Expression of genes involved in anthocyanin biosynthesis in relation to anthocyanin,proanthocyanidin, and flavonol levels during bilberry fruit development

The production of anthocyanins in fruit tissues is highly controlled at the developmental level. We have studied the expression of flavonoid biosynthesis genes during the development of bilberry (Vaccinium myrtillus) fruit in relation to the accumulation of anthocyanins, proanthocyanidins, and flavonols in wild berries and in color mutants of bilberry. The cDNA fragments of five genes from the flavonoid pathway, phenylalanine ammonia-lyase, chalcone synthase, flavanone 3-hydroxylase, dihydroflavonol 4-reductase, and anthocyanidin synthase, were isolated from bilberry using the polymerase chain reaction technique, sequenced, and labeled with a digoxigenin-dUTP label. These homologous probes were used for determining the expression of the flavonoid pathway genes in bilberries. The contents of anthocyanins, proanthocyanidins, and flavonols in ripening bilberries were analyzed with high-performance liquid chromatography-diode array detector and were identified using a mass spectrometry interface. Our results demonstrate a correlation between anthocyanin accumulation and expression of the flavonoid pathway genes during the ripening of berries. At the early stages of berry development, procyanidins and quercetin were the major flavonoids, but the levels decreased dramatically during the progress of ripening. During the later stages of ripening, the content of anthocyanins increased strongly and they were the major flavonoids in the ripe berry. The expression of flavonoid pathway genes in the color mutants of bilberry was reduced. A connection between flavonol and anthocyanin synthesis in bilberry was detected in this study and also in previous data collected from flavonol and anthocyanin analyses from other fruits. In accordance with this, models for the connection between flavonol and anthocyanin syntheses in fruit tissues are presented.     

Flavonoids in flowers of 16 Kalanchoë blossfeldiana varieties

Kalancho? blossfeldiana varieties with orange, pink, red and magenta flowers were found to contain 3,5-O-beta-D-diglucosides of pelargonidin, cyanidin, peonidin, delphinidin, petunidin and malvidin. Pink, red and magenta varieties contained relatively high amounts of quercetin based flavonols. Four distinct quercetin flavonols were identified, namely quercetin 3-O-beta-D-glucoside and three that were quercetin 3-O-alpha-L-rhamnoside based, with either glucose, xylose or arabinose attached to position 2 of the rhamnose. In addition, the presence of at least three kaempferol based diglycosides was suggested from LC-MS analyses. Orange varieties contained very low amounts of flavonol co-pigments and of delphinidin derivatives. The flower extracts of the varieties 'Diva' (magenta) and 'Molly' (red) had identical anthocyanin ratios but differed significantly in flavonol content. The magenta variety contained four times as much quercetin relative to anthocyanidin as the red variety. This difference was mainly due to a larger content of quercetin 3-O-(2'-O-beta-D-glucopyranosyl-alpha-L-rhamnopyranoside). Based on pigment and co-pigment analyses, approaches for molecular breeding towards blue flower colour are discussed.     

Flavonol glycosides of Warburgia ugandensis leaves

Four new flavonol gycosides: kaempferide 3-O-beta-xylosyl (1-->2)-beta-glucoside, kaempferol 3-O-alpha-rhamnoside-7,4'-di-O-beta-galactoside, kaempferol 3,7,4'-tri-O-beta-glucoside and quercetin 3-O-[alpha-rhamnosyl (1-->6)] [beta-glucosyl (1-->2)]-beta-glucoside-7-O-alpha-rhamnoside, were characterized from a methanolic leaf extract of Warburgia ugandensis. The known flavonols: kaempferol, kaempferol 3-rhamnoside, kaempferol 3-rutinoside, myricetin, quercetin 3-rhamnoside, kaempferol 3-arabinoside, quercetin 3-glucoside, quercetin, kaempferol 3-rhamnoside-4'-galactoside, myricetin 3-galactoside and kaempferol 3-glucoside were also isolated. Structures were established by spectroscopic and chemical methods and by comparison with authentic samples.     

