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The blue petal color of the cornflower (Centaurea cyanus) is caused by protocyanin, a kind of metalloanthocyanin, which is a self-assembled supramolecular metal complex pigment. Protocyanin is composed of six molecules of anthocyanin, six molecules of flavone, one ferric ion, and one magnesium ion. The ferric ion is essential for blue color development. Here, we identify the vacuolar iron transporter gene (CcVIT) from the blue petals of C. cyanus and its function is identified and characterized. The CcVIT transcript was observed only in the petals. Its amino acid sequence is highly homologous to the Arabidopsis thaliana (AtVIT1) and Tulipa gesneriana (TgVit1) vacuolar iron transporters. Heterologous expression of the CcVIT gene in yeast indicated that the corresponding gene product transports ferrous ion into vacuoles. Analysis of purple mutant-line petals clarified that the anthocyanin and flavone components were the same as those found in plants with blue petals, but the amount of iron ions in the colored cells decreased, and consequently the amount of blue protocyanin was reduced. The CcVIT gene was expressed even in purple mutant petals, however, an amino acid substitution (A236E) occurred in that case. This change in the CcVIT gene sequence also resulted in loss of iron transport activity. The CcVIT protein thus plays a critical role in the blue coloration of cornflower petals.

The sepal color of a chameleon hydrangea, Hydrangea macrophylla cv. Hovariatrade mark 'Homigo' changes in four stages, from colorless to blue, then to green, and finally to red, during maturation and the senescence periods. To clarify the chemical mechanism of the color change, we analyzed the components of the sepals at each stage. Blue-colored sepals contained 3-O-sambubiosyl- and 3-O-glucosyldelphinidin along with three co-pigments, 5-O-p-coumaroyl-, 5-O-caffeoyl- and 3-O-caffeoylquinic acids. The contents of glycosyldelphinidins decreased toward the green-colored stage, with a coincident increase in the number of chloroplasts. During the last red colored stage, the two species of 3-O-glycosyldelphinidin almost disappeared, and another two anthocyanins, 3-O-sambubiosyl- and 3-O-glucosylcyanidin, increased in amounts. Mixing of 3-O-glycosylcyanidins, co-pigments, and Al3+ in a buffered solution at pH 3.0-3.5 gave not a blue, but a red, colored solution that was the same as that of the sepal color of the 4th stage. Sepals of hydrangea grown in an highland area also turned red in autumn, and contained the same cyanidin glycosides. The red coloration of the hydrangea during senescence was due to a change in anthocyanin biosynthesis.

A metalloanthocyanin, cyanosalvianin, was found in blue petals of Salvia uliginosa . Cyanosalvianin consisted of 3- O -(6- O - p -coumaroylglucopyranosyl)-5- O -(4- O -acetyl-6- O -malonylglucopyranosyl) delphinidin, 7,4′-di- O -glucopyranosylapigenin and magnesium ion. We reproduced the same blue color as the petals by mixing the three components together. An ESI-MS measurement gave a molecular weight of 9014 indicating the composition of cyanosalvianin to be six molecules of the anthocyanin component, six molecules of the flavone component and two magnesium ions. The special arrangement of the organic components in cyanosalvianin was analyzed by CD and 2D-NMR spectroscopy. It was clarified that cyanosalvianin has a similar structure to that of commelinin, a metalloanthocyanin isolated from blue dayflower, Commelina communis .

The Himalayan blue poppy, Meconopsis grandis, has sky blue-colored petals, although the anthocyanidin nucleus of the petal pigment is cyanidin. The blue color development in this blue poppy involving ferric ions was therefore studied. We analyzed the vacuolar pH, and the organic and inorganic components of the colored cells. A direct measurement by a proton-selective microelectrode revealed that the vacuolar pH value was 4.8. The concentrations of the total anthocyanins in the colored cells were around 5mM, and ca. three times more concentrated flavonols were detected. Fe was detected by atomic analysis of the colored cells, and the ratio of Fe to anthocyanins was ca. 0.8 eq. By mixing the anthocyanin, flavonol and metal ion components in a buffered aq. solution at pH 5.0, we were able to reproduce the same blue color; the visible absorption spectrum and CD were identical to those in the petals, with Fe(3+), Mg(2+) and flavonol being essential for the blue color. The blue pigment in Meconopsis should be a new type of metal complex pigment that is different from a stoichiometric supramolecular pigment such as commelinin or protocyanin.

