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 <i>CitUGT</i> gene family was conducted to identify <i>CitUGTs</i> that contribute to flavonoid 4'-<i>O</i>-glucosides biosynthesis. Our analysis identified 136 CitUGTs in the <i>Citrus clementina</i> 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 <i>CitUGT</i> gene (<i>Ciclev10025462m</i>, designated <i>CitUGT72AZ4</i>) that encodes flavonoid 4'-<i>O</i>-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 <i>in vitro</i>. Virus-induced gene silencing of <i>CitUGT72AZ4</i> significantly decreased the accumulation of flavonoid 4'-<i>O</i>-glucosides. These results indicated that <i>CitUGT72AZ4</i> participated in the biosynthesis of flavonoid 4'-<i>O</i>-glucoside in citrus. Overall, our findings provide valuable insights into the <i>CitUGT</i> gene family and contribute to its functional characterization.