The domestication syndrome within Hybrid Perpetuals roses: the effect of unconscious selection on flavonoids

Cultivated GallicanaexChinenses hybrids of roses, namely Hybrid Perpetuals, were compared with their parents as to morphology, petal colour, flavonol and anthocyanin metabolism. Morphology exhibited clear patterns of hybridity. An objective measure of petal lightness (L) indicated that Hybrid Perpetuals were submitted to a selection pressure favouring dark-flowered cultivars. When compared to the parental flavonoid metabolisms, Hybrid Perpetuals exhibited increased synthesis of anthocyanin and quercetin. High amounts of anthocyanin in Hybrid Perpetuals resulted from the selection of deeper-coloured flowers. High amounts of quercetin were correlated with enhanced anthocyanin synthesis, so that this originality of the flavonol metabolism was interpreted on biogenetic ground as a repercussion of this same selection pressure. Finally, the patterns of variation of flavonol glycosides within the Hybrid Perpetuals reflected the indirect selection pressure for the quercetin end-products, and with the ancestral hybridizations for the kaempferol derivatives.     

The flavonoids of Stenosiphon (Onagraceae)

Stenosiphon linifolius is a monotypic genus of the tribe Onagreae of the Onagraceae. The species is widespread in, but restricted to, the Great Plains of the United States. Three flavonol glycosides, kaempferol 3-O-rhamnoside, quercetin 3-O-rhamnoside and myricetin 3-O-rhamnoside, were found to occur in methanolic extracts of Stenosiphon leaves. Similar compounds are found in the leaves of such related genera as Oenothera and Gaura, but in the latter genera, additional flavonols exhibiting greater substitutional variation also are found.     

Analysis of flavonoids in flower petals of soybean near-isogenic lines for flower and pubescence color genes

W1, W3, W4, and Wm genes control flower color, whereas T and Td genes control pubescence color in soybean. W1, W3, Wm, and T are presumed to encode flavonoid 3'5'-hydroxylase (EC 1.14.13.88), dihydroflavonol 4-reductase (EC 1.1.1.219), flavonol synthase (EC 1.14.11.23), and flavonoid 3'-hydroxylase (EC 1.14.13.21), respectively. The objective of this study was to determine the structure of the primary anthocyanin, flavonol, and dihydroflavonol in flower petals. Primary component of anthocyanin in purple flower cultivars Clark (W1W1 w3w3 W4W4 WmWm TT TdTd) and Harosoy (W1W1 w3w3 W4W4 WmWm tt TdTd) was malvidin 3,5-di-O-glucoside with delphinidin 3,5-di-O-glucoside as a minor compound. Primary flavonol and dihydroflavonol were kaempferol 3-O-gentiobioside and aromadendrin 3-O-glucoside, respectively. Quantitative analysis of near-isogenic lines (NILs) for flower or pubescence color genes, Clark-w1 (white flower), Clark-w4 (near-white flower), Clark-W3w4 (dilute purple flower), Clark-t (gray pubescence), Clark-td (near-gray pubescence), Harosoy-wm (magenta flower), and Harosoy-T (tawny pubescence) was carried out. No anthocyanins were detected in Clark-w1 and Clark-w4, whereas a trace amount was detected in Clark-W3w4. Amount of flavonols and dihydroflavonol in NILs with w1 or w4 were largely similar to the NILs with purple flower suggesting that W1 and W4 affect only anthocyanin biosynthesis. Amount of flavonol glycosides was substantially reduced and dihydroflavonol was increased in Harosoy-wm suggesting that Wm is responsible for the production of flavonol from dihydroflavonol. The recessive wm allele reduces flavonol amount and inhibits co-pigmentation between anthocyanins and flavonols resulting in less bluer (magenta) flower color. Pubescence color genes, T or Td, had no apparent effect on flavonoid biosynthesis in flower petals.     

