Spatial distribution of marker compounds in Epimedium sagittatum leaves and the role of UGT72B113 in generating kaempferol glycoside diversity.

Epimedium sagittatum is an important medicinal plant whose main bioactive flavonol glycosides, such as epimedin C and icariin, are derived from kaempferol. However, the spatial distribution of these compounds within the plant and the genetic basis for their structural diversity remain unclear. Here, we employed MALDI-TOF mass spectrometry imaging to visualize the distribution of epimedin C and icariin in E. sagittatum leaves, revealing their predominant accumulation in nonleaf vein mesophyll part. Integrated metabolomic and transcriptomic analyses of leaves, petioles, and rhizomes highlighted glycosylation as a key contributor to kaempferol derivative diversity. We identified five candidate UDP-glycosyltransferase genes, among which UGT72B113 exhibited remarkable functional versatility. In vitro enzymatic assays demonstrated that UGT72B113 accepts multiple kaempferol-type aglycones and various sugar donors, catalyzing the formation of monoglycosylate, diglycosylate, and even triglycosylated products from a single substrate. Our findings reveal that UGT72B113 is involved in generating flavonol glycoside diversity in E. sagittatum via its broad substrate promiscuity, multi-glycosylation capability, and sugar donor flexibility. This study provides new insights into the spatial regulation of flavonol accumulation and the enzymatic mechanisms underlying phytochemical diversity in medicinal plant.