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bioRxiv · 10.1101/2025.11.01.685562

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

Porri, A.·Sudhakar, S.·Noguera, M.·Betz, M.·Lerchl, J.·Dayan, F.·Norsworthy, J. K. E.
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Abstract

Glufosinate-ammonium (GFA) is a key non-selective herbicide for controlling Amaranthus palmeri and other weeds by targeting glutamine synthetase (GS). GS copy number, expression, sequence polymorphisms, and enzymatic properties in a GFA-resistant population (CCR) were studied. Digital PCR revealed no major GS amplification or target upregulation: most CCR plants had copy numbers and expression comparable to the susceptible reference, with only minor increases in GS2.1 and GS2.2 in a few individuals. Sequencing identified a non-synonymous substitution, G255D, in GS2.2 within a conserved region adjacent to the GFA-binding site. G255D retained [~]58% of wild-type activity in vitro assays, but was completely insensitive to GFA, with no measurable inhibition at tested concentrations. However, ectopic expression of G255D in Arabidopsis thaliana did not confer GFA tolerance, indicating the mutation alone is insufficient for resistance. In vitro analysis of the Eleusine indica GS1.1 S59G substitution revealed increased catalytic activity without affecting GFA sensitivity. A mutational panel of GS1.1 variants showed that substitutions at E131, E192, G245, H249, R291, R311, and R332 abolished enzyme activity or inhibitor sensitivity, with most variants retaining <2% of wild-type function. Among a broader set of predicted GS1.1 variants, high resistance indices were consistently linked to strong reductions in catalytic efficiency, underscoring the fitness costs of target-site alterations. Collectively, GS2.2 G255D appears to be a rare substitution combining substantial residual activity with complete GFA insensitivity and suggest that resistance via target-site modifications studied is constrained by trade-offs between catalytic function and herbicide binding.

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Porri, A., Sudhakar, S., Noguera, M., Betz, M., Lerchl, J., Dayan, F., Norsworthy, J. K. E.. 2025-11-01. Analysis of glutamine synthetase target-site mutations and their role in endowing glufosinate-ammonium resistance. https://doi.org/10.1101/2025.11.01.685562

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