Phytochrome-induced flavonoid biosynthesis in mustard (Sinapis alba L.) cotyledons. Enzymic control and differential regulation of anthocyanin and quercetin formation

Phytochrome-induced increases in enzyme activities for phenylalanine ammonia-lyase (EC 4.3.1.5) and chalcone isomerase (EC 5.5.1.6), and in amounts of the related end products, anthocyanin and the flavonol, quercetin, were measured in cotyledons of mustard (Sinapis alba L.). There was no correlation between the activities of these enzymes and the rate of anthocyanin accumulation; however, some correlation was found with the quercetin accumulation rate. Since anthocyanin and flavonol accumulation is spatially separated in mustard (flavonols in the upper epidermis, anthocyanin in the lower epidermis), it was possible to measure anthocyanin-associated phenylalanine ammonia-lyase independently. This activity correlated well with the accumulation rate for anthocyanin during the first few hours after induction. The phytochrome effect on anthocyanin formation differed from that on quercetin formation: anthocyanin was strongly induced by continuous far-red light and by both continuous red light and red light pulses, whereas quercetin was only effectively induced by continuous far-red light.Abbreviations CHI chalcone isomerase - PAL phenylalanine ammonia-lyase     

BIOCHEMICAL BASIS OF FLORAL COLOR POLYMORPHISM IN A HETEROCYANIC POPULATION OF TRILLIUM SESSILE (LILIACEAE)

A study of flavonoids occurring within a heterocyanic population of Trillium sessile was made to determine the chemical basis of a common floral color polymorphism in this species. In the study population, three floral color phenotypes (red, pink, yellow) are determined primarily by the presence or absence of anthocyanin compounds in the petal tissue, and secondarily by quantitative differences in the concentration of several flavonol glycosides. Petals of red phenotypes contain both cyanidin 3-arabinoside and 3-diarabinoside, petals of pink phenotypes contain only cyanidin 3-arabinoside, and petals of yellow phenotypes lack cyanidin entirely. Quercetin 3-0-glucoside, quercetin 3-0-arabinoglucoside, quercetin 3–0-arabinogalactoside, and quercetin 3-0-arabinogalactosyl, 7-0-glucoside occur in petals of all three phenotypes but differ in relative amounts. Petals of the red phenotype have mostly 3-0-biosides, but lesser amounts of both quercetin 3-0-glucoside and the 3,7-0-triglycoside. Petals of the pink phenotype contain relatively equal amounts of quercetin mono-, di-, and triglycosides. Petals of the yellow phenotypes contain mostly quercetin 3,7-0-triglycosides, and less mono- and di-glycosides. Small amounts of a quercetin tetraglycoside were detected in petals of both yellow and pink phenotypes, but not in red phenotypes. The enhancement of quercetin polyglycoside biosynthesis in yellow petal phenotypes is attributed to the shunting of dihydroflavonol precursors to synthesis of quercetin compounds when their conversion to anthocyanins is blocked genetically.     

Flavonoids of Heterogaura (Onagraceae)

Heterogaura is a monotypic genus of the tribe Onagreae of the Onagraceae. It is endemic to south western Oregon and California. Four flavonol glycosides, kaempferol 3-O-rhamnoside, quercetin 3-O-glucoside, quercetin 3-O-rhamnoglucoside and myricetin 3-O-glucoside, were found to occur in methanolic leaf extracts of each of the populations sampled. The presence of only flavonols is consistent with flavonoid analyses from other genera of the Onagreae, including Clarkia, the closest relative of Heterogaura.     

Flavonol glycosides of Carya pecan

The flavonol glycosides characterized from the branches of Carya pecan include three new compounds, azaleatin 3-glucoside azaleatin 3-diglycoside and caryatin 3′- (or 4′-) rhamnoglucoside. together with azaleatin 3-rhamnoside. In the leaf tissue, quercetin 3-glucoside, quercetin 3-galactoside, quercetin 3-rhamnoside, quercetin 3-arabinoside and a small amount of kaempferol 3-monomethyl ether were identified.     

Regulation of gene expression involved in flavonol and anthocyanin biosynthesis during petal development in lisianthus (Eustoma grandiflorum)

