Characterization of anthocyanic vacuolar inclusions in Vitis vinifera L. cell suspension cultures

Anthocyanic vacuolar inclusions (AVIs) are intra-vacuolar structures capable of concentrating anthocyanins and are present in over 50 of the highest anthocyanin-accumulating plant species. Presence of AVIs alters pigment intensity, total anthocyanin levels, pigment hue and causes bathochromic shifts in a spatio-temporal manner within various flowers, vegetables and fruits. A year-long study on Vitis vinifera cell suspension cultures found a strong correlation between AVI prevalence and anthocyanin content, but not the number of pigmented cells, growth rate or stilbene content. Furthermore, enhancement of the prevalence of AVIs and anthocyanins was achieved by treatment of V. vinifera cell suspension cultures with sucrose, jasmonic acid and white light. A unique autofluorescence of anthocyanins was used to demonstrate microscopically that AVIs proceed from the cytosol across the tonoplast and were able to coalesce intravacuolarly, with fewer, larger AVIs predominating as cells mature. Purification and characterisation of these bodies were performed, showing that they were dense, highly organic structures, with a lipid component indicative of membrane-encasement. These purified AVIs were also shown to comprise long-chain tannins and possessed an increased affinity for binding acylated anthocyanins, though no unique protein component was detected.     

Anthocyanic vacuolar inclusions--their nature and significance in flower colouration

The petals of a number of flowers are shown to contain similar intensely coloured intravacuolar bodies referred to herein as anthocyanic vacuolar inclusions (AVIs). The AVIs in a blue-grey carnation and in purple lisianthus have been studied in detail. AVIs occur predominantly in the adaxial epidermal cells and their presence is shown to have a major influence on flower colour by enhancing both intensity and blueness. The latter effect is especially dramatic in the carnation where the normally pink pelargonidin pigments produce a blue-grey colouration. In lisianthus, the presence of large AVIs produces marked colour intensification in the inner zone of the petal by concentrating anthocyanins above levels that would be possible in vacuolar solution. Electron microscopy studies on lisianthus epidermal tissue failed to detect a membrane boundary in AVI bodies. AVIs isolated from lisianthus cells are shown to have a protein matrix. Bound to this matrix are four cyanidin and delphinidin acylated 3,5-diglycosides (three, new to lisianthus), which are relatively minor anthocyanins in whole petal extracts where acylated delphinidin triglycosides predominate. Flavonol glycosides were not bound. A high level of anthocyanin structural specificity in this association is thus implied. The specificity and effectiveness of this anthocyanin "trapping" is confirmed by the presence in the surrounding vacuolar solution of only delphinidin triglycosides, accompanied by the full range of flavonol glycosides. "Trapped" anthocyanins are shown to differ from solution anthocyanins only in that they lack a terminal rhamnose on the 3-linked galactose. The results of this study define for the first time the substantial effect AVIs have on flower colour, and provide insights into their nature and their specificity as vacuolar anthocyanin traps.     

Detection of 1-O-malylglucose: pelargonidin 3-O-glucose-6'-O-malyltransferase activity in carnation (Dianthus caryophyllus)

Carnations have anthocyanins acylated with malate. Although anthocyanin acyltransferases have been reported in several plant species, anthocyanin malyltransferase (AMalT) activity in carnation has not been identified. Here, an acyl donor substance of AMalT, 1-O-β-d-malylglucose, was extracted and partially purified from the petals of carnation. This was synthesized chemically to analyze AMalT activity in a crude extract from carnation. Changes in the AMalT activity showed close correlation to the accumulation of pelargonidin 3-malylglucoside (Pel 3-malGlc) during the development of red petals of carnation, but neither AMalT activity nor Pel 3-malGlc accumulation was detectable in roots, stems and leaves.     

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.     

Short communication. Do anthocyanins play a role in UV protection of the red juvenile leaves of Syzygium?

