Single-cell analysis clarifies mosaic color development in purple hydrangea sepal.

Hydrangea sepals exhibit a wide range of colors, from red, through purple, to blue; the purple color is a color mosaic. However, all of these colors are derived from the same components: simple anthocyanins, 3-O-glycosyldelphinidins, three co-pigment components, acylquinic acids and aluminum ions (Al3+ ). We show the color mosaic is a result of graded differences in intravacuolar factors. In order to clarify the mechanisms of mosaic color, we performed single-cell analyses of vacuolar pH, and anthocyanin, co-pigment and Al3+ content. From the sepals, a protoplast mixture of various colors was obtained. The cell color was evaluated by microspectrophotometry and vacuolar pH then was recorded by using a pH microelectrode. The organic and Al3+ contents were quantified by micro-HPLC. We found that the bluer the cell, the greater the ratio of 5-O-acylquinic acids and Al3+ to anthocyanins. Furthermore, reproducing experiments were conducted by mixing the components under various pH condition; all the colors could be reproduced in the various mixing conditions. Based on the above, we provide experimental evidence for cell color variation in hydrangea. Our study demonstrates the expression of phenotypic differences without any direct genomic control.

Blue flower coloration of Corydalis ambigua requires ferric ion and kaempferol glycoside.

Corydalis ambigua (Japanese name, Ezoengosaku) flowers bloom with blue to purplish petals in early spring in Hokkaido prefecture. In this study, a mechanism for blue petal coloration by ferric ions and keampferol glycoside was elucidated. Blue petals and cell sap exhibited similar visible (Vis) spectra, with λmax at approximately 600 nm and circular dichroism (CD) with positive exciton-type Cotton effects in the Vis region. Analysis of the organic components of the petals confirmed cyanidin 3-O-sambubioside and kaempferol 3-O-sambubioside as the major flavonoids. Mg, Al, and Fe were detected in petals using atomic emission spectroscopy. Color, Vis absorption, and CD consistent with those of blue petals were reproduced by mixing cyanidin 3-O-sambubioside, kaempferol 3-O-sambubioside, and Fe3+ in a buffered aqueous solution at pH 6.5. Both Fe3+ and flavonol were essential for blue coloration.

Insight into chemical mechanisms of sepal color development and variation in hydrangea.

Hydrangea (Hydrangea macrophylla) is a unique flower because it is composed of sepals rather than true petals that have the ability to change color. In the early 20th century, it was known that soil acidity and Al3+ content could intensify the blue hue of the sepals. In the mid-20th century, the anthocyanin component 3-O-glucosyldelphinidin (1) and the copigment components 5-O-caffeoylquinic, 5-O-p-coumaroylquinic, and 3-O-caffeoylquinic acids (2-4) were reported. Interestingly, all hydrangea colors from red to purple to blue are produced by the same organic components. We were interested in this phenomenon and the chemical mechanisms underlying hydrangea color variation. In this review, we summarize our recent studies on the chemical mechanisms underlying hydrangea sepal color development, including the structure of the blue complex, transporters involved in accumulation of aluminum ion (Al3+), and distribution of the blue complex and aluminum ions in living sepal tissue.

Polyacylated Anthocyanins in Bluish-Purple Petals of Chinese Bellflower, Platycodon grandiflorum.

The bluish-purple petals of Chinese bellflower, Platycodon grandiflorum (kikyo in Japanese), contain platyconin (1) as the major anthocyanin. Platyconin (1) is a polyacylated anthocyanin with two caffeoyl residues at the 7-position, and its color is stable in a diluted, weakly acidic aqueous solutions. HPLC analysis of the fresh petal extract showed the presence of several minor pigments. Photo-diode array detection of minor pigments suggested that some of these were polyacylated anthocyanins. To establish the relationship between structure and stability of the acylated anthocyanins and to obtain information on their biosynthetic pathways, minor pigments were isolated from the petals, and their structures were determined by MS and NMR analyses. Four known (2-5) and three new anthocyanins (6-8) were identified, which contained a delphinidin chromophore, and four of these (5-8) were diacylated anthocyanins, in which the acyl-glucosyl-acyl-glucosyl chain was attached at the 7-O-position of the delphinidin chromophore. These diacylated anthocyanins exhibited a bluish-purple color at pH 6, which was stable for more than a week.

Determination of absolute configuration of photo-degraded catechinopyranocyanidin A by modified Mosher's method.

