Citrus fruits have abundant flavonoid glycosides (FGs), an important class of natural functional and flavor components. However, there have been few reports about the modification of UDP-glycosyltransferases (UGTs) on flavonoids in citrus. Notably, in flavonoid biosynthesis, 7-O-glucosylation is the initial and essential step of glycosylation prior to the synthesis of flavanone disaccharides, the most abundant and iconic FGs in citrus fruits. Here, based on the accumulation of FGs observed at the very early fruit development stage of two pummelo varieties, we screened six novel flavonoid 7-O-glucosyltransferase genes (7GlcTs) via transcriptomic analysis and then characterized them in vitro. The results revealed that four Cg7GlcTs possess wide catalytic activities towards various flavonoid substrates, with CgUGT89AK1 exhibiting the highest catalytic efficiency. Transient overexpression of CgUGT90A31 and CgUGT89AK1 led to increases in FG synthesis in pummelo leaves. Interestingly, these two genes had conserved sequences and consistent functions across different germplasms. Moreover, CitUGT89AK1 was found to play a role in the response of citrus to Huanglongbing infection by promoting FG production. The findings improve our understanding of flavonoid 7-O-glucosylation by identifying the key genes, and may help improve the benefits of flavonoid biosynthesis for plants and humans in the future.

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