The biological function of juvenile leaves pigmented with anthocyanin is poorly understood. The role anthocyanins play in UV protection was assessed in juvenile leaves of two Syzygium species (S. luehmannii and S. wilsonii) which contain high anthocyanin concentrations. HPLC was used to separate UV-absorbing anthocyanins from other soluble UV-absorbing phenolic compounds. The isolated anthocyanins (predominantly malvidin-3,5-diglucoside) contributed little to the total absorbance of UV-A and UV-8 radiation. This was because the non-acylated anthocyanins only effectively absorbed shortwave UV-B radiation and the strong absorbance by other compounds. These results suggest that the UV protection hypothesis is not valid for anthocyanins in juvenile Syzygium leaves.     

Manipulating anthocyanin composition in Vitis vinifera suspension cultures by elicitation with jasmonic acid and light irradiation

Jasmonic acid altered the accumulation of major anthocyanins in Vitis vinifera cell culture. Peonidin 3-glucoside content at day three was increased from 0.3 to 1.7 mg g^–1 dry cell wt while other major anthocyanins were increased by smaller increments. By day 14, the content of methylated and acylated anthocyanins (peonidin 3-p-coumaroylglucoside and malvidin 3-p-coumaroylglucoside) was 6.3 mg g^–1 DCW, in response to treatment with jasmonic acid, and comprising 45% (w/w) of total anthocyanins. In comparison, the untreated control culture contained 1.2 mg g^–1 DCW which made up 32% (w/w) of total anthocyanins. Light further enhanced anthocyanin accumulation induced by jasmonic acid elicitation. The content of peonidin 3-glucoside at day 3 was 6.6 mg g^–1 DCW, 22-fold higher than control cultures while the content in response to light irradiation alone was 0.6 mg g^–1 DCW. When a highly pigmented cell line was elicited with jasmonic acid total anthocyanins increased from 9.2 to 20.7 mg g^–1 DCW, but there was no change in the anthocyanin composition.     

Anthocyanins from red cabbage--stability to simulated gastrointestinal digestion

Anthocyanins were the main polyphenol components in extracts of fresh and pickled red cabbage. The composition of anthocyanins in red cabbage was studied using liquid chromatography mass-spectrometry. Eleven major peaks absorbing at 520 nm were discerned, which represented 18 different anthocyanin structures. Another five minor anthocyanin components could be identified by searching at their respective m/z values but only in anthocyanin-enriched concentrates produced by sorption to solid phase extraction matrices. The predominant anthocyanins were constructed of cyanidin-3-diglucoside-5-glucoside "cores" which were non-acylated, mono-acylated or di-acylated with p-coumaric, caffeic, ferulic and sinapic acids. Pelargonidin-3-glucoside and novel forms of cyanidin-3-O-triglucoside-5-O-glucoside di-acylated with hydroxycinnamic acids were also detected in extracts of raw red cabbage, commercially pickled red cabbage and anthocyanin-enriched concentrates. The stability of the anthocyanins to simulated gastrointestinal digestion was assessed. The anthocyanins were effectively stable in the acidic gastric digestion conditions but the total recovery after simulated pancreatic digestion was around 25% compared to around 100% recovery of phenol content. As anthocyanins make up the majority of red cabbage polyphenols, this suggested that anthocyanins broke down to form new phenolic components. The recovery of the individual anthocyanins was monitored by LC-MS(n). All of the anthocyanins were reduced in content after pancreatic digestion but acylated forms were notably more stable than non-acylated forms. There was also a relationship between the type of acylated hydroxycinnamic acid and stability to pancreatic digestion.     

Grapevine MATE-Type Proteins Act as Vacuolar H+-Dependent Acylated Anthocyanin Transporters

In grapevine (Vitis vinifera), anthocyanins are responsible for most of the red, blue, and purple pigmentation found in the skin of berries. In cells, anthocyanins are synthesized in the cytoplasm and accumulated into the vacuole. However, little is known about the transport of these compounds through the tonoplast. Recently, the sequencing of the grapevine genome allowed us to identify genes encoding proteins with high sequence similarity to the Multidrug And Toxic Extrusion (MATE) family. Among them, we selected two genes as anthocyanin transporter candidates and named them anthoMATE1 (AM1) and AM3. The expression of both genes was mainly fruit specific and concomitant with the accumulation of anthocyanin pigment. Subcellular localization assays in grapevine hairy roots stably transformed with AM1 or AM3green fluorescent protein fusion protein revealed that AM1 and AM3 are primarily localized to the tonoplast. Yeast vesicles expressing anthoMATEs transported acylated anthocyanins in the presence of MgATP. Inhibitor studies demonstrated that AM1 and AM3 proteins act in vitro as vacuolar H⁺-dependent acylated anthocyanin transporters. By contrast, under our experimental conditions, anthoMATEs could not transport malvidin 3-O-glucoside or cyanidin 3-O-glucoside, suggesting that the acyl conjugation was essential for the uptake. Taken together, these results provide evidence that in vitro the two grapevine AM1 and AM3 proteins mediate specifically acylated anthocyanin transport.     