The mechanism of dusky reddish-brown “kaki” color development of morning glory, Ipomoea nil cv. Danjuro, was studied. Three major known anthocyanins were isolated as glucosylated pelargonidin derivatives. Measurement of the vacuolar pH with proton-selective microelectrodes revealed the vacuolar pH of the colored cell of open flowers to be 6.8, while that of buds was 5.8. Mixing of the three anthocyanins according to the composition ratio in petals at pH 6.8 allowed the identical color to that of petals to be reproduced. The typical “kaki” color development was mostly caused by 5-OH free acylated anthocyanins, which have two λmax around 435 and 535 nm in the visible region.

Two novel diacylated anthocyanins and two known monoacylated anthocyanins were isolated from violet petals of Petunia hybrida cv. Surfinia Violet Mini. Their structures were elucidated by spectral methods. The major novel pigment was identified as malvidin 3- O -(6- O -(4- O -(4- O -(6- O -caffeoyl- β -d-glucopyranosyl)- p -coumaroyl)- α -l-rhamnosyl)- β -d-glucopyranoside)-5- O - β -d-glucopyranoside and the minor novel pigment was determined as malvidin 3- O -(6- O -(4- O -(4- O -(6- O -caffeoyl- β -d-glucopyranosyl)-caffeoyl)- α -l-rhamnosyl)- β -d-glucopyranoside)-5- O - β - d-glucopyranoside.

The dicaffeoyl anthocyanin, phacelianin, was isolated from blue petals of Phacelia campanularia. Its structure was determined to be 3-O-(6-O-(4'-O-(6-O-(4'-O-beta-d-glucopyranosyl-(E)-caffeoyl)-beta-d-glucopyranosyl)-(E)-caffeoyl)-beta-d-glucopyranosyl)-5-O-(6-O-malonyl-beta-d-glucopyranosyl)delphinidin. The CD of the blue petals of the phacelia showed a strong negative Cotton effect and that of the suspension of the colored protoplasts was the same, indicating that the chromophores of phacelianin may stack intermolecularly in an anti-clockwise stacking manner in the blue-colored vacuoles. In a weakly acidic aqueous solution, phacelianin displayed the same blue color and negative Cotton effect in CD as those of the petals. However, blue-black colored precipitates gradually formed without metal ions. A very small amount of Al(3+) or Fe(3+) may be required to stabilize the blue solution. Phacelianin may take both an inter- and intramolecular stacking form and shows the blue petal color by molecular association and the co-existence of a small amount of metal ions. We also isolated a major anthocyanin from the blue petals of Evolvulus pilosus and revised the structure identical to phacelianin.

To clarify the function of each caffeoyl residue in the diacylated anthocyanin gentiodelphin, a pigment from the blue flower of Gentiana makinoi, two mono-deacyl derivatives were compared for both color development and stability. In neutral solution, 3,5-di-O-beta-D-glucopyranosyl-3'-O-(6-O-caffeoyl-beta-D- glucopyranosyl)delphinidin was both bluer and more stable than 3,3'-di-O-beta-D-glucopyranosyl-5-O-(6-O-caffeoyl-beta-D- glucopyranosyl)delphinidin. Conformational analysis of each derivative under acidic conditions revealed only the 3'-O-caffeoylglucopyranosyl derivative to demonstrate intramolecular stacking. Additionally, the acyl residue in the B-ring contributed more to blue color development than that in the A-ring.

Three flavones from purple leaves of Perilla ocimoides var. crispa were characterized as the 7-O-diglucuronides of apigenin, luteolin, and scutellarein. The latter is a new natural compound.