Cloning and characterization of soybean gene Fg4 encoding a novel flavonol 3-O-glucoside/galactoside...

The glycosylation patterns of flavonol glycosides (FGs) in soybean leaves are associated with productivity and insect resistance. The structure of FGs is conditioned by four flavonoid glycoside glycosyltransferase (FGG) genes, Fg1 (6"-glucoside present), Fg2 (6"-rhamnoside present), Fg3 (2"-glucoside present), and Fg4 (2"-rhamnoside present). We previously cloned and characterized the Fg1 to Fg3 genes. This study was conducted to comprehensively identify FGG genes by isolating the Fg4 gene. Leaves of the cultivar, Clark (Fg4 allele), contained FGs with rhamnose at the 4"-position, whereas those of the cultivar A.K. (fg4 allele), were devoid of 4"-rhamnoside. Their F₂ population segregated in three Clark-type: 1 A.K.-type ratios, suggesting that a single gene controlled the existence of 4"-rhamnoside. Based on genetic mapping, we cloned a candidate gene, Glyma.06G235000, with three amino acid substitutions between cultivars. Recombinant protein of Clark acted on kaempferol 3-O-rhamnosyl-(1→6)-galactoside and generated a product with a similar retention time to kaempferol 3-O-rhamnosyl-(1→4)-[rhamnosyl-(1→6)-glalactoside]. Furthermore, it acted on kaempferol 3-O-glucoside/galactoside and generated products with similar retention times to kaempferol 3-O-rhamnosyl-(1→2)-glucoside/galactoside. The A.K. protein did not function presumably due to G293R mutation. The cleaved amplified polymorphic sequence (CAPS) marker to detect the mutation co-segregated with FG composition. These results suggest that Glyma.06G235000 corresponds to the Fg4 gene and that the protein attaches rhamnose to the 2"- or 4"-position, depending on the substrates. Information of all FGG genes may be useful for developing cultivars with the desired FGG composition.