Catechinopyranocyanidins A and B (cpcA and cpcB) are two purple pigments present in the seed-coat of red adzuki bean, Vigna angularis, of which cpcA is the major pigment, containing two chiral carbons in the catechin part. Their absolute configurations were determined by comparison of their experimental and quantum chemical calculated electronic circular dichroisms (ECDs). These purple pigments are labile on light irradiation and easily decompose to photo-degraded catechinopyranocyanidins A and B (pdcpcA and pdcpcB), while retaining the stereostructure of the catechin residue. We applied modified Mosher's method for determining the chirality of the secondary alcohol in pdcpcA. Hexamethylation of pdcpcA by diazomethane followed by esterification using (S)- and (R)-MTPACl gave (R)- and (S)-MTPA esters, respectively. By analysis of the NMR spectra of (R)- and (S)-MTPA esters of tetramethylated (+)-catechin, the chirality of pdcpcA was determined to be 2R, 3S, same as the absolute configuration of cpcA.

Determination of Anthocyanins and Antioxidants in 'Titanbicus' Edible Flowers In Vitro and In Vivo.

Titanbicus (TB), a hybrid of Hibiscus moscheutos × H. coccineus (Medic.) Walt., has potential to be used as an edible flower. In this study, proximate nutritional content, anthocyanin content, total polyphenol content (TPC), and antioxidant activities in vitro and in vivo were investigated. Three cultivars of TB, namely Artemis (AR), Rhea (R), and Adonis (AD), were used as materials. Protein and carbohydrates were the primary macronutrients, while crude fat and ash were detected in trace amounts. Cyanidin 3-glucoside (Cy3-G) and cyanidin 3-sambubioside (Cy3-Sam), were identified in all TBs. The highest anthocyanin content was observed in AD (47.09 ± 1.45 mg/g extract), followed by R and AR (6.04 ± 0.20 and 2.72 ± 0.11 mg/g extract, respectively). The TPC of AD (225.01 ± 1.97 mg/g extract) was greater than that of AR and R (185.41 ± 3.24 and 144.10 ± 1.71 mg/g extract, respectively). AD exhibited the strongest in vitro antioxidant activity in hydrophilic oxygen radical absorbance capacity, compared to the other two TBs. In addition, AD extract suppressed the generation of reactive oxygen species in caudal fin of wounded zebrafish. Antioxidant activities of AD appeared to be related to its total anthocyanin content, Cy3-G, Cy3-Sam, and TPC. Our findings indicate that TB, particularly the AD cultivar, would be an attractive source of bioactive compounds with antioxidant activities, and can improve both nutritional value and appearance of food.

Change of Petals' Color and Chemical Components in Oenothera Flowers during Senescence.

Oenothera flower petals change color during senescence. When in full bloom, the flowers of O. tetraptera are white and those of O. laciniata and O. stricta are yellow. However, the colors change to pink and orange, respectively, when the petals fade. We analyzed the flavonoid components in these petals as a function of senescence using HPLC-DAD and LC-MS. In all three species, cyanidin 3-glucoside (Cy3G) was found in faded petals. The content of Cy3G increased in senescence. In full bloom (0 h), no Cy3G was detected in any of the petals. However, after 12 h, the content of Cy3G in O. tetraptera was 0.97 µmol/g fresh weight (FW) and the content of Cy3G in O. laciniata was 1.82 µmol/g FW. Together with anthocyanins, major flavonoid components in petals were identified. Quercitrin was detected in the petals of O. tetraptera and isosalipurposide was found in the petals of O. laciniata and O. stricta. The content of quercitrin did not change during senescence, but the content of isosalipurposide in O. laciniata increased from 3.4 µmol/g FW at 0 h to 4.8 µmol/g FW at 12 h. The color change in all three Oenothera flowers was confirmed to be due to the de novo biosynthesis of Cy3G.

Intermolecular Copigmentation Between Delphinidin 3-O-Glucoside and Chlorogenic Acid: Taking into Account the Existence of Neutral and Negatively Charged Forms of the Copigment.