Accumulation of peonidin 3-glucoside enhanced by osmotic stress in grape (Vitis vinifera L.) cell suspension

Cell cultures of grapes, Vitis vinifera L. cv Gamay Fréaux were grown under different conditions of external osmotic potential induced by an increase of sucrose concentration or by the addition of mannitol to the culture medium. Addition of 82 mM mannitol or increasing sucrose concentration to 132 mM had similar effects on repressing growth. Cyanidin 3-glucoside, peonidin 3-glucoside and peonidin 3-p-coumaroylglucoside are three main anthocyanins of Vitis cells. Increasing osmotic potential from –0.43 MPa to –0.8 MPa in the medium resulted in a significant intracellular accumulation of anthocyanin especially peonidin 3-glucoside in the pigmented cells. High osmotic potential appears to stimulate the methylation of anthocyanins. Osmotic potential is an important culture factor and may be useful in the controlling of anthocyanin production and composition.     

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.     

Anthocyanin production in selected cell lines of grape (Vitis vinifera L.)

Summary Anthocyanin production of two lines ofVitis vinifera cell cultures, i.e., 5.4 and 13.1, which were obtained from the same starting material after 20 and 37 mo. of clonal selection, respectively, was investigated. Cell suspension cultures of lines 5.4 and 13.1 maintained an anthocyanin content of 0.44 ± 0.15 and 1.02 ± 0.31 mg·g⁻¹ fresh weight during 50 and 32 weekly maintenance subcultures, respectively. Under anthocyanin-promoting culture conditions, both lines showed an enhancement of their anthocyanin level by approximately fourfold. While line 5.4 accumulated peonidin 3-glucoside and cyanidin 3-glucoside in decreasing order, line 13.1 accumulated primarily peonidin 3-p-coumaroylglucoside with lesser amounts of malvidin monoglucoside. Results show that while the anthocyanin content was improved during the course of repeated selections, the anthocyanin composition was modified markedly favoring the accumulation of more metabolically-advanced anthocyanins.     

高等植物花色苷在液泡中的存在状态及其着色效应

综述了花色苷被摄入液泡的原因、花色苷在液泡中的存在状态及其对植物细胞的着色效应。花色苷在植物细胞质中合成后转运到液泡里是为了解除其对蛋白质和DNA等细胞功能分子的毒性。花色苷的液泡区隔化是花色苷在植物细胞中发挥正常功能的前提。在大多数植物中,花色苷在绝大多数情况下完全溶解在液泡里。但是,花色苷也能在液泡里形成颗粒,这些颗粒可以划分为花色苷体和花色苷液泡包涵体两类。花色苷体由膜包裹,其形成是液泡中小的有色囊泡逐渐合并的结果,发育完全的花色苷体为典型的球状、具比液泡更深的红色;液泡里的花色苷体具高密度,呈现为含高浓度花色苷的不溶性小球;花色苷体的存在可导致液泡的强烈色彩。花色苷液泡包涵体可能具备蛋白质基质,既无膜包裹又无内部结构,其形成是转运进液泡的花色苷与蛋白质基质结合的结果;液泡里的花色苷液泡包涵体形状不规则,象果冻;在花色苷液泡包涵体中,花色苷可能通过氢键连接于蛋白质基质的一个有限空间位点;花色苷液泡包涵体被认为是液泡中花色苷的"陷阱",优先摄取花色素3,5-二糖苷或酰化的花色苷;花色苷液泡包涵体的存在可增加液泡色彩的强度并导致"蓝化"。     

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.     

