Molecular cloning and biochemical characterization of a novel anthocyanin 5-O-glucosyltransferase by mRNA differential display for plant forms regarding anthocyanin

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-O-glucosides 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, Km 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.