Heterologous production of four flavonoid C-glycosides corresponding to major bamboo leaf flavonoids in Nicotiana tabacum

Vitexin, isovitexin, orientin, and isoorientin are representative flavonoid C- glycosides and major bioactive constituents of bamboo leaf flavonoids, but their reliable production remains limited by variable accumulation in natural sources and the difficulty of selective C- glycoside synthesis. Here, we reconstructed a flavonoid C- glycoside pathway in stable transgenic tobacco by introducing EbCHS, EbCHI, PhF2H, PhF3’H, and ZmUGT708A6. LC-MS/MS analysis confirmed the production of all four target compounds in the full-pathway S5 lines, with vitexin, isovitexin, orientin, and isoorientin reaching 40.70 ± 16.49, 57.02 ± 17.81, 23.04 ± 8.86, and 17.33 ± 7.67 μg/g dry weight in leaves, respectively. In floral tissues, the total content of the four target flavonoid C- glycosides reached approximately 1.17 mg/g dry weight in S5 plants. Metabolic profiling showed that pathway reconstruction also remodeled endogenous flavonoid metabolism in tobacco. Flavonoid-enriched extracts from T 1 engineered leaves showed low cytotoxicity in RAW264.7 macrophages and stronger inhibition of LPS-induced nitric oxide production than wild-type extracts. These results establish transgenic tobacco as a plant-based platform for producing major bamboo-leaf-flavonoid C- glycosides and generating bioactive flavonoid-enriched materials for preliminary functional evaluation.