Flavonoid composition related to petal color in different lines of Clitoria ternatea

Flavonoids in the petals of several C. ternatea lines with different petal colors were investigated with LC/MS/MS. Delphinidin 3-O-(2"-O-alpha-rhamnosyl-6"-O-malonyl)-beta-glucoside was newly isolated from the petals of a mauve line (wm) together with three known anthocyanins. They were identified structurally using UV, MS, and NMR spectroscopy. Although ternatins, a group of 15 (poly)acylated delphinidin glucosides, were identified in all the blue petal lines (WB, BM-1, 'Double Blue' and 'Albiflora'), WM accumulated delphinidin 3-O-(6"-O-malonyl)-beta-glucoside instead. The white petal line (WW) did not contain anthocyanins. Quantitative data showed that the total anthocyanin contents in WB and 'Double Blue' were ca. 8- and 10-fold higher than that in BM-1, a bud mutant of 'Double Blue', respectively. The total anthocyanin content in 'Albiflora' was less than 2 x 10(-3) times those in WB or 'Double Blue'. While all the lines contained the same set of 15 flavonol glycosides in similar relative ratios, the relative ratio of myricetin glycosides in ww and 'Albiflora' was ca. 30-70 times greater than those in the other lines. The change in flower color from blue to mauve was not due to a change in the structure of an anthocyanidin from delphinidin, but to the lack of (polyacylated) glucosyl group substitutions at both the 3'- and 5'-positions of ternatins. This implies that glucosylation at the 3'- and 5'-positions of anthocyanin is a critical step in producing blue petals in C. ternatea.     

Structure of anthocyanin from the blue petals of Phacelia campanularia and its blue flower color development

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.     

The involvement of tonoplast proton pumps and Na+(K+)/H+ exchangers in the change of petal color during flower opening of Morning Glory, Ipomoea tricolor cv. Heavenly Blue

The petal color of morning glory, Ipomoea tricolor cv. Heavenly Blue, changes from purplish red to blue during flower opening. This color change is caused by an unusual increase in vacuolar pH from 6.6 to 7.7 in the colored adaxial and abaxial cells. To clarify the mechanism underlying the alkalization of epidermal vacuoles in the open petals, we focused on vacuolar H+-ATPase (V-ATPase), H+-pyrophosphatase (V-PPase) and an isoform of Na+/H+ exchanger (NHX1). We isolated red and blue protoplasts from the petals in bud and fully open flower, respectively, and purified vacuolar membranes. The membranes contained V-ATPase, V-PPase and NHX1, which were immunochemically detected, with relatively high transport activity. NHX1 could be detected only in the vacuolar membranes prepared from flower petals and its protein level was the highest in the colored petal epidermis of the open flower. These results suggest that the increase of vacuolar pH in the petals during flower opening is due to active transport of Na+ and/or K+ from the cytosol into vacuoles through a sodium- or potassium-driven Na+(K+)/H+ exchanger NXH1 and that V-PPase and V-ATPase may prevent the over-alkalization. This systematic ion transport maintains the weakly alkaline vacuolar pH, producing the sky-blue petals.     

The role of light in the regulation of anthocyanin accumulation in <Emphasis Type="Italic">Gerbera hybrida</Emphasis>

In the present work, the pigmentation regulated by light was investigated in ray floret (rf) of Gerbera hybrida. When inflorescences from stage 1 were covered with aluminium foil in vivo the pigmentation of the rf petals was strongly blocked and the gene expression of CHS (Chalcone synthase) and DFR (Dihydroflavonol-4-reductase) was inhibited. Similar results were obtained when the detached rfs were cultured in vitro. Covering of the leaves on the plants resulted in reduced pigmentation compared with the covering of inflorescences in vivo. Removal of the green bracts did not affect the pigmentation significantly and the anthocyanin concentration was maintained at a level similar to that of the control. The ultrastructure of the plastids in rf petals was examined to investigate the possible role of photosynthesis in light regulation of flower pigmentation. Plastids within rf epidermal cells showed a characteristic chloroplast morphology in flowers at stage 2, which deteriorated by stage 3. They then changed to a chromoplast-like structure in fully opened rf petals (stage 6). Similar chromoplast-like structures were observed in the plastids of the rf petals from inflorescences both shaded in vivo and in vitro. Additionally, DCMU, a photosynthetic inhibitor, did not show a significant effect on light-induced anthocyanin accumulation. Our data suggest that light is an important factor for pigmentation of rf petal in Gerbera and the petal itself acts as a light sensor site to perceive the light signal. From the different light qualities evaluated, blue light promoted gene expression of CHS and DFR, and red light enhanced the gene expression of CHS, indicating the photoreceptors responding to blue and red light involved in the photoregulation of flower pigmentation in Gerbera.     

