Biochemical and Molecular Characterization of a Novel UDP-Glucose:Anthocyanin 3′-O-Glucosyltransferase, a Key Enzyme for Blue Anthocyanin Biosynthesis, from Gentian

• Published in: Plant physiology (Bethesda) Vol. 132; no. 3; pp. 1652 - 1663
• Main Authors: Masako Fukuchi-Mizutani, Hiroaki Okuhara, Yuko Fukui, Masahiro Nakao, Katsumoto, Yukihisa, Keiko Yonekura-Sakakibara, Takaaki Kusumi, Hase, Toshiharu, Yoshikazu Tanaka
• Format: Journal Article
• Language: English
• Published: Rockville, MD American Society of Plant Biologists 01-07-2003; American Society of Plant Physiologists
• Subjects: Amino Acid Sequence; Amino acids; Anthocyanins - biosynthesis; Anthocyanins - chemistry; Base Sequence; Betacyanins; Biochemical Processes and Macromolecular Structures; Biological and medical sciences; Biosynthesis; Cloning, Molecular; Complementary DNA; DNA, Complementary - genetics; Enzymes; Flavonoids; Flowers; Flowers - enzymology; Fundamental and applied biological sciences. Psychology; Gene Expression; Gene Expression Profiling; Gentiana - enzymology; Gentiana - genetics; Gentiana - metabolism; Gentiana triflora; Glucosyltransferases - chemistry; Glucosyltransferases - genetics; Glucosyltransferases - isolation & purification; Glucosyltransferases - metabolism; Magnetic Resonance Spectroscopy; Metabolism; Molecular Sequence Data; Molecular Structure; Petals; Petunia - genetics; Phylogeny; Plant physiology and development; Plants; Plants, Genetically Modified; RNA; RNA, Messenger - genetics; RNA, Messenger - metabolism; Storage and secretion, pigments, phytochrome; Anthocyanin; Characterization; Enzyme; Dicotyledones; Transferases; Angiospermae; Gentianaceae; Glycosyltransferases; Spermatophyta; Biosynthesis; Glycosylation
• ISSN: 0032-0889; 1532-2548
• DOI: 10.1104/pp.102.018242

Gentian (Gentiana triflora) blue petals predominantly contain an unusually blue and stable anthocyanin, delphinidin 3-O-glucosyl-5-O-(6-O-caffeoyl-glucosyl)-3′-O-(6-O-caffeoyl-glucoside) (gentiodelphin). Glucosylation and the subsequent acylation of the 3′-hydroxy group of the B-ring of anthocyanins are important to the stabilization of and the imparting of bluer color to these anthocyanins. The enzymes and their genes involved in these modifications of the B-ring, however, have not been characterized, purified, or isolated to date. In this study, we purified a UDP-glucose (Glc):anthocyanin 3′-O-glucosyltransferase (3′GT) enzyme to homogeneity from gentian blue petals and isolated a cDNA encoding a 3′GT based on the internal amino acid sequences of the purified 3′GT. The deduced amino acid sequence indicates that 3′GT belongs to the same subfamily as a flavonoid 7-O-glucosyltransferase from Schutellaria baicalensis in the plant glucosyltransferase super-family. Characterization of the enzymatic properties using the recombinant 3′GT protein revealed that, in contrast to most of flavonoid glucosyltransferases, it has strict substrate specificity: 3′GT specifically glucosylates the 3′-hydroxy group of delphinidin-type anthocyanins containing Glc groups at 3 and 5 positions. The enzyme specifically uses UDP-Glc as the sugar donor. The specificity was confirmed by expression of the 3′GT cDNA in transgenic petunia (Petunia hybrida). This is the first report of the gene isolation of a B-ring-specific glucosyltransferase of anthocyanins, which paves the way to modification of flower color by production of blue anthocyanins.