A combination of elicitation and precursor feeding leads to increased anthocyanin synthesis in cell suspension cultures of <Emphasis Type="Italic">Vitis vinifera</Emphasis>

Both elicitation and precursor feeding are effective strategies for improving secondary metabolite production in plant cell suspension cultures. In this study, cell suspension cultures of Vitis vinifera subjected to methyl jasmonate treatment resulted in a significant increase in levels of anthocyanin production. Moreover, a combination of 5 mg/L phenylalanine and 50 mg/L methyl jasmonate promoted the highest level of anthocyanin biosynthesis, resulting in 4.6- and 3.4-fold increases in anthocyanin content and yield, respectively, over the control. The optimum period for elicitation of anthocyanin synthesis was 4 days following incubation in the presence of elicitors, at the beginning of the exponential growth phase. V. vinifera cell lines of different anthocyanin-producing capabilities responded differently to elicitation and precursor feeding. Anthocyanin production of a low-producing cell line, VV06, could be enhanced with addition of elicitors and precursor feeding. Methyl jasmonate was the only elicitor that increased anthocyanin production of the high-producing cell line VV05, but contributed to moderate enhancement of anthocyanin production compared with VV06. For cell line VV06, synergistic effects were observed for all treatment combinations of methyl jasmonate along with other elicitors and precursors. In addition, 6.1- and 4.6-fold increases in anthocyanin content and yield, respectively, were obtained in the presence of 5 mg/L phenylalanine, 50 mg/L methyl jasmonate, and 1 mg/L dextran. However, none of these treatment combinations exhibited synergistic effects in cell line VV05.     

Anthocyanin production by plant cell cultures on media based on milk whey

Summary Selected callus cultures ofAjuga reptans produce anthocyanins in the dark on Murashige-Skoog medium with sucrose as carbon source. From these cultures we could isolate, by a two-stage selection procedure, new lines, which produce anthocyanins on media based on milk whey with lactose as the only carbon source. The anthocyanin production of the cell lines on milk whey is comparable with the production of the cell lines on MS-medium. Our results prove that it is possible to lower medium costs of plant cell culture by using cheap raw materials.     

二氢黄酮醇 4 还原酶在花青素合成中的功能及调控研究进展

植物色素主要有花青素、类胡萝卜素和生物碱类色素三大类,其中花青素是决定大部分被子植物组织或器官颜色的重要色素。花青素通过类黄酮途径合成,该途径是生物学上研究较多且较为清楚的代谢途径之一。近年来的研究表明,在该途径中除了查尔酮合成酶(chalcone synthase,CHS)、查尔酮异构酶(chalcone isomerase,CHI)和黄烷酮-3-羟化酶(flavanone-3-hydrolase,F3H)起着关键作用外,二氢黄酮醇-4-还原酶(dihydroflavonol 4-reductase,DFR)对花青素的合成也至关重要。DFR可催化3种二氢黄酮醇和2种黄烷酮生成5种不同的花青素前体,且DFR基因家族不同成员对各个底物的催化效率不同,因此它在一定程度上决定着植物中花青素的种类和含量,从而影响植物组织或器官的颜色。该文对近年来国内外有关DFR在花青素合成过程中的生物学功能与调控,包括DFR的特征、作用机制和系统进化以及环境、转录因子和一些结构基因与DFR的关系等方面的研究进展进行了综述,以期为DFR今后的研究和利用基因工程改变植物组织或器官的颜色提供理论依据。     

基于花青素代谢培育蓝色花卉的研究进展

花色是植物吸引昆虫传播花粉的主要因素,对于植物在自然界的生存必不可少,也是观赏植物最重要的性状之一。在蓬勃发展的花卉产业中,色彩各异花卉的培育,可以弥补自然花色的匮乏,但是令人垂涎的蓝色花比较难培育。花色的多样性主要是由花青素及其衍生物的种类和含量等因素决定的,飞燕草色素的合成是形成蓝色花的关键因素,许多植物体内缺少合成飞燕草色素的结构基因。近年来,利用基因工程技术培育蓝色花的研究也时有报道。文中以常见的观赏植物为例,基于花青素代谢调控,从影响飞燕草色素合成的关键因素和不同分子改良途径培育蓝色花等几个方面对植物花朵呈色的机制进行了综述,并展示不同分子育种策略可能在其他领域的应用,为其他植物或经济作物的色泽改良如彩色棉蓝色纤维的培育等提供参考和技术支持。     

