Components of protocyanin, a blue pigment from the blue flowers of Centaurea cyanus

The components involved in the formation of protocyanin, a stable blue complex pigment from the blue cornflower, Centaurea cyanus, were investigated. Reconstruction experiments using highly purified anthocyanin [centaurocyanin, cyanidin 3-O-(6-O-succinylglucoside)-5-O-glucoside], flavone glycoside [apigenin 7-O-glucuronide-4'-O-(6-O-malonylglucoside)] and metals, Fe and Mg, showed the presence of another factor essential for the formation of protocyanin. The unknown factor was revealed to be Ca. Reconstructed protocyanin using anthocyanin, flavone, Fe, Mg, and Ca was identical with protocyanin from nature in UV-Vis and CD spectra, and was isolated as crystals for the first time. In addition, substitution of the metal components in protocyanin with other metals was also examined.     

Ferric ions involved in the flower color development of the Himalayan blue poppy, Meconopsis grandis

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.     

ANTHOCYANIN SYNTHESIS IN SALPIGLOSSIS SINUATA

Measurements were made of the growth and pigment content of developing flower buds of Salpiglossis sinuata. From the time the buds were approximately 10 mm long they grew in length exponentially until they reached their final length. The logarithm of bud length increased linearly with time and served as a convenient morphological index on which to relate the progress of anthocyanin synthesis. Buds shorter than about 42 mm had no anthocyanin, but when buds reached this length, anthocyanin production was initiated and proceeded rapidly. The maximum relative pigment concentration (pigment/mg fresh weight) was attained by the buds about 17 hr after the initiation of pigment synthesis. In the mahogany-colored variety used in these studies, two anthocyanidins were found and identified as cyanidin and delphinidin. Buds excised from the plants could be cultured in vitro. Buds started in culture at a length of 30–35 mm when they contained no anthocyanins developed pigment during their growth. The amount of pigment formed increased with increasing light intensity, while only small amounts of pigment could be formed in buds cultured in darkness. The anthocyanidins of these cultured buds were the same as those of the intact flowers, but the ratio of delphinidin to cyanidin decreased with decreasing light intensity. Brief daily irradiation of dark-grown buds with red, far-red or blue light did not increase pigment synthesis nor change the anthocyanidin ratio. If buds were placed in culture at 20–25 mm and grown in darkness, they developed a third anthocyanidin, identified as malvidin, which was not present in intact flowers, light-grown buds or 30–35-mm buds cultured in darkness.     

Cyanidin 3-[6-(p-coumaroyl)-2-(xylosyl)-glucoside]-5-glucoside and other anthocyanins from fruits of sambucus canadensis

From the fruits of Sambucus canadensis four anthocyanin glycosides have been isolated by successive application of an ion-exchange resin, droplet-counter chromatography and gel filtration. The structure of the novel, major (69.8%) pigment, cyanidin 3-O-[6-O-(E-p-coumaroyl-2-O-(β- -xylopyranosyl)-β- -glucopyranoside]-5-O-β- -glucopyranoside, was determined by means of chemical degradation, chromatography and spectroscopy, especially homo- and heteronuclear two-dimensional NMR techniques. The other anthocyanins were identified as cyanidin 3-sambubioside-5-glucoside (22.7%), cyanidin 3-sambubioside (2.3 %) and cyanidin 3-glucoside (2.1 %).     

Concerning the structure of cinerarin,a blue anthocyanin from garden cineraria

A purplish-blue anthocyanin was isolated from the flower of garden cineraria (Senecio cruentus DC.). The pigment retains a stable blue color within the range of pH 3.5-7; but it differs in other characteristics from the known blue anthocyanins. This pigment is composed of delphinidin, glucose and caffeic acid in a molecular ratio of 1∶3∶2, respectively and is tentatively called “cinerarin”. The blue flower color of cineraria seems to be manifested solely by cinerarin, and it becomes likely that the caffeic acid involved in the molecule plays an essential role in the blueness of this pigment. Part LXV: Bot. Mag. Tokyo85: 303–306 (1972).     