To elucidate gene regulation of flower colour formation, the gene expressions of the enzymes involved in flavonoid biosynthesis were investigated in correlation with their product during floral development in lisianthus. Full-length cDNA clones of major responsible genes in the central flavonoid biosynthetic pathway, including chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavonoid 3',5'-hydroxylase (F3'5'H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), and flavonol synthase (FLS), were isolated and characterized. In lisianthus, the stage of the accumulation of flavonols and anthocyanins was shown to be divided clearly. The flavonol content increased prior to anthocyanin accumulation during floral development and declined when anthocyanin began to accumulate. CHS, CHI, and F3H were necessary for both flavonol and anthocyanin biosynthesis and were coordinately expressed throughout all stages of floral development; their expressions were activated independently at the stages corresponding to flavonol accumulation and anthocyanin accumulation, respectively. Consistent with flavonol and anthocyanin accumulation patterns, FLS, a key enzyme in flavonol biosynthesis, was expressed prior to the expression of the genes involved in anthocyanin biosynthesis. The genes encoding F3'5'H, DFR, and ANS were expressed at later stages, just before pigmentation. The genes responsible for the flavonoid pathways branching to anthocyanins and flavonols were strictly regulated and were coordinated temporally to correspond to the biosynthetic order of their respective enzymes in the pathways, as well as in specific organs. In lisianthus, FLS and DFR, at the position of branching to flavonols and anthocyanins, were supposed to play a critical role in regulation of each biosynthesis.     

Genetic control of the conversion of dihydroflavonols into flavonols and anthocyanins in flowers of Petunia hybrida

Petunia hybrida mutants, homozygous recessive for one of the genes An1, An2, An6, or An9 do not show anthocyanin synthesis in in vitro complementation experiments per se (see also Kho et al. 1977). Extracts of flowers of these mutants all provoke anthocyanin synthesis in isolated petals of an an3an3 mutant. Mutants homozygous recessive for one of the genes An1, An2, An6, or An9 and homozygous recessive for F1 accumulate dihydroflavonols in comparable amounts. The synthesis of dihydromyricetin is blocked in an1an1 mutants, which indicates a regulating effect of the gene An1 on the gene Hfl. Similar mutants, but dominant for F1, accumulate flavonols (kaempferol and quercetin) instead of dihydroflavonols. Myricetin is accumulated in minor amounts and not at all in an1an1 mutant. Furthermore, the synthesis of this flavonol is not controlled by the gene F1. The synthesis of cyanidin (derivatives) is greatly reduced when flavonols are synthesized (F1 dominant). In mutants dominant for Ht1 and Hf1 and thus able to synthesize cyanidin (derivatives) and delphinidin (derivatives), predominantly delphinidin (derivatives) are synthesized. The results indicate that kaempferol (derivatives), quercetin (derivatives), and delphinidin (derivatives) are the main endproducts of flavonoid biosynthesis in Petunia hybrida.     

Anthocyanin,flavonol copigments,and pH responsible for larkspur flower color

The anthocyanin and flavonol glycosides in Larkspur flowers (cv. Dark Blue Supreme) are delphinidin 3-di(p-hydroxybenzoyl)glucosylglucoside, kaempferol 3-robinobioside-7-rhamnoside (robinin), kaempferol 3-rutinoside, kaempferol 7-rhamnoside, and kaempferol 3-(caffeylgalactosylxyloside)-7-rhamnoside. As young flowers age the pH of epidermal tissue increases from 5·5 to 6·6 and the color of many of the cells changes from moderate reddish-purple to light purplish-blue. Many of the older cells also contain blue crystals. Visible absorption spectra of moderate reddish-purple and light purplish-blue cells were simulated with a solution of the anthocyanin (10⁻² M) plus robinin (5 × 10⁻³ M) at pH 5·6 and 7·1, respectively. Changes in the absorption spectra of living tissue with heating or cooling and of concentrated solutions of the anthocyanin with dilution or moderate heat, indicate that in the natural state the pigment is present in an associated form.     

Flavonoid patterns and phytogeography: the genus Rhododendron section Vireya

A survey of leaf hydrolysates of 52 species in section Vireya of Rhododendron showed that the flavonols kaempferol, quercetin and myricetin were commonly present. However, other more characteristic Rhododendron flavonol derivatives were either rare (caryatin, azaleatin) or absent (gossypetin). A study of the glycosides in four representative species showed that quercetin and kaempferol were present as the 3-rhamnoside, 3-glucoside, 3-galactoside or 3-rutinoside. The pattern in section Vireya is thus simpler than that in other Rhododendron species. This is in keeping with its geographical isolation, most species being endemic to the mountains of the Malesian-Australian region.     

