cDNA Cloning, Heterologous Expressions, and Functional Characterization of Malonyl-Coenzyme A:Anthocyanidin 3-O-Glucoside-6"-O-Malonyltransferase from Dahlia Flowers

• Published in: Plant physiology (Bethesda) 2002-12, Vol.130 (4), p.2142-2151
• Main Authors: Hirokazu Suzuki, Toru Nakayama, Keiko Yonekura-Sakakibara, Yuko Fukui, Nakamura, Noriko, Masa-atsu Yamaguchi, Yoshikazu Tanaka, Takaaki Kusumi, Nishino, Tokuzo
• Format: Article
• Language: English
• Subjects: Acyltransferases - genetics; Acyltransferases - metabolism; Agronomy. Soil science and plant productions; Amino Acid Sequence; Amino acids; Anthocyanins - metabolism; Biochemical Processes and Macromolecular Structures; Biological and medical sciences; Biosynthesis; Classical and quantitative genetics. Population genetics. Molecular genetics; Cloning, Molecular; Complementary DNA; Dahlia - enzymology; Dahlia - genetics; Dahlia variabilis; DNA, Complementary - chemistry; DNA, Complementary - genetics; Economic plant physiology; Enzymes; Escherichia coli - genetics; Escherichia coli - metabolism; Flowers; Flowers - enzymology; Flowers - genetics; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Plant; Generalities. Genetics. Plant material; Genes. Genome; Genetics and breeding of economic plants; Metabolism; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Nutrition. Photosynthesis. Respiration. Metabolism; Petals; Petunia - genetics; Petunia - metabolism; Petunia hybrida; Pigments; Plant physiology and development; Plants; Plants, Genetically Modified; Polymerase Chain Reaction; Reserves and secretions. Pigments. Phytochromes; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Storage and secretion, pigments, phytochrome; Substrate Specificity; Transgenic animals
• ISSN: 0032-0889; 1532-2548
• DOI: 10.1104/pp.010447

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 dehlia (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 (Km, 18.8 μM) to pelargonidin 3-O-glucoside (Km, 46.7 μM) to produce pelargonidin 3-O-6"-O-malonylglucoside with a kcat 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 β-glucosidase. These results suggest that malonylation should serve as a strategy for pigment stabilization in the flowers.