Identification and distribution of malonated anthocyanins in plants of the compositae

A survey of 31 species from 28 genera in the Compositae showed the presence of zwitterionic anthocyanins in petals or stems of 27 species. Detailed investigations, including the use of FAB-MS, showed that mono- and dimalonated esters of pelargonin and cyanin occurred in Dahlia variabilis cultivars. The corresponding delphin mono- and dimalonates occur in blue flowers Cichorium intybus. A cyanidin 3-dimalonylglucoside was identified in stems of Coleostephus myconis while pelargonidin 3-(6″-malonylg]ucoside) was found in Callistephus petals. A further malonated cyanidin derivative in flowers of Helenium cv. Bruno was found to be the 3-glucuronosylglucoside; this is the first report of an anthocyanin with glucuronic acid. Overall, the results confirm that malonated anthocyanins are widespread in the family and that many pigments previously reported in the Compositae as being unacylated probably contain these labile organic acid attachments.     

Factors determining Petal Colour of Baccara Roses III: EFFECT OF THE RATIO BETWEEN CYANIN AND PELARGONIN

The changes in colour and in the pigment concentration of thetwo sides of Baccara rose petals which occur when plants aregrown under various temperature regimes, were examined. Theinner side of the petal is redder and the predominant pigmentis pelargonin whereas the outer petal surface tends to ‘blue’,and, the predominant pigment on this side is cyanin. The cyanin:pelargonin ratio on the outer side of petals increased three-foldunder the influence of low temperatures. The outer surface of petals growing for a long period underlow tempertaures was ‘blue’ when compared with thered petals which had been subjected to low temperatures fora short period. The cyanin: pelargonin ratio of ‘blue’petals was higher than that of red petals. Total pigment contentwas similar in both types of petal. Flowers grown under hightemperatures ‘blued’ without a concomitant fallin the cyaninpel: argonin ratio. Examination of colour solutions in which the ratio between cyaninand pelargonin was varied revealed that the colour of the solutionbecame bluer as this ratio increased. We suggest that the ‘blueing’ of Baccara rose petalsis caused primarily by a dilution of the cyanin content, butwhen the ratio between cyaniri and pelargonin increases sharply,‘blueing’ may also occur in dark flowers in whichthe total pigment content did not diminish.     

Active anthocyanin degradation in <Emphasis Type="Italic">Brunfelsia calycina</Emphasis> (yesterday–today–tomorrow) flowers

Anthocyanins are the largest group of plant pigments responsible for colors ranging from red to violet and blue. The biosynthesis of anthocyanins, as part of the larger phenylpropanoid pathway, has been characterized in great detail. In contrast to the detailed molecular knowledge available on anthocyanin synthesis, very little is known about the stability and catabolism of anthocyanins in plants. In this study we present a preliminary characterization of active in planta degradation of anthocyanins, requiring novel mRNA and protein synthesis, in Brunfelsia calycina flowers. Brunfelsia is a unique system for this study, since the decrease in pigment concentration in its flowers (from dark purple to white) is extreme and rapid, and occurs at a specific and well-defined stage of flower development. Treatment of detached flowers with protein and mRNA synthesis inhibitors, at specific stages of flower development, prevented degradation. In addition, treatment of detached flowers with cytokinins delayed senescence without changing the rate of anthocyanin degradation, suggesting that degradation of anthocyanins is not part of the general senescence process of the flowers but rather a distinctive and specific pathway. Based on studies on anthocyanin degradation in wine and juices, peroxidases are reasonable candidates for the in vivo degradation. A significant increase in peroxidase activity was shown to correlate in time with the rate of anthocyanin degradation. An additional indication that oxidative enzymes are involved in the process is the fact that treatment of flowers with reducing agents, such as DTT and glutathione, caused inhibition of degradation. This study represents the first step in the elucidation of the molecular mechanism behind in vivo anthocyanin degradation in plants.     

