A novel glucosylation reaction on anthocyanins catalyzed by acyl-glucose-dependent glucosyltransferase in the petals of carnation and delphinium

Glucosylation of anthocyanin in carnations (Dianthus caryophyllus) and delphiniums (Delphinium grandiflorum) involves novel sugar donors, aromatic acyl-glucoses, in a reaction catalyzed by the enzymes acyl-glucose-dependent anthocyanin 5(7)-O-glucosyltransferase (AA5GT and AA7GT). The AA5GT enzyme was purified from carnation petals, and cDNAs encoding carnation Dc AA5GT and the delphinium homolog Dg AA7GT were isolated. Recombinant Dc AA5GT and Dg AA7GT proteins showed AA5GT and AA7GT activities in vitro. Although expression of Dc AA5GT in developing carnation petals was highest at early stages, AA5GT activity and anthocyanin accumulation continued to increase during later stages. Neither Dc AA5GT expression nor AA5GT activity was observed in the petals of mutant carnations; these petals accumulated anthocyanin lacking the glucosyl moiety at the 5 position. Transient expression of Dc AA5GT in petal cells of mutant carnations is expected to result in the transfer of a glucose moiety to the 5 position of anthocyanin. The amino acid sequences of Dc AA5GT and Dg AA7GT showed high similarity to glycoside hydrolase family 1 proteins, which typically act as β-glycosidases. A phylogenetic analysis of the amino acid sequences suggested that other plant species are likely to have similar acyl-glucose-dependent glucosyltransferases.     

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.     

Anthocyanins in Catharanthus roseus in vivo and in vitro: a review

The production of anthocyanin in Catharanthus roseus flowers from both field-grown and regenerated by somatic embryogenesis plants and cell cultures was described. The anthocyanins were identified as the 3-O-glucosides, and the 3-O-(6-O-p-coumaroyl) glucosides of hirsutidin, malvidin and petunidin, respectively both in vivo and in vitro. The influence of environmental conditions on in vitro anthocyanin accumulation is described. The relationship between in vivo and in vitro anthocyanin production is discussed.     

The expression level of anthocyanidin synthase determines the anthocyanin content of crabapple (<Emphasis Type="Italic">Malus</Emphasis> sp.) petals

In addition to contributing to the coloration of plant organs and their defense against herbivores, the consumption of anthocyanins in the human diet has a number of health benefits. Crabapple (Malus sp.) represents a valuable experimental model system to research the mechanisms and regulation of anthocyanin accumulation, in part due to the often vivid and varied petal and leaf coloration that is exhibited by various cultivars. The enzyme anthocyanidin synthase (ANS) plays a pivotal role in anthocyanin biosynthesis; however, the relationship between ANS expression and petal pigmentation has yet to be established in crabapple. To illuminate the mechanism of anthocyanin accumulation in crabapple petals, we evaluated the expression of two crabapple ANS allelic genes (McANS-1 and McANS-2) and the levels of anthocyanins in petals from cultivars with dark red (‘Royalty’) and white (‘Flame’) petals, as well as another (‘Radiant’) whose petals have an intermediate pink color. We determined that the expression of McANS in the three cultivars correlated with the variation of anthocyanin accumulation during different petal developmental stages. Furthermore, transgenic tobacco plants constitutively overexpressing one of the two McANS genes, McANS-1, had showed elevated anthocyanin accumulation and a deeper red coloration in their petals than those from untransformed control lines. In conclusion, we propose that McANS are responsible for anthocyanin accumulation during petal coloration in different crabapple cultivars.     

Heterologous Expression of Two Gentian Cytochrome P450 Genes can Modulate the Intensity of Flower Pigmentation in Transgenic Tobacco Plants

Two different heterologous expression systems, microsomal fractions of Saccharomyces cerevisiae and transgenic tobacco plants, were used to investigate the enzymatic activities of flavonoid 3′-hydroxylase (GtF3′H) and flavone synthase II (GtFSII) homologues isolated from gentian petals. Recombinant GtF3′H expressed in yeast showed hydroxylation activities in the 3′ position with several flavonoid substrates, while recombinant GtFSII was able to produce flavone from flavanone. GtF3′ H-expressing transgenic tobacco plants showed a slight increase in anthocyanin content and flower color intensity, and conversion of the flavonol quercetin from kaempferol. On the other hand, GtFSII-expressing plants showed a remarkable reduction in anthocyanin content and flower color intensity, and additional accumulation of flavone, especially luteolin derivatives. We demonstrated that two cytochrome P450s from gentian petals have F3′H and FSII enzymatic activities both in vitro and in vivo, and might therefore be useful in modification of flower color using genetic engineering.     