Stopped flow corroborated by UV-vis measurements allowed for the calculation of the copigmentation constants of delphinidin 3-O-glucoside with the neutral (CP) and negatively charged CP(-) forms of chlorogenic acid. Solutions of delphinidin 3-O-glucoside in the absence and presence of the copigment were equilibrated at several pH values in the acidic region, pH < 6, and reverse pH jumps monitored by stopped flow were carried out by adding sufficient acid to give flavylium cation at pH ≤ 1. This procedure allows for the separation of three contributions: (i) all flavylium cation and quinoidal base species, (ii) all hemiketal species, and (iii) all cis-chalcone species. Reverse pH jumps can also be performed at fixed pH versus copigment addition. The contribution of trans-chalcone, minor species in the present system, requires reverse pH jumps from the equilibrium followed by a common spectrophotometer. The system was also studied by UV-vis as a function of the copigment addition at different pH values. A global fitting of all experimental data allowed for determination of the copigmentation constants with flavylium cation, KAH+CP = 167 M-1, KAH+CP(-) = 338 M-1; and quinoidal base, KACP = 1041 M-1, KACP(-)= 221 M-1. No significant copigmentation was observed for hemiketal and chalcones. Computational calculations confirm different geometries for the interactions of flavylium cation and quinoidal base with the neutral or the negatively charged forms of the copigment as well as predict identical relative order for the binding energies of the four adducts.

Urakunoside, a new tetraglycosyl kaempferol from petals of the Wabisuke camellia cv. Tarokaja.

A new tetraglycosyl flavonol, 3-O-[2-O-xylosyl-6-O-(3-O-glucosyl-rhamnosyl) glucosyl] kaempferol was isolated from pale purplish-pink petals of Wabisuke camellia cv. Tarokaja with three known flavonols. It was named urakunoside after the species name of Tarokaja, Camellia uraku. Urakunoside was a major flavonol component in the Tarokaja petals, but was not detected in petals of Tarokaja's presumed ancestor species.

Protecting group-free method for synthesis of N-glycosyl carbamates and an assessment of the anomeric effect of nitrogen in the carbamate group.

The first protecting group-free synthesis of N-glycosyl carbamates has been developed through reaction of d-glucose with n-butyl carbamate in acidic aqueous media. The structures of the N-glucosyl carbamates were unambiguously determined by comparison with authentic samples, prepared using the isocyanide method. With this protective group-free method for synthesis of N-glycosyl carbamates in hand, an anomeric pair of N-xylopyranosyl carbamates were prepared and used to assess the anomeric effect of nitrogen in the carbamate group.

Discovery of a natural cyan blue: A unique food-sourced anthocyanin could replace synthetic brilliant blue.

The color of food is critical to the food and beverage industries, as it influences many properties beyond eye-pleasing visuals including flavor, safety, and nutritional value. Blue is one of the rarest colors in nature's food palette-especially a cyan blue-giving scientists few sources for natural blue food colorants. Finding a natural cyan blue dye equivalent to FD&C Blue No. 1 remains an industry-wide challenge and the subject of several research programs worldwide. Computational simulations and large-array spectroscopic techniques were used to determine the 3D chemical structure, color expression, and stability of this previously uncharacterized cyan blue anthocyanin-based colorant. Synthetic biology and computational protein design tools were leveraged to develop an enzymatic transformation of red cabbage anthocyanins into the desired anthocyanin. More broadly, this research demonstrates the power of a multidisciplinary strategy to solve a long-standing challenge in the food industry.

5,7,3',4'-Tetrahydroxyflav-2-en-3-ol 3-O-glucoside, a new biosynthetic precursor of cyanidin 3-O-glucoside in the seed coat of black soybean, Glycine max.

The seed coat of mature black soybean, Glycine max, accumulates a high amount of cyanidin 3-O-glucoside (Cy3G), which is the most abundant anthocyanin in nature. In the pod, it takes two months for the seed coat color change from green to black. However, immature green beans rapidly adopt a black color within one day when the shell is removed. We analyzed the components involved in the color change of the seed coat and detected a new precursor of Cy3G, namely 5,7,3',4'-tetrahydroxyflav-2-en-3-ol 3-O-glucoside (2F3G). Through quantitative analysis using purified and synthetic standard compounds, it was clarified that during this rapid color change, an increase in the Cy3G content was observed along with the corresponding decrease in the 2F3G content. Chemical conversion from 2F3G to Cy3G at pH 5 with air and ferrous ion was observed. Our findings allowed us to propose a new biosynthetic pathway of Cy3G via a colorless glucosylated compound, 2F3G, which was oxidized to give Cy3G.

