cDNA cloning, heterologous expressions, and functional characterization of malonyl-coenzyme a:anthocyanidin 3-o-glucoside-6"-o-malonyltransferase from dahlia flowers.
In the flowers of important ornamental Compositae plants, anthocyanins generally carry malonyl group(s) at their 3-glucosyl moiety. In this study, for the first time to our knowledge, we have identified a cDNA coding for this 3-glucoside-specific malonyltransferase for anthocyanins, i.e. malonyl-coenzyme A:anthocyanidin 3-O-glucoside-6"-O-malonyltransferase, from dahlia (Dahlia variabilis) flowers. We isolated a full-length cDNA (Dv3MaT) on the basis of amino acid sequences specifically conserved among anthocyanin acyltransferases of the versatile plant acyltransferase family. Dv3MaT coded for a protein of 460 amino acids. Quantitative real-time PCR analyses of Dv3MaT showed that the transcript was present in accordance with the distribution of 3MaT activities and the anthocyanin accumulation pattern in the dahlia plant. The Dv3MaT cDNA was expressed in Escherichia coli, and the recombinant enzyme was purified to homogeneity and characterized. The recombinant Dv3MaT catalyzed the regiospecific transfer of the malonyl group from malonyl-coenzyme A (K(m), 18.8 microM) to pelargonidin 3-O-glucoside (K(m), 46.7 microM) to produce pelargonidin 3-O-6"-O-malonylglucoside with a k(cat) value of 7.3 s(-1). The other enzymatic profiles of the recombinant Dv3MaT were closely related to those of native anthocyanin malonyltransferase activity in the extracts of dahlia flowers. Dv3MaT cDNA was introduced into petunia (Petunia hybrida) plants whose red floral color is exclusively provided by cyanidin 3-O-glucoside and 3,5-O-diglucoside. Thirteen transgenic lines of petunia were found to produce malonylated products of these anthocyanins (11-63 mol % of total anthocyanins in the flower). The spectral stability of cyanidin 3-O-6"-O-malonylglucoside at the pHs of intracellular milieus of flowers was significantly higher than that of cyanidin 3-O-glucoside. Moreover, 6"-O-malonylation of cyanidin 3-O-glucoside effectively prevented the anthocyanin from attack of beta-glucosidase. These results suggest that malonylation should serve as a strategy for pigment stabilization in the flowers.
Suzuki
Hirokazu
H
Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aoba-yama 07, Sendai 980-8579, Japan.
Nakayama
Toru
T
Yonekura-Sakakibara
Keiko
K
Fukui
Yuko
Y
Nakamura
Noriko
N
Yamaguchi
Masa-Atsu
MA
Tanaka
Yoshikazu
Y
Kusumi
Takaaki
T
Nishino
Tokuzo
T
D000217:Acyltransferases / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000872:Anthocyanins / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D031944:Dahlia / Q000201:enzymology / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D035264:Flowers / Q000201:enzymology / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D008969:Molecular Sequence Data; D032306:Petunia / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D000872:Anthocyanins; D018076:DNA, Complementary; D000217:Acyltransferases; C474250:malonyl-coenzyme A anthocyanidin 3-O-glucoside-6''-O-malonyltransferase