Relationship between the composition of flavonoids and flower colors variation in tropical water lily (Nymphaea) cultivars

Water lily, the member of the Nymphaeaceae family, is the symbol of Buddhism and Brahmanism in India. Despite its limited researches on flower color variations and formation mechanism, water lily has background of blue flowers and displays an exceptionally wide diversity of flower colors from purple, red, blue to yellow, in nature. In this study, 34 flavonoids were identified among 35 tropical cultivars by high-performance liquid chromatography (HPLC) with photodiode array detection (DAD) and electrospray ionization mass spectrometry (ESI-MS). Among them, four anthocyanins: delphinidin 3-O-rhamnosyl-5-O-galactoside (Dp3Rh5Ga), delphinidin 3-O-(2"-O-galloyl-6"-O-oxalyl-rhamnoside) (Dp3galloyl-oxalylRh), delphinidin 3-O-(6"-O-acetyl-β-glucopyranoside) (Dp3acetylG) and cyanidin 3- O-(2"-O-galloyl-galactopyranoside)-5-O-rhamnoside (Cy3galloylGa5Rh), one chalcone: chalcononaringenin 2'-O-galactoside (Chal2'Ga) and twelve flavonols: myricetin 7-O-rhamnosyl-(1 → 2)-rhamnoside (My7RhRh), quercetin 7-O-galactosyl-(1 → 2)-rhamnoside (Qu7GaRh), quercetin 7-O-galactoside (Qu7Ga), kaempferol 7-O-galactosyl-(1 → 2)-rhamnoside (Km7GaRh), myricetin 3-O-galactoside (My3Ga), kaempferol 7-O-galloylgalactosyl-(1 → 2)-rhamnoside (Km7galloylGaRh), myricetin 3-O-galloylrhamnoside (My3galloylRh), kaempferol 3-O-galactoside (Km3Ga), isorhamnetin 7-O-galactoside (Is7Ga), isorhamnetin 7-O-xyloside (Is7Xy), kaempferol 3-O-(3"-acetylrhamnoside) (Km3-3"acetylRh) and quercetin 3-O-acetylgalactoside (Qu3acetylGa) were identified in the petals of tropic water lily for the first time. Meanwhile a multivariate analysis was used to explore the relationship between pigments and flower color. By comparing, the cultivars which were detected delphinidin 3-galactoside (Dp3Ga) presented amaranth, and detected delphinidin 3'-galactoside (Dp3'Ga) presented blue. However, the derivatives of delphinidin and cyanidin were more complicated in red group. No anthocyanins were detected within white and yellow group. At the same time a possible flavonoid biosynthesis pathway of tropical water lily was presumed putatively. These studies will help to elucidate the evolution mechanism on the formation of flower colors and provide theoretical basis for outcross breeding and developing health care products from this plant.     

Spatiotemporal metabolic regulation of anthocyanin and related compounds during the development of marginal picotee petals in <Emphasis Type="Italic">Petunia hybrida</Emphasis> (Solanaceae)

Structures and levels of anthocyanin-related compounds were analyzed during the development of marginal picotee petals in white-center and white-marginal cultivars of Petunia hybrida. In the white site of a white-center cultivar, higher concentrations of quercetin derivatives possessing 7-O-glucoside and/or 3′-O-glucoside occurred than in the colored site, suggesting that these two quercetin glycosylation steps are site-specifically regulated. The boundary areas of petal coloration were composed of cells showing various color densities, whose uniformity among adjacent cells varied between these cultivars. These results indicate diversity in spatiotemporal regulation of anthocyanin biosynthesis and flavonol glycosylations between Petunia cultivars during marginal picotee formation. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Study on Degradation of Flavonols in Mutants of PoppyPapaver somniferumL.

