Abstract
Flavonols are a subclass of flavonoids widely found in plants and typically exist in glycosylated forms, decorated with various sugars at different positions on the flavonol aglycone. The composition and abundance of flavonol glycosides vary across species and among tissues within a species. Although flavonols are collectively known for their antioxidant activity, the specific physiological functions of individual flavonol structures remain poorly understood. Here, we show that 2 flavonol glycosides, kaempferol 3-O-glucosyl(1 → 2)galactoside (K2) and quercetin 3-O-glucosyl(1 → 2)galactoside (Q2), predominantly accumulate in the pollen of Solanaceae plants. K2 is evolutionarily conserved across Solanaceae, while Q2 has been lost in species such as tomato (Solanum lycopersicum). Our transcriptome profiling and biochemical analysis revealed SlUGT78D-B (78-B) as a pollen-specific flavonol 3-O-galactosyltransferase responsible for K2 production in tomato. Disruption of 78-B abolished K2 accumulation, leading to defective pollen tube growth in our in vitro assays. Supplementation with kaempferol 3-O-galactoside (K2 precursor) restores pollen tube growth, whereas quercetin 3-O-galactoside (Q2 precursor) or flavonol aglycones do not, suggesting distinct roles for individual flavonol structures. We further show that 3 key amino acid residues of 78-B dictate its sugar specificity, favoring galactosylation over glucosylation. Substitution of any one of these residues enables 78-B to acquire glucosyltransferase activity. However, 78-B remains evolutionarily constrained from gaining this activity, suggesting selective pressure to maintain flavonol galactoside accumulation in pollen. These findings indicate that individual flavonol glycosides can have specific physiological roles beyond enhancing solubility and stability.