Transient Expression of the Cytochrome p450 CYP78A2 Enhances Anthocyanin Production in Flowers

Transient expression of a cytochrome P450 gene (CYP78A2) cloned from Phalaenopsis was shown to enhance the anthocyanin contents in the petals of transformed Phalaenopsis. In this study, it was characterized further to understand the relationship between this P450 and the anthocyanin biosynthesis in flowers. The enhancement effect exerted by the P450 gene exhibits the following characteristics. First, its product seems to be able to effectively boost the existing pathway of biosynthesis without causing synthesis of any new anthocyanin. Second, the effect is not limited to Phalaenopsis, a monocotyledon, but also occurs in dicotyledons such as carnation and rose, indicating its wide range of action in heterologous plants. Third, the gene is not expressed in petals at any stage of flower development of Phalaenopsis, thus ruling out its direct participation in anthocyanin biosynthesis. It is possible that this P450 gene is associated with the biosynthesis of plant hormones or second metabolites, and through which to positively and indirectly influence the existing biosynthesis pathway of anthocyanins in petals.     

鸳鸯茉莉开花过程中花青素组成的变化

为了解鸳鸯茉莉(Brunfelsiaacuminata)花色变化的机理,采用高效液相色谱(HPLC)体系检测其开花过程中花青素组成的变化。结果表明,优化的HPLC体系为:流速为0.8 mL min–1,流动相A为7.5%甲酸乙腈,流动相B为7.5%甲酸水,洗脱程序为0 min,8%A;15 min,18%A;25 min,23%A;45 min,40%A;50 min,8%A。利用优化体系检测到鸳鸯茉莉花瓣中含有锦葵色素-3-O-葡萄糖苷、矮牵牛素葡萄糖苷和飞燕草素葡萄糖苷3种花青苷,其中锦葵色素-3-O-葡萄糖苷的含量最高,飞燕草素葡萄糖苷含量最低,且在花色由深变浅的过程中3种花青苷的含量均降低。因此,鸳鸯茉莉的呈色与这3种花青苷有关,且锦葵色素-3-O-葡萄糖苷起主导作用。     

Cytokinins in cut carnation flowers. II. Relationship between endogenous ethylene and cytokinin levels in the petals

Tentative identification using HPLC and RIA techniques indicated the presence of zeatin-O-glucoside, zeatin, ribosylzeatin, dihydrozeatin, iso-pentenyladenine and iso-pentenyladenosine in the petals of carnation flowers. Dihydrozeatin is apparently responsible for most of the biological activity. Within the petals most activity was detected in the basal parts which also senesced much slower than the upper parts of the petals. Treatment with AOA extended petal longevity and reduced ethylene production. This was associated with higher cytokinin-like activity in the basal parts of the petals.These higher levels of cytokinins were not observed in the petals of ACC treated flowers or in the detached control flowers. It is suggested that cytokinin transport and/or metabolism may play an important role in regulating ethylene production in cut carnations.     

Ethylene production and β-cyanoalanine synthase activity in carnation flowers

The relationship between ethylene production and the CN^--assimilating enzyme -cyanoalanine synthase (CAS; EC 4.4.1.9) was examined in the carnation (Dianthus caryophyllus L.) flower. In petals from cut flowers aged naturally or treated with ethylene to accelerate senescence the several hundred-fold increase in ethylene production which occurred during irreversible wilting was accompanied by a one- to twofold increase in CAS activity. The basal parts of the petal, which produced the most ethylene, had the highest CAS activity. Studies of flower parts (styles, ovaries, receptacles, petals) showed that the styles had a high level of CAS together with the ethylene-forming enzyme (EFE) system for converting 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene. The close association between CAS and EFE found in styles could also be observed in detached petals after induction by ACC or ethylene. Treatment of the cut flowers with cycloheximide reduced synthesis of CAS and EFE. The data indicate that CAS and ethylene production are associated, and are discussed in relation to the hypothesis that CN^- is formed during the conversion of ACC to ethylene.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglyoine - CAS -cyanoalanine synthase - CHI cycloheximide - EFE ethylene-forming enzyme     