We studied flavonol-degrading activity of cell-free extracts from petals of the flower color and structure mutants. The relationship between degradation of flavonols (kaempferol, quercetin, and myricetin) and biosynthesis of anthocyanins has been revealed. The white-flower mutant proved to have the highest flavonol-degrading activity toward all substrates, particularly quercetin. The mutations inhibiting synthesis of pelargonidin, an anthocyanin, provide for synthesis of various amounts of cyanidin in the petals. The flavonol-degrading activity considerably increases proportionally to the content of cyanidin. A similar relationship has been revealed in the mutants synthesizing both cyanidin and pelargonidin. The plants accumulating considerable amounts of pelargonidin in their petals have accordingly higher flavonol-degrading activity and predominantly hydrolyze kaempferol. The plants forming additional pods in their flower (pistillody) have higher flavonol-degrading activity as compared to the anther-in-petal and doubleness mutants     

Temporal lag between gene expression and metabolite accumulation in flavonol biosynthesis of Arabidopsis roots

The temporal lag between gene expression and metabolite accumulation has been estimated in flavonol biosynthesis, but the time difference between these events is unclear. In the present study, we investigated the expression of flavonol biosynthetic genes ELONGATED HYPOCOTYL5, MYELOBLASTOSIS PROYEIN12/PRODUCTION OF FLAVONOL GLYCOSYDES1, CHALCONE SYNTHASE, CHALCONE ISOMERASE, FLAVANONE 3-HYDROXYLASE, and FLAVONOL SYNTHASE1, and the accumulation of flavonol glycosides (kaempferol and quercetin glycosides) in time-series samples of Arabidopsis thaliana roots. All genes started to be expressed within 3 h after sequential light irradiation (HAS) and reached their maximum expression levels at 12 HAS, and the accumulation of the flavonol glycosides started at 6 HAS. Metabolome analysis using liquid chromatography-mass spectrometry showed that the accumulation of kaempferol 3-O-glucoside-7-O-rhamnoside and kaempferol 3-O-rhamnosyl (1 → 2) glucoside-7-O-rhamnoside reached their maximum levels at 48 HAS, whereas other flavonol glycosides, such as kaempferol/quercetin 3-O-rhamnoside-7-O-rhamnoside, quercetin 3-O-glucoside-7-O-rhamnoside and quercetin 3-O-rhamnosyl (1 → 2) glucoside-7-O-rhamnoside, increased gradually until 96 HAS. These results show that the expression of the flavonol genes is an early response against light exposure, and that the accumulation of the flavonol glycosides is a late response.     

Temporal and spatial regulation of anthocyanin biosynthesis provide diverse flower colour intensities and patterning in <Emphasis Type="Italic">Cymbidium</Emphasis> orchid

Main conclusion

This study confirmed pigment profiles in different colour groups, isolated key anthocyanin biosynthetic genes and established a basis to examine the regulation of colour patterning in flowers of Cymbidium orchid. Cymbidium orchid (Cymbidium hybrida) has a range of flower colours, often classified into four colour groups; pink, white, yellow and green. In this study, the biochemical and molecular basis for the different colour types was investigated, and genes involved in flavonoid/anthocyanin synthesis were identified and characterised. Pigment analysis across selected cultivars confirmed cyanidin 3-O-rutinoside and peonidin 3-O-rutinoside as the major anthocyanins detected; the flavonols quercetin and kaempferol rutinoside and robinoside were also present in petal tissue. β-carotene was the major carotenoid in the yellow cultivars, whilst pheophytins were the major chlorophyll pigments in the green cultivars. Anthocyanin pigments were important across all eight cultivars because anthocyanin accumulated in the flower labellum, even if not in the other petals/sepals. Genes encoding the flavonoid biosynthetic pathway enzymes chalcone synthase, flavonol synthase, flavonoid 3′ hydroxylase (F3′H), dihydroflavonol 4-reductase (DFR) and anthocyanidin synthase (ANS) were isolated from petal tissue of a Cymbidium cultivar. Expression of these flavonoid genes was monitored across flower bud development in each cultivar, confirming that DFR and ANS were only expressed in tissues where anthocyanin accumulated. Phylogenetic analysis suggested a cytochrome P450 sequence as that of the Cymbidium F3′H, consistent with the accumulation of di-hydroxylated anthocyanins and flavonols in flower tissue. A separate polyketide synthase, identified as a bibenzyl synthase, was isolated from petal tissue but was not associated with pigment accumulation. Our analyses show the diversity in flower colour of Cymbidium orchid derives not from different individual pigments but from subtle variations in concentration and pattern of pigment accumulation.