Non-target-site resistance mechanisms to glyphosate in Eleusine indica from Xinjiang cotton fields

Glyphosate resistance in weeds is often mediated by target-site changes in 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), but non-target-site resistance (NTSR) mechanisms are increasingly recognized as important contributors to field-evolved resistance. Here, we characterized a glyphosate-resistant Eleusine indica population collected from cotton fields in Xinjiang, China. Whole-plant dose-response analysis showed that the resistant population GR-R had a 3.36-fold higher GR₅₀ than the susceptible population GR-S. GR-R also accumulated substantially less shikimic acid after glyphosate treatment (approximately two-fold lower than GR-S at 24 and 48 hours after treatment), indicating reduced impact on the shikimate pathway. However, sequencing of a conserved EPSPS region revealed no substitutions at known resistance-associated residues, and EPSPS expression did not differ significantly between GR-R and GR-S. RNA-seq analysis identified 364 differentially expressed genes, with enrichment of oxidative stress, carbohydrate metabolism, amino acid metabolism, hormone signaling, and transporter-related processes. Four upregulated genes associated with herbicide metabolic resistance, CYP94C1, ABCA7, ABCG37, and ABCG39, were validated by qPCR. Structural modeling and molecular docking suggested potential interactions between glyphosate and the encoded proteins, with ABCG39 showing the strongest predicted binding among the ABC transporters. These results suggest that glyphosate resistance in GR-R is not driven by common EPSPS-based mechanisms but is associated with NTSR involving cytochrome P450 and ABC transporter candidates. Our findings provide a foundation for functional studies of P450- and transporter-mediated glyphosate resistance in E. indica.