Two flavonoid glucosyltransferases from Petunia hybrida: molecular cloning, biochemical properties and developmentally regulated expression.
Two flavonoid glucosyltransferases, UDP-glucose:flavonoid 3-0-glucosyltransferase (3-GT) and UDP-glucose: anthocyanin 5-O-glucosyltransferase (5-GT), are responsible for the glucosylation of anthocyani(di)ns to produce stable molecules in the anthocyanin biosynthetic pathway. The cDNAs encoding 3-GT and 5-GT were isolated from Petunia hybrida by hybridization screening with heterologous probes. The cDNA clones of 3-GT, PGT8, and 5-GT, PH1, encode putative polypeptides of 448 and 468 amino acids, respectively. A phylogenetic tree based on amino acid sequences of the family of glycosyltransferases from various plants shows that PGT8 belongs to the 3-GT subfamily and PH1 belongs to the 5-GT subfamily. The function of isolated cDNAs was identified by the catalytic activities for 3-GT and 5-GT exhibited by the recombinant proteins produced in yeast. The recombinant PGT8 protein could convert not only anthocyanidins but also flavonols into the corresponding 3-O-glucosides. In contrast, the recombinant PH1 protein exhibited a strict substrate specificity towards anthocyanidin 3-acylrutinoside, comparing with other 5-GTs from Perilla frutescens and Verbena hybrida, which showed broad substrate specificities towards several anthocyanidin 3-glucosides. The mRNA expression of both 3-GT and 5-GT increased in the early developmental stages of P. hybrida flower, reaching the maximum at the stage before flower opening. Southern blotting analysis of genomic DNA indicates that both 3-GT and 5-GT genes exist in two copies in P. hybrida, respectively. The results are discussed in relation to the molecular evolution of flavonoid glycosyltransferases.
Yamazaki
Mami
M
Laboratory of Molecular Biology and Biotechnology, Graduate School of Pharmaceutical Sciences, Chiba University, Japan.
Yamagishi
Emiko
E
Gong
Zhizhong
Z
Fukuchi-Mizutani
Masako
M
Fukui
Yuko
Y
Tanaka
Yoshikazu
Y
Kusumi
Takaaki
T
Yamaguchi
Masaatsu
M
Saito
Kazuki
K
D000595:Amino Acid Sequence; D000872:Anthocyanins / Q000096:biosynthesis; D015152:Blotting, Northern; D015139:Blotting, Southern; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D018744:DNA, Plant / Q000235:genetics; D018507:Gene Expression Regulation, Developmental; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D005964:Glucosyltransferases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D010802:Phylogeny; D018749:RNA, Plant / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D019657:Solanaceae / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development
D000872:Anthocyanins; D018076:DNA, Complementary; D018744:DNA, Plant; D018749:RNA, Plant; D005964:Glucosyltransferases