:p>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\u2010specific screening to develop herbicide\u2010resistant peanut varieties. \u00a9 2026 Society of Chemical Industry.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1002\/ps.70798","type":"journal-article","created":{"date-parts":[[2026,4,8]],"date-time":"2026-04-08T12:01:01Z","timestamp":1775649661000},"page":"7308-7320","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Isoform\u2010specific function underlies differential herbicide resistance of the\n                    <scp>W574L<\/scp>\n                    mutation in peanut\n                    <scp>AhALS<\/scp>\n                    isozymes"],"prefix":"10.1002","volume":"82","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-6653-9119","authenticated-orcid":false,"given":"Xian","family":"Xu","sequence":"first","affiliation":[{"name":"College of Food Science and Biology Hebei University of Science and Technology  Shijiazhuang China"},{"name":"Key Laboratory of Crop Cultivation Physiology and Green Production of Hebei Province Institute of Cereal and Oil, Hebei Academy of Agriculture and Forestry Sciences  Shijiazhuang China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jingyi","family":"Xu","sequence":"additional","affiliation":[{"name":"College of Food Science and Biology Hebei University of Science and Technology  Shijiazhuang China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fang","family":"Xue","sequence":"additional","affiliation":[{"name":"College of Food Science and Biology Hebei University of Science and Technology  Shijiazhuang China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Weijia","family":"Li","sequence":"additional","affiliation":[{"name":"College of Food Science and Biology Hebei University of Science and Technology  Shijiazhuang China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Huixuan","family":"Liu","sequence":"additional","affiliation":[{"name":"College of Food Science and Biology Hebei University of Science and Technology  Shijiazhuang China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhaocong","family":"Zhang","sequence":"additional","affiliation":[{"name":"College of Food Science and Biology Hebei University of Science and Technology  Shijiazhuang China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9962-3686","authenticated-orcid":false,"given":"Silong","family":"Chen","sequence":"additional","affiliation":[{"name":"College of Food Science and Biology Hebei University of Science and Technology  Shijiazhuang China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,4,8]]},"reference":[{"key":"e_1_2_9_2_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.indcrop.2023.117231"},{"key":"e_1_2_9_3_1","first-page":"1","article-title":"Progress on genetic and genomic research for enhanced oil content and quality in peanut","volume":"1","author":"Liao B","year":"2016","journal-title":"Oil Crop Sci"},{"key":"e_1_2_9_4_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.indcrop.2016.04.050"},{"key":"e_1_2_9_5_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.ultsonch.2020.105419"},{"key":"e_1_2_9_6_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.bcab.2019.101206"},{"key":"e_1_2_9_7_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.indcrop.2025.121439"},{"key":"e_1_2_9_8_1","unstructured":"FAOSTAT Groundnut production in 2023; Crops\/Regions\/World List\/Production Quantity (pick lists). 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