Cloning and characterization of soybean gene Fg1 encoding flavonol 3-O-glucoside/galactoside (1→6) glucosyltransferase

Abstract

Flavonoids are important secondary metabolites in plants. Sugar-sugar glycosyltransferases are involved in the final step of flavonoid biosynthesis and contribute to the structural diversity of flavonoids. This manuscript describes the first cloning of a sugar-sugar glucosyltransferase gene in the UGT family that attaches glucose to the 6″-position of sugar bound to a flavonol. The results provide a glimpse on the possible evolution of sugar-sugar glycosyltransferase genes and identify putative amino acids responsible for the recognition of the hydroxyl group of the sugar moiety and specification of sugar. A scheme for the genetic control of flavonol glycoside biosynthesis is proposed. Flavonol glycosides (FGs) are predominant in soybean leaves and they show substantial differences among genotypes. In previous studies, we identified two flavonoid glycoside glycosyltransferase genes that segregated in recombinant inbred lines developed from a cross between cultivars Nezumisaya and Harosoy; one was responsible for the attachment of glucose to the 2″-position of glucose or galactose that is bound to the 3-position of kaempferol and the other was involved in the attachment of glucose to the 6″-position. This study was conducted to clone and characterize the 6″-glucosyltransferase gene. Linkage mapping indicated that the gene was located in the molecular linkage group I (chromosome 20). Based on the genome sequence, we cloned a candidate cDNA, GmF3G6"Gt from Harosoy but the corresponding cDNA could not be amplified by PCR from Nezumisaya. The coding region of GmF3G6″Gt in Harosoy is 1386 bp long encoding 462 amino acids. This gene was not expressed in leaves of Nezumisaya. The GmF3G6″Gt recombinant protein converted UDP-glucose and kaempferol 3-O-glucoside or kaempferol 3-O-galactoside to kaempferol 3-O-glucosyl-(1→6)-glucoside or kaempferol 3-O-glucosyl-(1→6)-galactoside, respectively. These results indicate that GmF3G6″Gt encodes a flavonol 3-O-glucoside/galactoside (1→6) glucosyltransferase and corresponds to the Fg1 gene. GmF3G6″Gt had an amino acid similarity of 82 % with GmF3G6″Rt encoding flavonol 3-O-glucoside/galactoside (1→6) rhamnosyltransferase, suggesting a recent evolutionary divergence of the two genes. This may be the first cloning of a sugar-sugar glucosyltransferase gene in the UGT family that attaches glucose to the 6″-position of sugar bound to a flavonol. A scheme for the control of FG biosynthesis is proposed.

Identification and characterization of a rhamnosyltransferase involved in rutin biosynthesis in Fagopyrum esculentum (common buckwheat)

“…Several flavonoid 3-O-(6″-O-rhamnosyl) glycosides, such as rutin, quercetin 3-O-robinobioside [34], and kaempferol 3-O-rutinoside [35] have been found in buckwheat plants, and we detected rutin and a compound predicted to be quercetin 3-O-robinobioside in the common buckwheat cultivar Shinano No.1 used in this study ( Figure S7); these compounds are likely to be produced by FeF3G6″RhaT. The enzymatic property of FeF3G6″RhaT is similar to that of 6″-Orhamnosyltransferase from Glycine max (GmF3G6″ RT) [17], which showed 52% identity with FeF3G6″ RhaT. In contrast, 6″-O-rhamnosyltransferases from Citrus (Cs1,6RhaT and Cm1,6RhaT), which shared 57% identity with FeF3G6″RhaT, reacted with flavonoid 7-O-glucosides as major substrates and reacted with flavonol 3-O-glucosides as minor substrates [12].…”

“…At present, some members of rhamnosyltransferases, which catalyze rhamnosylation of the sugar moiety of flavonoid glycosides, have been characterized. These include flavanone 7-O-glucoside 6″-O-rhamnosyltransferase from Citrus sinensis and C. maxima [12,13], flavanone 7-O-glucoside 2″-O-rhamnosyltransferase from C. maxima [14], anthocyanidin-3-O-glucoside 6″-O-rhamnosyltransferase from Petunia x hybrida [15] and Lobelia erinus [16], and flavonoid 3-O-glucoside 6″-O-rhamnosyltransferase from Glycine max [17].…”

GmMYB176 Regulates Multiple Steps in Isoflavonoid Biosynthesis in Soybean

“…The key isoflavonoid gene GmIFS (Jung et al, 2000) was also downregulated in GmMYB176-OE. No known glycosyltransferase (Dhaubhadel et al, 2008; Di et al, 2015; Rojas Rodas et al, 2016) were found differentially expressed in GmMYB176-OE or GmMYB176-Si compared to control.…”

UGT79B31 is responsible for the final modification step of pollen-specific flavonoid biosynthesis in Petunia hybrida

“…On the other hand, kaempferol/quercetin 3- O -glucosyl(1 → 2)glucoside derivatives were identified in petals and leaves of the Petunia cultivar ‘Mitchell’ (Bloor et al 1998 ; Davies et al 1998 ) and petals of P. × hybrida clone RL01 (Griesbach 1993 ). Flavonol 3- O -glucoside: 2″- O -glucosyltransferase/6″- O -glucosyltransferase also recognizes flavonoid 3- O -galactoside as a sugar acceptor at a comparable level (Yonekura-Sakakibara et al 2014 ; Di et al 2015 ; Rodas et al 2016 ). These data suggest that the target gene encoding flavonol 3- O -galactoside: 2″- O -glucosyltransferase may be expressed in tissues other than pollen and may be involved in the 2″- O -glucosylation of flavonol 3- O -glucosides.…”