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 dis play was carried out for mRNA from the leaves of reddish-purple and green f orms of P. frutescens, resulting in the isolation of five cDNA clones predo minantly expressed in the red form. The cDNA encoded a polypeptide of 460 a mino acids, exhibiting a low homology with the sequences of several glucosy ltransferases 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 f unction of the encoded proteins, these cDNAs were expressed in Saccharomyce s cerevisiae cells. The recombinant proteins in the yeast extracts catalyze d the conversion of anthocyanidin 3-O-glucosides into the corresponding ant hocyanidin 3,5-di-O-glucosides using UDP-glucose as a cofactor, indicating the identity of the cDNAs encoding 5-GT, Several biochemical properties (op timum pH, K-m values, and sensitivity to inhibitors) were similar to those reported previously for 5-GTs, Southern blot analysis indicated the presenc e 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 l eaves 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 anthocya nin biosynthesis in P, frutescens.