继代周期和接种量对葡萄细胞培养的影响

在每种不同的继代周期和接种量条件下,葡萄细胞在连续10次继代培养过程中的生物量、花青素含量、胞内糖、胞内蛋白及胞内总磷均表现出不同程度的波动。不同接种量对培养不稳定性的影响比不同继代周期大;在所考察的条件中,7d继代周期与1.60g接种量组合的继代条件下花青素合成相对稳定;花青素合成与胞内蔗糖或胞内总磷水平呈负相关。     

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.     

激素等外源物质对马铃薯愈伤组织花色苷积累的影响

从来源于马铃薯(Solanum tuberosum cv.Chieftain)茎的愈伤组织中分离到白色和红色2种愈伤组织,用鲜重法和分光光度法分别测量愈伤组织的生长量和花色苷的含量,并对激素、抗菌素和糖对马铃薯愈伤组织生长和花色苷积累的影响进行研究。结果表明:低浓度的2,4-D有利于红色愈伤组织的花色苷积累,高浓度的2,4-D促进其生长而不利于花色苷的积累;高浓度的6-BA能促进红色愈伤组织中花色苷的积累并诱导白色愈伤组织花色苷的合成,但抑制其生长;卡那霉素能使白色愈伤组织变红并积累花色苷,高浓度的卡那霉素严重抑制愈伤组织的生长并最终变褐死亡;提高蔗糖浓度能促进愈伤组织花色苷的产生和积累,但超过70g/L时抑制生长。实验结果为今后花色苷生物合成机理研究和花色苷的工厂化生产奠定了基础。     

Diversity in plant red pigments: anthocyanins and betacyanins

Plant pigments are of interest for research into questions of basic biology as well as for purposes of applied biology. Red colors in flowers are mainly produced by two types of pigments: anthocyanins and betacyanins. Though anthocyanins are broadly distributed among plants, betacyanins have replaced anthocyanins in the Caryophyllales. Red plant pigments are good indicator metabolites for evolutionary studies of plant diversity as well as for metabolic studies of plant cell growth and differentiation. In this review, we focus on the biosynthesis of anthocyanins and betacyanins and the possible mechanisms underlying the mutual exclusion of betalains and anthocyanins based on the regulation of the biosynthesis of these red pigments.     

园艺作物花青素合成调控研究进展

花青素是一类保护植物免受生物和非生物胁迫的重要次生代谢产物,因其赋予植物丰富的色彩和对人体的保健功能而受到广泛关注。花青素合成调控机理的相关研究是目前园艺作物分子生物学研究的前沿课题,对于园艺作物花青素含量的提高、种质品质的提升等具有重要的意义。结合国内外园艺作物中花青素生物合成调控方面的最新研究进展,介绍了环境因素、酶与激素、DNA甲基化与泛素化和调控基因等对花青素生物合成的作用,以及花青素抵御外界胁迫的功能机制,综述了近年来园艺作物中花青素生物合成调控的研究成果,以期利用基因工程为提升园艺作物的色彩丰富度提供理论参考。     

Increased accumulation and decreased catabolism of anthocyanins in red grape cell suspension culture following magnesium treatment