A carnation anthocyanin mutant is complemented by the glutathione<Emphasis Type="Italic"> S</Emphasis>-transferases encoded by maize<Emphasis Type="Italic"> Bz2</Emphasis> and petunia<Emphasis Type="Italic"> An9</Emphasis>

Particle bombardment was used to elucidate the function of Flavonoid3, a late-acting anthocyanin gene of the ornamental plant, carnation ( Dianthus caryophyllus L.). The fl3 mutation conditions dilute anthocyanin coloration that closely resembles phenotypes produced by the anthocyanin mutants bz2 of maize and an9 of petunia. Bz2 and An9 encode glutathione S-transferases (GSTs) involved in vacuolar sequestration of anthocyanins. Constructs containing either of these or another late-function maize gene, Bronze1 (UDPglucose:flavonol 3- O-glucosyltransferase), were introduced via microprojectile bombardment into fl3 petals. Complementation resulted only from Bz2 and An9, indicating that Fl3 encodes a GST involved in the transport of anthocyanins to the vacuole. The observed result in carnation, an angiosperm phylogenetically distant from maize and petunia, indicates that GST activity might be a universal step in the anthocyanin pathway. Microprojectile bombardment was used to identify late-pathway anthocyanin mutations, which may be responsible for the pale anthocyanin coloration of important cultivars in many species but which can be difficult to characterize by other means.     

Optimisation of the second phase of a two phase growth system for anthocyanin accumulation in callus cultures of Rudbeckia hirta

A two-phase growth system composed of modified Schenk-Hildebrandt medium and enriched Miller's medium was used to grow callus of R. hirta L. The second phase of the system has been optimised and the callus synthesised a 12-compound set of anthocyanins, comprising 4.97% of tissue mass (tubular flowers of the plant in nature only contain one compound comprising 0.28%). The beneficial effect of high content of cysteine on anthocyanin accumulation was noted. The predominant element of the anthocyanin set obtained in vitro is cyanidin-3-O-(6-O-malonyl-β-D-glucopyranoside), a relatively stable compound. This revised version was published online in June 2006 with corrections to the Cover Date.     

Characteristics of the inhibitory action of 1,1-dimethyl-4-(phenylsulfonyl)semicarbazide (DPSS) on ethylene production in carnation (Dianthus caryophyllus L.) flowers

1,1-Dimethyl-4-(phenylsulfonyl)semicarbazide (DPSS)inhibited ethylene productionin carnation flowers during natural senescence, butdid not inhibit the ethyleneproduction induced by exogenous ethylene in carnationflowers, by indole-3-acetic acid (IAA) in mungbean hypocotylsegments and by wounding in winter squashmesocarp tissue. These findings suggested that DPSSdoes not directly inhibit ethylene biosynthesis fromL-methionine to ethylenevia S-adenosyl-L-methionine and1-aminocyclopropane-1-carboxylate. During naturalsenescence of carnation flowers, abscisic acid (ABA)was accumulated in the pistil and petals 2 days beforethe onset of ethylene production in the flower, andthe ABA content remained elevated until the onset ofethylene production. Application of exogenousABA to cut flowers from the cut stem end caused arapid increase in the ABA content in flower tissuesand promoted ethylene production in the flowers. These results were in agreement with the previousproposal that ABA plays a crucial role in theinduction of ethylene production during natural senescence incarnation flowers. DPSS preventedthe accumulation of ABA in both the pistil and petals,suggesting that DPSS exerted its inhibitory action onethylene production in naturally-senescing carnationflowers through the effect on the ABA-related process.     

New insight into the structures and formation of anthocyanic vacuolar inclusions in flower petals

Background  

Although the biosynthetic pathways for anthocyanins and their regulation have been well studied, the mechanism of anthocyanin accumulation in the cell is still poorly understood. Different models have been proposed to explain the transport of anthocyanins from biosynthetic sites to the central vacuole, but cellular and subcellular information is still lacking for reconciliation of different lines of evidence in various anthocyanin sequestration studies. Here, we used light and electron microscopy to investigate the structures and the formation of anthocyanic vacuolar inclusions (AVIs) in lisianthus (Eustoma grandiflorum) 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.     

