Identification and Functional Analysis of Two UGT84 Glycosyltransferases in Flavonoid Biosynthesis of Carthamus tinctorius

Safflower (<i>Carthamus tinctorius</i> L.) is a multipurpose economic crop. Flavonoid glycosides are its key bioactive constituents, and several glycosyltransferases involved in their biosynthesis have been identified. The glycosyltransferase 84 subfamily represents a specialized branch with diverse functions, involved not only in catalyzing flavonoid glycosylation but also in the biosynthesis of auxins, tannins, and other compounds. However, this subfamily remains poorly characterized in safflower. In this study, two UGT84 subfamily genes, <i>UGT84A28</i> and <i>UGT84B3</i>, were screened based on expression patterns and phylogenetic evolution analysis. Recombinant proteins were induced and purified using prokaryotic expression systems. Functional characterization was subsequently conducted through enzymatic assays in vitro and transient expression in tobacco leaves. Molecular docking was employed to investigate the binding modes of UGTs with UDP-glucose. The results indicated that both UGTs demonstrated glycosylation activity at the flavonoid 7-OH position. Notably, when luteolin was employed as the aglycone, both enzymes also exhibited 3'-<i>O</i>-glycosylation activity. Combined with amino acid sequence alignment, we propose that residues A351/T343 and G263/F254, which affect spatial conformation and hydrogen bonding ability, may be one of the reasons for the functional differences between these two enzymes. These findings provide new insights into the catalytic diversity of glycosyltransferases.