Anthocyanins are the largest and best studied group of plant pigments. However, not very much is known about the fate of these phenolic pigments after they have accumulated in the cell vacuoles of plant tissues. We have previously shown that magnesium treatment of ornamentals during the synthesis of anthocyanins in the flowers or foliage caused an increase in the pigment concentration. In this study, we characterized the effect of magnesium on the accumulation of anthocyanin in red cell suspension originating from Vitis vinifera cv. Gamay Red grapes. Magnesium treatment of the cells caused a 2.5- to 4.5-fold increase in anthocyanin concentration, with no substantial induction of the biosynthetic genes. This treatment inhibited the degradation of anthocyanins occurring in the cells, and changed the ratio between different anthocyanins determining cell color, with an increase in the relative concentration of the less stable pigment molecules. The process by which magnesium treatment affects anthocyanin accumulation is still not clear. However, the results presented suggest at least part of its effect on anthocyanin accumulation stems from inhibition of the pigments’ catabolism. When anthocyanin biosynthesis was inhibited, magnesium treatments prevented the constant degradation of anthocyanins in the cell suspension. Future understanding of the catabolic processes undergone by anthocyanins in plants may enable more efficient inhibition of this process and increased accumulation of these pigments, and possibly of additional phenolic compounds.     

Hairy Roots, their Multiple Applications and Recent Patents

In the last years, hairy root (HR) cultures are gaining attention in the biotechnology industry. This particular plant cell culture derives from explants infected with Agrobacterium rhizogenes. They constitute a relatively new approach to in vitro plant biotechnology and modern HR cultures are far away from the valuables findings performed by Philip R. White in the 1930?s, who obtained indefinite growth of excised root tips. HR cultures are characterized by genetic and biochemical stability and high growth rate without expensive exogenous hormones source. HR cultures have allowed a deep study of plant metabolic pathways and the production of valuable secondary metabolites and enzymes, with therapeutic or industrial application. Furthermore, the potential of HR cultures is increasing continuously since different biotechnological strategies such as genetic engineering, elicitation and metabolic traps are currently being explored for discovery of new metabolites and pathways, as well as for increasing metabolites biosynthesis and/or secretion. Advances in design of proper bioreactors for HR growth are being of great interest, since scale up of metabolite production will allow the integration of this technology to industrial processes. Another application of HR cultures is related to their capabilities to biotransform and to degrade different xenobiotics. In this context, removal assays using this plant model system are useful tools for phytoremediation assays, previous to the application in the field. This review highlights the more recent application of HRs and those new patents which show their multiple utilities.     

Effect of sucrose and methyl jasmonate on biomass and anthocyanin production in cell suspension culture of Melastoma malabathricum (Melastomaceae)

Melastoma malabathricum, belongs to the Melastomaceae family, is an important medicinal plant widely distributed from Madagascar to Australia, that is used in traditional remedies for the treatment of various ailments. Besides its medicinal properties, it has been identified as a potential source of anthocyanin production. The present study was carried out to investigate the effect of sucrose and methyl jasmonate and feeding time on cell biomass yield and anthocyanin production in cell suspension culture of M. malabathricum. Addition of different concentrations of sucrose into the cell culture of M. malabathricum influenced cell biomass and pigment accumulation. The addition of methyl jasmonate was found to have no effect on cell biomass but the presence of higher amount (12.5-50 mg/L) had caused a reduction in anthocyanin production and accumulation. MS medium supplemented with 30 g/L sucrose and 3.5 mg/L of MeJA added on cero day and 3rd day produced high fresh cell mass at the end of nine days of culture but did not support the production of anthocyanins. However, cells cultured in the medium supplemented with 45 g/L sucrose without MeJA showed the highest pigment content (0.69 +/- 0.22 CV/g-FCM). The cells cultured in MS medium supplemented with 30 g/L sucrose with 3.5 mg/L MeJA added on the 3rd and 6th day of culture, showed the lowest pigment content (0.37-0.40 CV/g-FCM). This study indicated that MeJA was not necessary but sucrose was needed for the enhancement of cell growth and anthocyanin production in M. malabathricum cell cultures.     

The COI1 and DFR Genes are Essential for Regulation of Jasmonate-Induced Anthocyanin Accumulation in Arabidopsis

Jasmonates(JAs)are a class of plant hormones that play important roles in the regulation of plant development and plantdefense.It has been shown that Arabidopsis plants produce much higher levels of anthocyanins when treated exogenouslywith methyl jasmonate(MeJA).However,a molecular link between the JA response and anthocyanin production hasnot been determined.The CORONATINE INSENTITIVE1(COI1)gene is a key player in the regulation of many JA-relatedresponses.In the present study,we demonstrate that the COI1 gene is also required for the JA-induced accumulation ofanthocyanins in Arabidopsis.Furthermore,the MeJA-inducible expression of DIHYDROFLAVONOL REDUCTASE(DFR),anessential component in the anthocyanin biosynthesis pathway,was completely eliminated in the coil mutant.Jasmonate-induced anthocyanin accumulation was found to be independent of auxin signaling.The present results indicate that theexpression of both COI1 and DFR genes is required for the regulation of JA-induced anthocyanin accumulation and thatDFR may be a key downstream regulator for this process.     