Anthocyanins and their distribution in the genusEpimedium

Anthocyanins contained in plants belonging to the genusEpimedium in Japan are discussed in this study. Two kinds of anthocyanin, delphinidin 3-p-coumaroyl-sophoroside-5-glucoside (cayratinin) and cyanidin 3-p-coumaroylsophoroside, were identified, and the latter is new to the literature. Only cayratinin was found in the colored petals of theEpimedium species, but cayratinin and cyanidin glucoside were contained in the stems, young leaves and autumn leaves of all the species surveyed.     

Wirkungsspektren der Anthocyansynthese in Gewebekulturen und Keimlingen von Haplopappus gracilis

Summary The biosynthesis of anthocyanin in tissue cultures and intact seedlings of Haplopappus gracilis is a light-dependent reaction which can be induced by blue light only. Anthocyanin appeared in all organs of the seedling.Wounding of the plant led to an increase in the content of anthocyanin due to increased anthocyanin synthesis in the cotyledons.The action spectra of anthocyanin formation in tissue cultures and intact seedlings have two peaks, one at 438 nm and the other at 372 nm. The limit of activity in the direction of longer wavelengths lies between 474 and 493 nm. Red light of short and long wavelength is ineffective in the induction of pigment synthesis. The photoreceptor of the light reaction is supposed to be a yellow pigment (flavoprotein or carotinoid). In contrast to the intact plants, isolated cotyledons and wounded seedlings are able to form anthocyanin not only in the blue region but also during irradiation with red light of high intensity. The action spectrum of anthocyanin synthesis in the isolated cotyledons has a marked maximum at about 440 nm and a second one at about 660 nm. A little activity can be observed throughout the visible spectrum. The pigment synthesis induced by red light can be completely suppressed by DCMU, an inhibitor of photosynthesis. This indicates that in the case of the activity in the red light caused by wounding chlorophyll serves as photoreceptor.The anthocyanin synthesis in tissue cultures and seedlings could not be influenced by low energy radiation in the red or in the far red region, even after induction of anthocyanin synthesis by blue light of high intensity. Therefore it seems that the phytochrome system is not involved in anthocyanin synthesis in Haplopappus gracilis.     

Mechanisms underlying flower color variation in Asian species of Meconopsis: A preliminary phylogenetic analysis based on chloroplast DNA and anthocyanin biosynthesis genes

The Qinghai–Tibet Plateau (QTP) harbors the highest species diversity of alpine plants in the world, with a spectacular diversity of flower colors. Among these QTP plants, the genus Meconopsis comprises more than 50 species, for which flower color is a key diagnostic character. However, the mechanisms underlying flower color variation have rarely been investigated. In the present study, we used three chloroplast (cp) DNA fragments and two anthocyanin biosynthesis genes (F3H andF3′H) for phylogenetic reconstruction of Meconopsis. We revealed the presence of three well-supported clades and/or subclades in the cpDNA and nuclear gene trees; further, flower color transition occurred in each lineage. The results of selection tests and preliminary expression analyses of the anthocyanin biosynthesis genes indicate that the pigment pathway leading to cyanidin is active in blue and red flowers of Meconopsis; further, a blue–red color shift is not attributable to an on/off switching of the anthocyanin biosynthetic pathway (ABP) branches. Together with the results of previous flower pigment analyses, our findings suggest that blue–red flower color transitions in Meconopsis are attributable to modification of cyanidin. Our molecular dating results indicate that the lineage diversification inMeconopsis is closely related to the QTP uplift; thus, it is likely that environmental changes arising from the QTP uplift have played important roles in driving the diversification of flavonoids, through which species of Meconopsis have adapted physiologically to diverse habitats.     