Molecular characterization of an anthocyanin-related glutathione S-transferase gene in cyclamen

Anthocyanins are a subclass of flavonoids and are a major contributor to flower colors ranging from red to blue and purple. Previous studies in model and ornamental plants indicate a member of the glutathione S-transferase (GST) gene family is involved in vacuolar accumulation of anthocyanins. In order to identify the anthocyanin-related GST in cyclamen, degenerate PCR was performed using total RNA from immature young petals. Four candidates of GSTs (CkmGST1 to CkmGST4) were isolated. Phylogenetic analysis indicated that CkmGST3 was closely related to PhAN9, an anthocyanin-related GST of petunia, and this clade was clustered with other known anthocyanin-related GSTs. Expression analysis at different developmental stages of petals revealed that CkmGST3 was strongly expressed in paler pigmented petals than in fully pigmented petals, in contrast to the constitutive expression of the other three candidates during petal development. This expression pattern of CkmGST3 was correlated with those of other anthocyanin biosynthetic genes such as CkmF3'5'H and CkmDFR2. Molecular complementation of Arabidopsis tt19, a knockout mutant of an anthocyanin-related GST gene, demonstrated that CkmGST3 could complement the anthocyanin-less phenotype of tt19. Transgenic plants that expressed the other three CkmGSTs did not show anthocyanin accumulation. These results indicate CkmGST3 functions in anthocyanin accumulation in cyclamen.     

5种野牡丹属植物花色素成分及影响花色呈色因子分析

为明确野牡丹属(Melastoma L.)植物花瓣的色素成分和呈色机理,为花色育种提供参考。以野牡丹(M.candidum)、白花野牡丹(M.candidum f.albiflorum)、印度野牡丹(M.malabathiricum)、白花印度野牡丹(M. malabathricumvar.alba)、毛稔(M.sanguinrum)5种野牡丹属植物材料,采用目测法、RHSCC比色法和色差仪测定花瓣表型,应用化学显色法、紫外分光光度法对花色素成分及含量进行初步分析与测定,通过徒手切片组织切片法观察花瓣表皮细胞的显微结构和分布特点,测定花瓣pH值、可溶性糖及可溶性蛋白含量等生理指标分析对花色的影响。结果显示,野牡丹属植物花瓣不含叶绿素和类胡萝卜素,紫罗兰色系主要含花青素苷和黄酮类化合物,白色系主要含黄酮类化合物。野牡丹和毛稔花色素分布于上、下表皮,印度野牡丹花色素分布于上、下表皮和栅栏组织,白花野牡丹和白花印度野牡丹花瓣没有发现色素积累;紫罗兰色系野牡丹上表皮细胞呈圆锥形突起,白色系野牡丹上表皮细胞呈不规则的扁平状,它们下表皮细胞全呈不规则的扁平状。野牡丹属植物花色明度L*随花瓣颜色变深而降低,明度L*与红度a*呈极显著负相关、与蓝度b*呈极显著的正相关。花瓣中花青素苷含量与其明度L*和蓝度b*呈显著负相关,pH值与花瓣红度a*呈现显著的负相关。研究表明,野牡丹属植物花色主要由花青素苷决定,花青素苷含量、色素分布、上表皮细胞形状等是引起花色呈现多样的主要因子。     

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.     

Effect of Aluminum Ions on the Bluing of Petal Color in Cut Chinese Bellflower, Platycodon grandiflorum

Petal color of Chinese bellflower, Platycodon grandiflorum A.DC,changed from blueviolet to blue in a short period after cutflowers were placed in a solution containing AI ions. The absorptionmaximum of Al-treated fresh petals shifted to longer wavelength(bathochromic shift) and increased in intensity. These phenomenawere also observed upon addition of Al ions to anthocyanin solution.The addition of EDTA to the anthocyanin solution treated withAl ions decreased the bluing effect due to Al addition. Thus, the flower color change of cut Chinese bellflowers byAl uptake seems to be due to the chelation of anthocyanin withAl ions. (Received December 2, 1982; Accepted March 31, 1983)     

Novel aspects of the flowers and floral pigmentation of two Cleome species (Cleomaceae), C. hassleriana and C. serrulata