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。     

Anthocyanase and Anthocyanins Occurring in Eggplant,Solanum melongena L. (I)

Anthocyanins present in eggplant were decolorized by anthocyanase from flesh of eggplant. The anthocyanins consisted of at least three different anthocyanins containing delphinidin as common aglycone, and that a main component of those was nasunin, delphinidin-3-diglucoside acylated with p-coumaric acid.

Using the anthocyanin as substrate, the anthocyanase action was optimal at pH 6.0 and 35^°C, and was inhibited by potassium cyanide, thiourea, and sodium chloride. The data obtained so far show that anthocyanase acts on the following anthocyanidin derivatives in order of increasing rate of decolorization; pelargonidin-=peonidin-<cyanidin-<delphinidin-<delphinidin-glucoside acylated with p-coumaric acid.     

A gene controlling rate of anthocyanin synthesis and mutation frequency of the gene An1 in Petunia hybrida

Summary The difference in colour intensity between flowers of sporogenic revertants of the white flowering lines W17 and W28 is caused by an incompletely dominant gene Inl. This gene is not linked to the anthocyanin gene Anl. In the dominant state Inl causes a 50% decrease in colour intensity of selfcoloured red flowers.Chromatographic analysis of anthocyanins of plants homozygous recessive or dominant for Inl showed that the same anthocyanins are produced in both genotypes (cyanidin-3-glucoside and cyanidin-3-diglucoside). Anthocyanin synthesis starts at the same stage of development of the flower in both genotypes. When the bud reaches a length of approximately 45 mm, however, anthocyanin synthesis in the Inl Inl line slows down.No influence of the gene Inl on the concentration of dihydroquercetin-7-glucoside in buds and flowers could be observed, which indicates that the influence of Inl on flower colour development is restricted to the last part of the biosynthesis of anthocyanins, i.e. the conversion of dihydroflavonols into anthocyanins.In addition to Inl having a decreasing effect on flower colour intensity, evidence is produced that the gene Inl also influences the reversion frequency of unstable alleles of the gene Anl.     

The stabilizing effect of the acyl group on the co-pigmentation of acylated anthocyanins with C-glucosylflavones

The stability of the complex formed between acylated anthocyanins and flavocommelin was compared with that between unacylated anthocyanins and the flavone. At low anthocyanin concentrations (5 × 10^?4 M), the complex involving acylated anthocyanins was much stabler than that involving ordinary anthocyanins. This extra stability is due to the acyl moiety in the acylated anthocyanins. The co-pigmentation constant (Kc) is defined as the affinity between an anthocyanin and its co-pigment.     

Anthocyanin Profiles in Grape Berry Skins of Different Species of Wine Grapes in Shanxi,China

To understand the anthocyanin characteristics of wine grape varieties, the anthocyanin composition and content of 31 wine grape varieties were analyzed to explore the use of anthocyanins as chemical fingerprints to distinguish varieties. Results showed that a total of 21 anthocyanins were detected in the skins, including cyanidin, delphinidin, petunidin, peonidin and malvidin 3-monoglucosides (or 3,5-diglucosides) along with the corresponding acetyl and p-coumaroyl derivatives. The highest and lowest total amount of anthocyanins were detected in ‘Ruby Cabernet’ and ‘Muscat Rouge’, respectively. In the 21 Vitis vinifera grapes, there were 3~11 monoglucoside anthocyanins detected, however, there were 4 to 9 monoglucoside anthocyanins and 1~7 diglucoside anthocyanins detected in the 10 other species of grapes. Except for ‘Zhesexiang’ ‘Seibel Noir’, ‘44-6-7-1’ and ‘Beibinghong’, the contents of diglucoside anthocyanins in the other six varieties accounted for more than 52% of the total anthocyanins. Except for ‘Zhesexiang’, ‘Muscat Rouge’ and ‘Beibinghong’, the content of methylated anthocyanins accounted for more than 75% of total anthocyanins. There were significant differences in the anthocyanin types and contents in the skins among V. vinifera and other grapes. The results of the principal component analysis and the cluster classification of 31 grape varieties (lines) were nearly consistent, which suggested that anthocyanins can be used as chemical fingerprints to distinguish wine grape varieties.