Engineered EPSPS confers glyphosate tolerance in tumorous stem mustard (Brassica juncea var. tumida)

Tumorous stem mustard (B. juncea var. tumida), the essential raw material for Chinese pickled mustard (zhacai), faces substantial yield and quality challenges due to weed competition. To address this issue, we developed glyphosate-resistant (GR) variants through strategic amino acid substitutions in two key B. juncea EPSPS genes, BjuA017481 and BjuA010725. qRT-PCR analysis revealed that BjuA010725 was highly expressed in siliques. Heterologous expression screening in E. coli identified the triple-mutant BjuA010725^TIAVPS exhibited exceptionally GR, surviving exposure to 0.3 % (w/v) glyphosate. Subcellular localization of the GFP-tagged BjuA010725^TIAVPS confirmed chloroplast targeting in tobacco cells. Transgenic A. thaliana lines expressing this variant achieved complete survival (100 %) following 0.1 % foliar glyphosate application at the three-leaf stage. Similarly, transgenic B. juncea demonstrated significantly higher survival rates (p < 0.05) compared to wild-type (WT) after a 0.2 % glyphosate treatment at the four-leaf stage. In field trials, transgenic plants achieved 65 % survival compared with 0 % in WT following 0.6 % glyphosate application during the stem-swelling phase (30-day observation). Physiological analyses revealed stable metabolic profiles in transgenic plants, including elevated chlorophyll content (2.8 mg/g vs. 1.2 mg/g in WT) and nitrogen levels (4.1 % vs. 2.3 %). Conversely, the WT displayed glyphosate-induced metabolic disruption, with a 3.4-fold increase in shikimate accumulation and a 2.8-fold rise in phenylalanine levels. Importantly, no significant differences in bolting time or tumorous stem size were observed between transgenic lines and WT. Structural modeling with AutoDock Vina predicted glyphosate binding interactions with BjuA010725, providing a preliminary mechanistic model of glyphosate action in tumorous stem mustard. Together, these findings offer both theoretical insight and practical strategies for enhancing weed management and improving B. juncea cultivation.