Cyanidin 3-glucoside accumulation in plane tree (Platanus acerifolia) cell-suspension cultures

The accumulation of only one anthocyanin, cyanidin 3-glucoside, in cell-suspension cultures of plane tree (Platanus aceriflia) is reported for the first time. During a time span of 6 years, no new anthocyanin was detected and cyanidin 3-glucoside was maintained at about 35 mg l^–1 cell culture medium. This stable cell culture system could therefore be used for the biotechnological production of cyanidin 3-glucoside.     

Inhibition of flavonoid biosynthesis by gibberellic acid in cell suspension cultures of Daucus carota L.

In carrot cells (Daucus carota L.), cultured in the presence of gibberellic acid, anthocyanin synthesis is blocked at the level of chalcone synthase. By feeding suitable precursors for anthocyanins (naringenin, eriodictyol, dihydroquercetin) biosynthesis of cyanidin glycosides can be restored. After addition of these substrates to the culture medium in the presence of gibberellic acid, the activity of chalcone synthase remained as low as in the control without precursors. The highest increase in anthocyanin content was achieved using dihydroquercetin as the added precursor. The time course of this supplementation showed a rapid response; within 4 h a substantial increase in anthocyanin could be observed. In contranst, the flavonol quercetin is not a precursor for cyanidin. The fact that naringenin was also accepted for cyanidin synthesis leads to the conclusion that hydroxylation in 3-position of ring B in Daucus carota takes place at the flavonoid stage.Abbreviations CHI Chalcone isomerase - CHS chalcone synthase - DMSO dimethylsulfoxide - GA₃ gibberellic acid - PAL phenylalanine ammonia-lyase     

New violet 3,3′-bipyridyl pigment purified from deep-sea microorganism <Emphasis Type="Italic">Shewanella violacea</Emphasis> DSS12

We have purified a new violet pigment derived from Shewanella violacea DSS12 to determine its chemical structure. The pigment colored blue in tetrahydrofuran (THF) or chloroform and showed a broad absorption spectrum from 500 to 700 nm. X-ray diffraction analysis of single crystals showed that the chemical structure of this pigment was 5,5′-didodecylamino-4,4′-dihydroxy-3,3′-diazodiphenoquinone-(2,2′), containing the same chromophore as an indigoidine known as microbial blue pigment. The violet color of this pigment was due to hypsochromic shift (blue shift) caused by the side-by-side orientation of this pigment molecule, revealed by X-ray structural analyses of a single crystal. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Antimutagenicity of Deacylated Anthocyanins in Purple-fleshed Sweetpotato

The antimutagenicity of the 3-sophoroside-5-glucoside of cyanidin and 3-sophoroside-5-glucoside of peonidin, the anthocyanin derivatives deacylated from the 3-(6,6'-caffeylferulylsophoroside)-5-glucoside of cyanidin (YGM-3) and 3-(6,6'-caffeylferulyl-sophoroside)-5-glucoside of peonidin (YGM-6) which had been purified from the sweetpotato with purple-colored flesh, was investigated by using Salmonella typhimurium TA 98. A comparison of the antimutagenicity between YGM-3 and YGM-6 and the deacylated derivatives showed that the activity of cyanidin was stronger than that of peonidin. Deacylation of the peonidin-type pigment markedly decreased this antimutagenicity. Caffeic acid showed the strongest antimutagenicity of the constituent organic acids of the anthocyanin pigments, caffeic acid, ferulic acid, and p-hydroxybenzoic acid. These results suggest that the cathecol structure plays an important role in the strong antimutagenicity of anthocyanin pigments.     

