AUTHOR=Li Bingjie , Chen Chen , Cui Mengmeng , Sun Yuhe , Lv Jing , Dai Changbo TITLE=Exploring the potential role of EPSPS mutations for enhanced glyphosate resistance in Nicotiana tabacum JOURNAL=Frontiers in Plant Science VOLUME=Volume 16 - 2025 YEAR=2025 URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2025.1516963 DOI=10.3389/fpls.2025.1516963 ISSN=1664-462X ABSTRACT=Glyphosate is a widely used non-selective, broad-spectrum, systemic herbicide by interfering with the biosynthesis of aromatic amino acids. However, the emergence of glyphosate-resistant weeds has driven the need for enhanced herbicide resistance in crops. In this study, we engineered two mutant variants of the tobacco EPSPS gene through amino acid substitution (TIPS-NtEPSPS and P180S-NtEPSPS). These mutated EPSPS genes were overexpressed in tobacco under the control of CaMV35S promoters. Our results demonstrate that overexpression of TIPS-NtEPSPS significantly enhances glyphosate tolerance, allowing plants to withstand up to four times the recommended dose without compromising their fitness. This research highlights the potential of the TIPS-NtEPSPS mutant to improve herbicide resistance in tobacco, offering a viable approach for effective weed management.