Engineering of flower color in forsythia by expression of two independently-transformed dihydroflavonol 4-reductase and anthocyanidin synthase genes of flavonoid pathway

Flower color was modified in forsythia (Forsythia x intermedia cv Spring Glory) by inducing anthocyanin synthesis in petals through sequential Agrobacterium-mediated transformation with dihydroflavonol 4-reductase from Antirrhinum majus (AmDFR) and anthocyanidin synthase from Matthiola incana (MiANS) genes. This is the second report of flower color modification of an ornamental shrub after rose, and the first time an ANS gene is used for this purpose. Double transformants (AmDFR+MiANS) displayed a novel bronze-orange petal color, caused by the de novo accumulation of cyanidin-derived anthocyanins over the carotenoid yellow background of wild type (wt), and intense pigmentation of vegetative organs. Transformation with single genes (either AmDFR or MiANS) produced no change in flower color, showing a multistep control of late anthocyanin pathway in petals of forsythia. Analysis of relevant late flavonoid pathway genes – an endogenous flavonoid glycosyltransferase (FiFGT) and transformed DFR and ANS genes – showed appropriate expression in flower organs. Functional characterization of FiFGT expressed in E. coli revealed its ability to metabolize both flavonols and anthocyanidin substrates, a prerequisite for effective anthocyanin accumulation in petals of plants transformed with constructs leading to anthocyanidin synthesis. Biochemical analyses of flavonoid compounds in petals and leaves showed that, besides anthocyanin induction in petals of double transformants, the accumulation pattern of flavan-3-ols was quantitatively and qualitatively modified in petals and leaves of transformants, in agreement with the most recent model proposed for flavan-3-ol synthesis. On the other hand, phenylpropanoid, flavone and flavonol pools were not quantitatively affected, indicating a tight regulation of early flavonoid pathway.     

1-aminocyclopropane-l-carboxylic acid (ACC)—The transmitted stimulus in pollinated flowers?

Ethylene production and senescence of petals of pollinated carnation flowers were not prevented by removal of the ethylene produced by the gynoecium, suggesting that these events are a response to movement from the gynoecium of some stimulus other than ethylene gas. Application of 1-aminocyclopropane-1-carboxylic acid (ACC) to the stigmas caused an initial increase in gynoecium and petal ethylene production similar to that reported for pollinated flowers. This response was not seen in flowers whose stigmas were treated with indoleacetic acid (IAA). When [2-¹⁴C]ACC was applied to the stigmas of carnation flowers, radioactive ethylene was produced both by the gynoecia and by the petals. The possibility that ACC, transported from the stigmas to the petals, is responsible for the postpollination changes in carnation flowers is discussed.     

Identification of Genes Associated with Chlorophyll Accumulation in Flower Petals

Plants have an ability to prevent chlorophyll accumulation, which would mask the bright flower color, in their petals. In contrast, leaves contain substantial amounts of chlorophyll, as it is essential for photosynthesis. The mechanisms of organ-specific chlorophyll accumulation are unknown. To identify factors that determine the chlorophyll content in petals, we compared the expression of genes related to chlorophyll metabolism in different stages of non-green (red and white) petals (very low chlorophyll content), pale-green petals (low chlorophyll content), and leaves (high chlorophyll content) of carnation (Dianthus caryophyllus L.). The expression of many genes encoding chlorophyll biosynthesis enzymes, in particular Mg-chelatase, was lower in non-green petals than in leaves. Non-green petals also showed higher expression of genes involved in chlorophyll degradation, including STAY-GREEN gene and pheophytinase. These data suggest that the absence of chlorophylls in carnation petals may be caused by the low rate of chlorophyll biosynthesis and high rate of degradation. Similar results were obtained by the analysis of Arabidopsis microarray data. In carnation, most genes related to chlorophyll biosynthesis were expressed at similar levels in pale-green petals and leaves, whereas the expression of chlorophyll catabolic genes was higher in pale-green petals than in leaves. Therefore, we hypothesize that the difference in chlorophyll content between non-green and pale-green petals is due to different levels of chlorophyll biosynthesis. Our study provides a basis for future molecular and genetic studies on organ-specific chlorophyll accumulation.