Body of Abstract: Flavonoids are the most diverse specialized metabolites found widely in plants. They are known to function in overall stress adaptation in plants, but evidence also indicates that some of them may be indispensable for plant survival as petunia flavonoid-deficient mutants are sterile. Yet, flavonoids responsible for fertility have not been identified, and our understanding of the physiological function of each flavonoid remains obscure.
Our metabolite analysis with various organs of tomato (Solanum lycopersicum) revealed that kaempferol-3-O-galactosides were exclusively accumulated in pollen, while flavonol-3-O-glucosides were ubiquitously detected in most organs. A phylogenetic analysis identified two flavonol 3-O-glycosyltransferases (F3GTs) in tomato, SlUGT78D-A and SlUGT78D-B. In vitro enzyme activity tests revealed that SlUGT78D-A has both flavonol 3-O-glucosyl and flavonol 3-O-galactosyltransferase activities, while SlUGT78D-B only has flavonol 3-O-galactosyltransferase activity. Predicted protein structures of SlUGT78D-A and SlUGT78D-B and amino acid sequence alignment led us to hypothesize that four amino acid residues may determine the sugar specificity of SlUGT78Ds. The substitution of four amino acids of SlUGT78D-B with corresponding amino acids of SlUGT78D-A enables SlUGT78D-B to gain glucosyltransferase activity without affecting its galactosyltransferase activity, suggesting that these four amino acids are indeed critical for sugar specificity.
These two tomato F3GTs showed distinctive organ-specific expression patterns. SlUGT78D-B predominantly expresses in pollen, while SlUGT78D-A exhibits ubiquitous expression except for pollen. To examine any biological roles of these organ-specific flavonol glycosides, we disrupted SlUGT78D-A and SlUGT78D-B using the CRISPR system. slugt78d-a contains about 30% of flavonol glucosides in most organs, while disruption of SlUGT78D-B completely abolishes kaempferol-3-O-galactosides in pollen, indicating that SlUGT78D-B is the only flavonol 3-O-galactosyltransferase in pollen. Furthermore, slugt78d-b single mutant and slugt78d-a/b double mutant show impaired pollen tube growth and reduced seed numbers. Taken together, galactosylation of kaempferol is required for proper pollen tube growth in tomato.