Palely pigmented inflorescences of cultivated Cleome hassleriana Chodat (spider flower) have a unique two-toned appearance shown in the present study to be due to a loss of petal pigmentation within 24 h of anthesis, accompanied by an equally unique loss of petal mass. A similar loss occurs in deeply pigmented petals but is less evident to the eye because of the high initial content due to the presence, in the petal mesophyll, of globular anthocyanic vacuolar inclusions (AVIs). Inflorescences of the wild species, Cleome serrulata Pursh. (Rocky Mountain bee flower) are also two-toned because of the deeper pink colour of the unopened bud. No AVIs were seen. The pink colour of the bee flower petals is due to the same five acylated cyanidin glycosides as those previously isolated from mauve petals of spider flower. The structural pattern of the spider flower anthocyanins is shared with at least three genera of the Brassicaceae.     

Ultraviolet A-specific induction of anthocyanin biosynthesis in the swollen hypocotyls of turnip (Brassica rapa)

Ultraviolet A (UV-A)-mediated regulation of anthocyanin biosynthesis was investigated in swollen hypocotyls of the red turnip 'Tsuda'. The shaded swollen hypocotyls which contained negligible anthocyanin were exposed to artificial light sources including low fluence UV-B, UV-A, blue, red, far-red, red plus UV-A, far-red plus UV-A, and blue plus red. Among these lights, only UV-A induced anthocyanin biosynthesis and co-irradiation of red or far-red with UV-A did not affect the extent of UV-A-induced anthocyanin accumulation. The expression of phenylalanine ammonia lyase (PAL; EC 4.3.1.5), chalcone synthase (CHS; EC 2.3.1.74), flavanone 3-hydroxylase (F3H; EC 1.14.11.9), dihydroflavonol 4-reductase (DFR; EC 1.1.1.219), and anthocyanidin synthase (ANS; EC 1.14.11.19) genes was increased with time during a 24 h exposure to UV-A. In contrast, irradiation with red, blue, UV-B, and a combination of blue with red failed to induce CHS expression. Microarray analysis showed that only a few genes, including CHS and F3H, were induced significantly by UV-A, while a separate set of many genes was induced by low fluence UV-B. The UV-A-specific induction of anthocyanin biosynthesis and the unique gene expression profile upon UV-A irradiation as compared with blue and UV-B demonstrated that the observed induction of anthocyanin biosynthesis in red turnips was mediated by a distinct UV-A-specific photoreceptor, but not by phytochromes, UV-A/blue photoreceptors, or UV-B photoreceptors.     

Factors determining Petal Colour of Baccara Roses I: THE CONTRIBUTION OF EPIDERMIS AND MESOPHYLL

The partition of light radiated on to the outer epidermis ofa Baccara rose petal or on to an intact petal was examined.Most of the red light was either reflected or transmitted whereasother wavelengths and especially the green range were absorbed.When the total amount of light transmitted (epidermis) or reflected(intact petal) increased, a rise in the blue range was recordedand the colour of the petal, determined objectively by CIE orMunsell's method, became more purple. Examination of the partition of light in the different layersof the petal revealed that light reflected from the outer epidermisis made up of two parts; one part is reflected directly andthe other part is first transmitted through the epidermis, reachesthe mesophyll, is reflected from it and is then transmittedthrough the epidermis. This latter part causes a shift in colourfrom purple to red. Colour differences between different petals on one flower anddifferent parts of the same petal were defined objectively.The change from red to purple colour was connected with vigorousgrowth of either the petal or epidermal cells, respectively. The contribution of the mesophyll in changing the reflectancecurve of petals is explained and it is suggested that althoughthe mesophyll is colourless, it contributes to a great extentto the changes occurring in petal colour.     

PLANTLETS FROM PETAL SEGMENTS,PETAL EPIDERMIS,AND SHOOT TIPS OF THE PERICLINAL CHIMERA,CHRYSANTHEMUM MORIFOLIUM ‘INDIANAPOLIS'

Cultivated chrysanthemums, especially the greenhouse series of ‘Indianapolis’ cultivars, are probably periclinal chimeras for flower color. Therefore, in vitro propagation of chrysanthemum, which has recently been described, might produce plants not true to type. To test this, plantlets were generated from cultures of petal segments, petal epidermis, and shoot tips; these plantlets were grown to flowering to determine whether chrysanthemums with two genetically different chimeral layers in the petals are stable in tissue culture. Layer I displaced layer II in the formation of new meristematic areas in shoot tip and petal culture, showing that such chimeras are unstable in culture. Many more abnormal morphological types were exhibited by the plants which were regenerated from petal cultures rather than those from shoot tip cultures. Abnormalities included quilled and incised petal forms, as well as lack of anthocyanin pigmentation, characteristics which may not be attributable to the rearrangement of chimeral layers. Paramutation, true mutation, and environmenal effects are offered as possible explanations for this phenomenon.     

