Cloning and Characterization of Vitis viniferaUDP-Glucose:Flavonoid 3-O-Glucosyltransferase, a Homologue of the Enzyme Encoded by the Maize Bronze-1Locus That May Primarily Serve to Glucosylate Anthocyanidins in Vivo

Abstract

We report here the cloning and optimized expression at 16°C and the characterization of a Vitis vinifera UDPglucose:flavonoid 3-O-glucosyltransferase, an enzyme responsible for a late step in grapevine anthocyanin biosynthesis. The properties of this and other UDP-glucose:flavonoid 3-O-glucosyltransferases, homologues of the product encoded by the maize Bronze-1 locus, are a matter of conjecture. The availability of a purified recombinant enzyme allowed for the unambiguous determination of the characteristics of a flavonoid 3-O-glucosyltransferase. Kinetic analyses showed that k cat for glucosylation of cyanidin, an anthocyanidin substrate, is 48 times higher than for glucosylation of the flavonol quercetin, whereas K m values are similar for both substrates. Activity toward other classes of substrates is absent. Cu 2؉ ions strongly inhibit the action of this and other glucosyltransferases; however, we suggest that this phenomenon in large part is due to Cu 2؉ -mediated substrate degradation rather than inhibition of the enzyme. Additional lines of complementary biochemical data also indicated that in the case of V. vinifera, the principal, if not only, role of UDP-glucose:flavonoid 3-Oglucosyltransferases is to glucosylate anthocyanidins in red fruit during ripening. Other glucosyltransferases with a much higher relative activity toward quercetin are suggested to glucosylate flavonols in a distinct spatial and temporal pattern. It should be considered whether gene products homologous to Bronze-1 in some cases more accurately should be referred to as UDPglucose:anthocyanidin 3-O-glucosyltransferases.Phytochemists have identified some 70,000 plant chemicals, many thousands of which are glycosylated (1). From a chemical point of view, two main properties differ between the glycosylated secondary products and their respective aglycones, as glycosylation invariably results in enhanced water solubility and lower chemical reactivity. Glycosylated compounds are therefore often thought of as transportable storage compounds or indeed waste/detoxification products assumed to lack physiological activity (2).Despite the widespread occurrence of glycosylated secondary metabolites, including flavonols (3), anthocyanins (4), monoterpenes (5), plant hormones (2), and metabolites of systemic fungicides (6), isolation and characterization of purified enzymes responsible for their metabolism have only been reported in a couple of select instances (7,8). The most widely studied groups of plant glycosyltransferases are those associated with the biosynthesis of flavonoid glucosides, including flavonol glucosides, flavanone glucosides, and anthocyanins (3, 4, 9, 10). Earliest reports included the detection of an anthocyanidin and flavonol glucosylating activity in endosperm extracts of maize (Zea mays), (11)(12)(13). This work formed the basis for identification by transposon tagging of the gene product of the maize Bronze-1 locus (14). cDNAs encoding flavonoid glucosyltransferases have been isolated from a number of plant species uti...

Functional Differentiation of the Glycosyltransferases That Contribute to the Chemical Diversity of Bioactive Flavonol Glycosides in Grapevines (Vitis vinifera)

“…The results were compared with those of Vv GT1 (Ford et al, 1998), which is responsible for anthocyanin biosynthesis in grapevine berry skin and Vv FLS1 (also termed Vv FLS4), which is responsible for flavonol biosynthesis (Fujita et al, 2006;Czemmel et al, 2009) (Figure 3, left panels). The Vv GT5 transcript was abundant in berry skin and increased at the onset of pigmentation (veraison), with the highest level of transcription detected in the fully pigmented skin (exocarp) of mature berries ( Figure 3, second panel).…”

“…Moreover, the differentiation of the sugar donor specificity of UGTs appears to have occurred in a lineage-specific manner after establishment of general regiospecificity for the glycosyl acceptor (Yonekura-Sakakibara et al, 2007;Noguchi et al, 2009a;Ono et al, 2010). The grapevine genome contains as many as 240 UGT genes (The French-Italian Public Consortium for Grapevine Genome Characterization, 2007), and of these, the only grapevine UGT that has been functionally characterized is Vv GT1, which catalyzes the 3-O-specific glucosylation of anthocyanidin to produce anthocyanidin 3-O-glucoside and is involved in the coloration of grape skin (Ford et al, 1998). This study aimed at identifying the UGTs that are responsible for the observed structural diversity of flavonol glycosides in grapevine.…”

“…The grapevine genome contains as many as 240 UGT genes (The French-Italian Public Consortium for Grapevine Genome Characterization, 2007), and of these, the only grapevine UGT that has been functionally characterized is Vv GT1, which catalyzes the 3-O-specific glucosylation of anthocyanidin to produce anthocyanidin 3-O-glucoside and is involved in the coloration of grape skin (Ford et al, 1998). This study aimed at identifying the UGTs that are responsible for the observed structural diversity of flavonol glycosides in grapevine.…”

Cloning and heterologous expression of cDNAs encoding flavonoid glucosyltransferases from Dianthus caryophyllus

“…It has been reported that anthocyanidin 3-GT has flavonol 3-GT activity in several plant species (Tanaka et al 1996;Ford et al 1998). To determine if clone DicGT1 has additional activity, the substrate specificity of DicGT1 was tested for anthocyanidins (pelargonidin and cyanidin), flavonols (kaempferol and quercetin) and betanidin.…”

Cloning and characterization of a glucosyltransferase that reacts on 7-hydroxyl group of flavonol and 3-hydroxyl group of coumarin from tobacco cells

“…The genes encoding flavonoid 3-O-glycosyltransferase (F3GT), anthocyanin 5-O-glycosyltransferase (A5GT), and flavonoid 7-O-glycosyltransferase (F7GT) were reported. The F3GT gene was first reported in maize [8], and later found in several plant species [9][10][11]. The amino acid sequences of F3GTs were highly conserved among these plant species, and many of them were reported to be associated with anthocyanin biosynthesis.…”