The COI1 and DFR Genes are Essential for Regulation of Jasmonate-Induced Anthocyanin Accumulation in Arabidopsis

Jasmonates （JAs） are a class of plant hormones that play important roles in the regulation of plant development and plant defense. It has been shown that Arabidopsis plants produce much higher levels of anthocyanins when treated exogenously with methyl jasmonate （MeJA）. However, a molecular link between the JA response and anthocyanin production has not been determined. The CORONATINE INSENTITIVE1 （COI1） gene is a key player in the regulation of many JA-related responses. In the present study, we demonstrate that the COI1 gene is also required for the JA-induced accumulation of anthocyanins in Arabidopsis. Furthermore, the MeJA-inducible expression of DIHYDROFLAVONOL REDUCTASE （DFR）, an essential component in the anthocyanin biosynthesis pathway, was completely eliminated in the coil mutant. Jasmonateinduced anthocyanin accumulation was found to be independent of auxin signaling. The present results indicate that the expression of both COI1 and DFR genes is required for the regulation of JA-induced anthocyanin accumulation and that DFR may be a key downstream regulator for this process.     

Light-dependent anthocyanin synthesis: A model system for the study of plant photomorphogenesis

The biosynthesis of anthocyanins in plant tissues either requires light or is enhanced by it. Light-dependent anthocyanin synthesis has been extensively used as a model system for studies of the mechanism of photoregulation of plant development. Two components can be distinguished in the action of light on anthocyanin production. The first component is the red-far red reversible, phytochrome-mediated response induced by short irradiations; the amount of anthocyanin formed in response to a single, short irradiation is small. The second component is the response to prolonged exposures; the formation of large amounts of anthocyanin requires prolonged exposures to high fluence rates of visible and near-visible radiation (290 to 750 nm) and shows the typical properties of the “High Irradiance Reaction” (HIR) of plant photomorphogenesis. Phytochrome is involved in the photoregulation of the HIR response and is the only photoreceptor mediating the action of prolonged red and far red irradiations. The response to prolonged ultraviolet and blue radiation is probably mediated, at least in some systems, by two photoreceptors: phytochrome and cryptochrome, the latter being a specific ultraviolet-blue-light photoreceptor. The nature of the interaction between phytochrome and cryptochrome in the regulation of plant photomorphogenic responses is still unclear.     

Genetic engineering of flavonoid pigments to modify flower color in floricultural plants

Recent advances in genetic transformation techniques enable the production of desirable and novel flower colors in some important floricultural plants. Genetic engineering of novel flower colors is now a practical technology as typified by commercialization of a transgenic blue rose and blue carnation. Many researchers exploit knowledge of flavonoid biosynthesis effectively to obtain unique flower colors. So far, the main pigments targeted for flower color modification are anthocyanins that contribute to a variety of colors such as red, pink and blue, but recent studies have also utilized colorless or faint-colored compounds. For example, chalcones and aurones have been successfully engineered to produce yellow flowers, and flavones and flavonols used to change flower color hues. In this review, we summarize examples of successful flower color modification in floricultural plants focusing on recent advances in techniques.     

植物花青素生物代谢调控

花青素是一类重要的天然色素物质,是植物的主要呈色物质之一,近年来花青素在保健方面的作用越来越受到人们的重视,利用基因工程改造植物花青素相关基因,提高花青素含量已成为研究的热点领域。综述花青素的合成途径、调控机理及转基因方面的研究,重点介绍近年来影响花青素合成的分子因素及外部环境因素的研究现状。     

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"。     

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.