Analysis of glutamine synthetase target-site mutations and their role in endowing glufosinate-ammonium resistance

Abstract

BACKGROUND. Glufosinate-ammonium (GFA) is a non-selective herbicide that inhibits glutamine synthetase (GS), a key enzyme in plant nitrogen metabolism. Although resistance to GFA has been reported in several weed species, confirmed cases of target-site resistance (TSR) via GS mutations remain rare. In Amaranthus palmeri, resistance is usually associated with GS gene amplification and overexpression, while the role of GS point mutations remains unclear. This study investigated GFA resistance in a population lacking GS amplification, and evaluated the functional relevance of putative resistance-conferring GS mutations. RESULTS. Digital PCR revealed no substantial GS amplification or overexpression in the CCR population. Sequencing identified a glycine-to-aspartic acid substitution at position 255 (G255D) in the chloroplastic GS2.2 isoform, detected in ~80% of CCR plants in heterozygous form. Notably, this mutation was exclusively in untreated plants. In vitro assays showed that GS2.2 G255D retained ~58% of wild-type catalytic activity and was completely insensitive to GFA inhibition. However, ectopic expression of G255D, as well as the homologous Arabidopsis GS2.2 G254D substitution, did not confer GFA tolerance in Arabidopsis. Analysis of GS1.1 variants demonstrated that mutations disrupting GFA binding generally caused severe reductions in catalytic efficiency. Re-evaluation of the previously reported Eleusine indica GS1.1 S59G mutation revealed no meaningful shift in GFA sensitivity. CONCLUSIONS. GFA resistance in the CCR population cannot be explained by GS amplification or GS2.2 G255D. These findings underscore the strong structural and functional constraints on GS target-site evolution and suggests that non-target-site mechanisms likely underlie GFA resistance in this population. © 2026 BASF SE and The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.