Anthocyanin components and mechanism for color development in blue Veronica flowers.

3-Di-p-coumaroylsophoroside-5-malonylglucoside and its demalonyl derivative were isolated from blue petals of Veronica persica Poiret. Blue, violet and purple cells coexist in the petal. These colors might be due to the varying pH of the vacuole between 5 and 7 unit. Only the demalonylated pigment was detected in the blue anthers.

Chemical studies on different color development in blue- and red-colored sepal cells of Hydrangea macrophylla.

To clarify the cause of the difference in blue and red color development of hydrangea sepals, Hydrangea macrophylla, we analyzed the organic and inorganic components in the colored cells. To obtain colored protoplasts, each blue and red sepal tissue was treated with a combination of cellulase and pectinase, and then from the suspension of the olored and colorless protoplast mixture colored cells of the same hue were collected with a micro-pipette. The content of organic components (delphinidin 3-glucoside, chlorogenic acid, neochlorogenic acid and 5-O-p-coumaroylquinic acid) and Al(3+) in each colored cell was quantified respectively by semimicro-HPLC and graphite furnace atomic absorption spectroscopy (GFAAS). In the blue cells 13 eq. of 5-O-acylquinic acids and 1.2 eq. of Al(3+) to anthocyanin were contained. Contrary to this result, in the red cells, only 3.6 eq. of 5-O-acylquinic acids and 0.03 eq. of Al(3+) were detected. A reproduction experiment of each blue and red sepal color by mixing those components concluded that, for blue coloration, both 5-O-acylquinic acids and Al(3+) were essential.

Synchrony between flower opening and petal-color change from red to blue in morning glory, Ipomoea tricolor cv. Heavenly Blue.

Petal color change in morning glory Ipomoea tricolor cv. Heavenly Blue, from red to blue, during the flower-opening period is due to an unusual increase in vacuolar pH (pHv) from 6.6 to 7.7 in colored epidermal cells. We clarified that this pHv increase is involved in tonoplast-localized Na+/H+ exchanger (NHX). However, the mechanism of pHv increase and the physiological role of NHX1 in petal cells have remained obscure. In this study, synchrony of petal-color change from red to blue, pHv increase, K+ accumulation, and cell expansion growth during flower-opening period were examined with special reference to ItNHX1. We concluded that ItNHX1 exchanges K+, but not Na+, with H+ to accumulate an ionic osmoticum in the vacuole, which is then followed by cell expansion growth. This function may lead to full opening of petals with a characteristic blue color.

Conversion of flavonol glycoside to anthocyanin: an interpretation of the oxidation-reduction relationship of biosynthetic flavonoid-intermediates.

An efficient conversion of rutin to the corresponding anthocyanin, cyanidin 3-O-rutinoside, was established. Clemmensen-type reduction of rutin gave a mixture of flav-2-en-3-ol and two flav-3-en-3-ols, which were easily oxidised by air to give the anthocyanin. The interconversion reactions of these flavonoids provide insight into their biosynthetic pathway.

Author Correction: Structure of two purple pigments, catechinopyranocyanidins A and B from the seed-coat of the small red bean, Vigna angularis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Structure of two purple pigments, catechiopyranocyanidins A and B from the seed-coat of the small red bean, Vigna angularis.

The small red bean, Vigna angularis, is primarily used to produce the "an-paste" component of Japanese sweets. Through the manufacturing process, the red seed-coat pigment is transferred to the colorless "an-particles", imparting a purple color. However, the major pigment in the seed coat has not yet been identified, although it is historically presumed to be an anthocyanin. Here, we report the isolation and structural determination of two hydrophobic purple pigments in the seed coat via instrumental analysis and derivatization. The new pigments, catechinopyranocyanidins A and B, contain a novel pyranoanthocyanidin skeleton condensed with a catechin and cyanidin ring system, and no sugar moieties. Catechinopyranocyanidins A and B are diastereomers with a different configuration at the catechin moiety, and both are purple in color in strongly acidic-to-neutral media. Catechinopyranocyanidins A and B are very stable under dark conditions, but, labile to light and decompose to colorless compounds. Thus, these pigments exhibit quite different chemical properties compared to simple anthocyanidins.

Cyanosalvianin, a supramolecular blue metalloanthocyanin, from petals of Salvia uliginosa.

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.

Change of color and components in sepals of chameleon hydrangea during maturation and senescence.

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.