Two distinctive anthocyanin patterns in the commelinaceae

A survey of lead and flower anthocyanins in a representative sample (28 spp./10 genera) of the Commelinaceae has shown that the dominant anthocyanin is cyanidin 3,7,3′-triglucoside, acylated with caffeic acid. Acylation with other hydroxycinnamic acids also occurs. As a flower pigment, this anthocyanin is stabilized at the pH of the cell sap by the presence of the three acyl substituents attached through glucose. In Gibasis, the related delphinidin triglucoside is also present. By contrast, the genus Commelina is distinguished by uniformly containing p-coumaroyl-delphinidin 3,5-diglucoside, which is stabilized in flowers as a copigment complex with glycoflavone. There are thus two distinctive sources of blue flower colour in the family. Furthermore, the presence of these rare acylated glucosides clearly separates the Commelinaceae from all other monocotyledonous groups.     

Triacylated cyanidin 3-(3X-glucosylsambubioside)-5-glucosides from the flowers of Malcolmia maritima

Three acylated cyanidin 3-(3(X)-glucosylsambubioside)-5-glucosides (1-3) and one non-acylated cyanidin 3-(3(X)-glucosylsambubioside)-5-glucoside (4) were isolated from the purple-violet or violet flowers and purple stems of Malcolmia maritima (L.) R. Br (the Cruciferae), and their structures were determined by chemical and spectroscopic methods. In the flowers of this plant, pigment 1 was determined to be cyanidin 3-O-[2-O-(2-O-(trans-sinapoyl)-3-O-(beta-D-glucopyranosyl)-beta-D-xylopyranosyl)-6-O-(trans-p-coumaroyl)-beta-D-glucopyranoside]-5-O-[6-O-(malonyl)-(beta-D-glucopyranoside) as a major pigment, and a minor pigment 2 was determined to be the cis-p-coumaroyl isomer of pigment 1. In the stems, pigment 3 was determined to be cyanidin 3-O-[2-O-(2-O-(trans-sinapoyl)-3-O-(beta-D-glucopyranosyl)-beta-D-xylopyranosyl)-6-O-(trans-p-coumaroyl)-beta-d-glucopyranoside]-5-O-(beta-D-glucopyranoside) as a major anthocyanin, and also a non-acylated anthocyanin, cyanidin 3-O-[2-O-(3-O-(beta-D-glucopyranosyl)-beta-D-xylopyranosyl)-beta-D-glucopyranoside]-5-O-(beta-D-glucopyranoside) was determined to be a minor pigment (pigment 4). In this study, it was established that the acylation-enzymes of malonic acid has important roles for the acylation of 5-glucose residues of these anthocyanins in the flower-tissues of M. maritima; however, the similar enzymatic reactions seemed to be inhibited or lacking in the stem-tissues.     

A stable reddish purple anthocyanin in the leaf ofGynura aurantiaca cv. ‘Purple Passion’

The anthocyanin (GAA) in the epidermis and hair of the leaf ofGynura aurantiaca cv. ‘Purple Passion’ was isolated and identified as cyanidin tetra-glucoside acylated by three molecules of caffeic acid and one molecule of malonic acid. GAA was also isolated from the lower epidermis of the leaf ofG. bicolor DC. GAA showed a very stable reddish purple color from weakly acid to neutral pH region, but the color of the deacylated compound disappeared rapidly in the same region. This indicated that the attached organic acids must play an essential role in the stabilization of the color. Comparison of the profiles of the visible absorption spectra of the intact epidermal peels and cells ofG. aurantiaca andG. bicolor with those of GAA dissolved in various pH solutions suggested that the pH of the epidermal vacuole containing GAA was nearly 4.3. GAA was indistinguishable from the anthocyanin (rubrocinerarin) which we had previously isolated from the purplish red flowers ofSenecio cruentus DC. by means of UV-Vis, NMR and Mass spectra. Deceased     

Cell cultures of Centaurea cyanus produce malonated anthocyanin in UV light

Callus cultures which produce anthocyanin under continuous irradiation of UV and white light were derived from the stem of blue-floweredCentaurea cyanus. From the callus a suspension culture, in which anthocyanin synthesis can be induced by UV light, was obtained. The pigment in the cell cultures was identified as cyanidin 3-(6″-malonylglucoside) which occurs in the leaf, but not the flowers, of the parent plant.