Identification and Characterization of CtUGT3 as the Key Player of Astragalin Biosynthesis in Carthamus tinctorius L

Safflower (Carthamus tinctorius L.) is a multipurpose economic crop that is distributed worldwide. Flavonoid glycosides are the main bioactive components in safflower, but only a few UDP-glycosyltransferases (UGT) have been identified. Three differentially expressed UGT genes related with the accumulation of 9 flavonoid O-glycosides were screened from metabolomics and transcriptome analysis. Safflower corolla protoplasts were used to confirm the glycosylation ability of UGT candidates in vivo for the first time. The astragalin content was significantly increased only when CtUGT3 was overexpressed. CtUGT3 also showed flavonoid 3-OH and 7-OH glycosylation activities in vitro. Molecular modeling and site-directed mutagenesis revealed that G15, T136, S276, and E384 were critical catalytic residues for the glycosylation ability of CtUGT3. These results demonstrate that CtUGT3 has a flavonoid 3-OH glycosylation function and is involved in the biosynthesis of astragalin in safflower. This study provides a reference for flavonoid biosynthesis genes research in nonmodel plants.