Functional Characterization of a Highly Efficient UDP-Glucosyltransferase CitUGT72AZ4 Involved in the Biosynthesis of Flavonoid Glycosides in Citrus

Citrus is an important dietary source of flavonoid glycosides, and UDP-glycosyltransferases (UGTs) are the key enzymes responsible for their glycosylation. In this study, a genome-wide analysis of the CitUGT gene family was conducted to identify CitUGTs that contribute to flavonoid 4'-O-glucosides biosynthesis. Our analysis identified 136 CitUGTs in the Citrus clementina genome, classifying them into 18 phylogenetic groups (A-R) and 25 families. This classification was strongly supported by consistent gene structures and motif patterns. Moreover, we identified a CitUGT gene (Ciclev10025462m, designated CitUGT72AZ4) that encodes flavonoid 4'-O-glucosyltransferase for the first time in citrus. This enzyme preferentially glycosylated the 4'-OH group of multiple flavonoids, exhibiting higher catalytic efficiency toward quercetin and three flavones in vitro. Virus-induced gene silencing of CitUGT72AZ4 significantly decreased the accumulation of flavonoid 4'-O-glucosides. These results indicated that CitUGT72AZ4 participated in the biosynthesis of flavonoid 4'-O-glucoside in citrus. Overall, our findings provide valuable insights into the CitUGT gene family and contribute to its functional characterization.