耐寒睡莲花瓣中花青素苷组成及其与花色的关系

睡莲(Nymphaea spp.)为多年生水生观赏花卉。以耐寒睡莲不同花色的119个栽培品种为材料, 利用高效液相色谱(HPLC-DAD)和液质联用技术(HPLC-ESI-MSⁿ)测定了其花瓣中的花青素苷成分。采用英国皇家园艺学会比色卡(RHSCC)和国际照明委员会(CIE)制定的CIEL^*a^*b^*表色系统测量了57个品种的花色, 运用多元线性回归方法分析花色与花青素苷组成之间的关系。结果表明: 耐寒睡莲花瓣中含有14种花青素苷, 其中飞燕草素-3-半乳糖-5-乙酰-半乳糖苷(Dp3Ga5acetylGa)、飞燕草素-3-鼠李糖-(1→2)-半乳糖苷(Dp3Rh(1→2)Ga)、矢车菊素-3-半乳糖-(1→2)-半乳糖苷(Cy3Ga(1→2)Ga)、矢车菊素-3-乙酰-半乳糖-(1→2)-半乳糖苷(Cy3acetylGa(1→2)Ga)、矢车菊素-3-没食子酰-半乳糖苷(Cy3galloylGa)、飞燕草素-3-乙酰-葡萄糖苷(Dp3acetylG)、飞燕草素-3-葡萄糖苷(Dp3G)和矢车菊素-3-半乳糖-半乳糖-半乳糖苷(Cy3GaGaGa)8个组分在耐寒睡莲中为首次报道。Dp3Ga、Dp3galloylGa、Cy3Ga(1→2)Ga和Cy3galloylGa是决定耐寒睡莲呈色的关键花青素苷。     

Relationship between petal abscission and programmed cell death in <Emphasis Type="Italic">Prunus yedoensis</Emphasis> and <Emphasis Type="Italic">Delphinium belladonna</Emphasis>

Depending on the species, the end of flower life span is characterized by petal wilting or by abscission of petals that are still fully turgid. Wilting at the end of petal life is due to programmed cell death (PCD). It is not known whether the abscission of turgid petals is preceded by PCD. We studied some parameters that indicate PCD: chromatin condensation, a decrease in nuclear diameter, DNA fragmentation, and DNA content per nucleus, using Prunus yedoensis and Delphinium belladonna which both show abscission of turgid petals at the end of floral life. No DNA degradation, no chromatin condensation, and no change in nuclear volume was observed in P. yedoensis petals, prior to abscission. In abscising D. belladonna petals, in contrast, considerable DNA degradation was found, chromatin was condensed and the nuclear volume considerably reduced. Following abscission, the nuclear area in both species drastically increased, and the chromatin became unevenly distributed. Similar chromatin changes were observed after dehydration (24 h at 60°C) of petals severed at the time of flower opening, and in dehydrated petals of Ipomoea nil and Petunia hybrida, severed at the time of flower opening. In these flowers the petal life span is terminated by wilting rather than abscission. It is concluded that the abscission of turgid petals in D. belladonna was preceded by a number of PCD indicators, whereas no such evidence for PCD was found at the time of P. yedoensis petal abscission. Dehydration of the petal cells, after abscission, was associated with a remarkable nuclear morphology which was also found in younger petals subjected to dehydration. This nuclear morphology has apparently not been described previously, for any organism.     

Development of Anthocyanin Pigmentation in Flowers of Lathyrus odoratus

A quantitative study has been made of developmental changesin the anthocyanins and a flavonol glycoside in the red/bluebicoloured flowers of Lathyrus odoratus L. Anthocyanin formationoccurs during the period of most rapid growth of the petals.At maturity about four times as much anthocyanin is presentin the standard petal as in the pair of wing petals, which aretogether comparable in fresh weight to the standard. The patternof development of flavonol glycoside is quite different; someare formed well before anthocyanin formation occurs and at maturityabout six times as much flavonol glycoside is present in thewings as in the standard per unit amount of anthocyanin. Somefurther evidence is thus provide that the flavonol glycosidemay be acting as a co-pigment which modifies the wing petalcolour to blue.