Molecular Cloning and Biochemical Characterization of a Novel Anthocyanin 5-O-Glucosyltransferase by mRNA Differential Display for Plant Forms Regarding Anthocyanin

Abstract

UDP-glucose: anthocyanin 5-O-glucosyltransferase (5-GT) is responsible for the modification of anthocyanins to more stable molecules in complexes for co-pigmentation, supposedly resulting in a purple hue. The cDNA encoding 5-GT was isolated by a differential display applied to two different forms of anthocyanin production in Perilla frutescens var. crispa. Differential display was carried out for mRNA from the leaves of reddish-purple and green forms of P. frutescens, resulting in the isolation of five cDNA clones predominantly expressed in the red form. The cDNA encoded a polypeptide of 460 amino acids, exhibiting a low homology with the sequences of several glucosyltransferases including UDP-glucose: anthocyanidin 3-O-glucosyltransferase. By using this cDNA as the probe, we also isolated a homologous cDNA clone from a petal cDNA library of Verbena hybrida. To identify the biochemical function of the encoded proteins, these cDNAs were expressed in Saccharomyces cerevisiae cells. The recombinant proteins in the yeast extracts catalyzed the conversion of anthocyanidin 3-Oglucosides into the corresponding anthocyanidin 3,5-di-O-glucosides using UDP-glucose as a cofactor, indicating the identity of the cDNAs encoding 5-GT. Several biochemical properties (optimum pH, K m values, and sensitivity to inhibitors) were similar to those reported previously for 5-GTs. Southern blot analysis indicated the presence of two copies of 5-GT genes in the genome of both red and green forms of P. frutescens. The mRNA accumulation of the 5-GT gene was detected in the leaves of the red form but not in those of the green form and was induced by illumination of light, as observed for other structural genes for anthocyanin biosynthesis in P. frutescens.

A Novel Mg2+-dependent O-Methyltransferase in the Phenylpropanoid Metabolism of Mesembryanthemum crystallinum

“…Unusually high sequence identities (90 to 95%) observed among the lignin monomer-specific CCoAOMTs may reflect the evolutionary need to methylate preferentially one substrate only, caffeoyl-CoA. A 50 to 70% identity, as observed in the novel PFOMT subcluster, appears to be more typical for enzymes with a broad substrate tolerance, such as glucosyl-or acyltransferases (51,52).…”

Identification of Glucosyltransferase Genes Involved in Sinapate Metabolism and Lignin Synthesis in Arabidopsis

“…B, sinapyl alcohol was used as substrate in the assay. rified from a wide range of different plant families (32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42), there have been no previous attempts to study this multigene family in a single species and therefore no possibility of directly comparing relative activities across family members. Access to the kinetic analysis of many family members from a single species can provide increased confidence of the range of substrates that may be used by each of these enzymes in vitro and thereby a foundation for exploring their substrates in vivo and their physiological roles in the plant.…”

UGT73C6 and UGT78D1, Glycosyltransferases Involved in Flavonol Glycoside Biosynthesis in Arabidopsis thaliana

“…The functional determination of Arabidopsis UGTs in vitro is still limited to a handful of genes (10 -13), whereas the in planta role of only three plant UGTs has been demonstrated to date (14 -16). Furthermore, whereas several flavonoid GTs, primarily acting on anthocyanidin and anthocyanin acceptor substrates, have been characterized at a molecular level in a range of species including Petunia hybrida (14,17), Vitis vinifera (18), and Perilla frutescens (19), none has as yet been reported from Arabidopsis. Although most anthocyanidin-GTs are capable of also glycosylating flavonols, closer examination of the enzyme kinetics has revealed a strong preference for anthocyanidins (18), and there is as yet only one report that describes a flavonol-exclusive GT (20).…”