Biochemical and molecular characterization of a novel UDP-glucose:anthocyanin 3'-O-glucosyltransferase, a key enzyme for blue anthocyanin biosynthesis, from gentian

Abstract

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 superfamily. 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.

Figure 1.

Structures of gentiodelphin and anthocyanins used in this study. The abbreviation for each compound is in parentheses. The enzymes involved in biosynthesis of gentiodelphin from DEL were indicated on the structure of gentiodelphin (top of figure). 3′AT, Hydroxycinnamoyl-CoA:anthocyanin 3′-O-glucoside-6′′′-O-hydroxycinnamoyltransferase.

Figure 2.

SDS-PAGE analysis of the 3′GT protein purified from gentian petals. The active fractions after each purification step were analyzed through a 10% to 20% (m/v) polyacrylamide gel and detected by silver-staining. Lane M, M_r marker; lane 1, fraction of 40% to 70% saturation with ammonium sulfate; lane 2, Sephadex G-25 elute; lane 3, Q-Sepharose elute; lane 4, Blue A elute; lane 5, Superose 12 elute. M_r for each band of the marker is indicated to the left of the panel.

Figure 3.

HPLC analysis of the reaction products of the purified gentian 3′GT. A, HPLC profiles of the substrate DEL 3G-5G (left) and the reaction product by the purified gentian 3′GT using DEL 3G-5G and UDP-Glc as substrates (right). A₅₂₀ was monitored. The retention times corresponding to DEL 3G-5G and the major product were approximately 14.6 and 11.4 min, respectively. B, UV-visible spectra of the DEL 3G-5G (solid line) and the major product detected at 11.4 min in HPLC analysis (dashed line). The absorbance maximum for DEL 3G-5G and the major product were 524 and 515 nm, respectively (as indicated).

Figure 4.

The nucleotide and deduced amino acid sequences of a gentian 3′GT cDNA. The sequences of five peptide fragments generated by lysylendopeptidase digestion of the purified gentian 3′Gt are underlined.

Figure 5.

Molecular phylogenetic tree of the amino acid sequences of the plant glycosyltransferase superfamily. The tree was obtained by ClustalW analysis program (Thompson et al., 1994). GenBank accession numbers of the GTs: Gentian 3′GT (AB076697); D. bellidiformis 5GT (Y18871); scuttellaria 7GT (BAA83484); verbena 5GT (BAA36423); perilla 5GT (AB013596); torenia 5GT (AB076698); petunia 5GT (AB027455); petunia RT (Z25802); maize 3GT (X13501); barley GT (X15694); petunia 3GT (AB027454); egg-plant 3GT (X77369); gentian 3GT (D85186); and grape 3GT (AF000371). The sequence of snapdragon 3GT appeared in Martin et al. (1991). The percent identity of amino acid residues between each glucosyltransferase and the gentian 3′GT is indicated in parentheses.

Figure 6.

SDS-PAGE analysis of the 3′GT protein expressed in E. coli. The active fraction after a Phenyl Sepharose column was analyzed through a 10% to 20% (m/v) polyacrylamide gel and stained by Coomassie Blue. Left lane, M_r marker; right lane, the active fraction after a Phenyl Sepharose. M_r for each band of the marker is indicated to the left of the panel. The arrow indicates the size for 3′GT.

Figure 7.

¹H- and ¹³C-NMR assignment and structure of the reaction produced by 3′GT. A, Assignment of the NMR spectra of the reaction product by the recombinant 3′GT protein using DEL 3G-5G and UDP-Glc as substrates. B, Structure of the reaction product confirmed by ROESY and heteronuclear multiple bond correlation (HMBC) analyses. Rotating frame overhauser enhancement (ROE) detected on ROESY are indicated by arrow curves and HMBC cross peaks are indicated by thick lines.

Figure 8.

Northern-blot analysis of the expression of the gentian 3′GT gene. Ten micrograms of total RNA from gentian petals at each developmental stage and from leaves was loaded on each lane and probed with DIG-labeled gentian 3′GT cDNA (middle panel) or gentian 5AT cDNA (bottom panel; Fujiwara et al., 1998) after electrophoresis and transfer to nylon membrane. The top panel shows RNA samples on an agarose gel before transfer to a nylon membrane. RNA was from petals of unpigmented buds tightly closed (lane 1), petals of pigmenting buds (lane 2), petals of fully pigmented buds before opening (lane 3), petals of fully opened flowers (lane 4), and leaves (lane 5).

Figure 9.

Northern-blot analysis of the expression of gentian 3′GT and torenia 5GT transgenes in petunia transformants. A, Schematic view of the binary plasmid pSPB1112 expression cassettes for both gentian 3′GT and torenia 5GT cDNAs. B, Ten micrograms of total RNA from petals of each transformant and host petunia cv Skr4 × Da was loaded on each lane, and the membrane was probed with ³²P-labeled gentian 3′GT cDNA (middle panel) and torenia 5GT cDNA (bottom panel). The top panel shows RNA samples on an agarose gel before transfer to a nylon membrane. The numbers above the panel identify each transformant. The content of DEL 3G-5G-3′G in fully opened flowers of each transformant (as a percentage of total anthocyanins analyzed by HPLC) are described below the bottom panel.

Figure 10.

Proposed modification pathway of DEL to gentiodelphin in gentian petals. G in an oval represents O-glucoside and Caf represent O-caffeic acid. 3′AT, Hydroxycinnamoyl-CoA:anthocyanin 3′-O-glucoside-6″-O-hydroxycinnamoyltransferase.