ABA信号通路对葡萄果皮花青苷生物合成的调控机制研究

以‘红巴拉多’葡萄为试验材料,在转色前期(约花后6周)用300 mg/L的ABA对果穗进行处理,以清水处理为对照;测定不同发育时期葡萄果实的单果重、可滴定酸、可溶性固性物等生理指标,同时测定果皮中总花青苷及ABA含量;检测不同发育时期果皮中ABA信号通路和花青苷生物合成相关基因表达量,克隆6个与花青苷生物合成相关基因的启动子,并预测启动子中的顺式作用元件,在转录调控水平上探讨ABA信号通路对葡萄果皮花青苷生物合成的调控作用。结果表明:(1)ABA处理的葡萄果实可溶性固形物含量明显提高、可滴定酸含量下降。(2)ABA处理显著提高了‘红巴拉多’葡萄果皮的着色水平以及总花青苷和ABA含量。(3)ABA处理后,9个ABA信号通路基因以及6个花青苷生物合成相关基因表达水平明显提高。(4)6个花青苷生物合成相关基因的启动子序列中均含有多个与ABA响应相关的ABRE作用元件。研究发现,9个ABA信号通路基因可能在葡萄果皮着色中发挥着重要作用,其中2个VvABFs转录因子可能直接作用于含有ABRE元件的花青苷生物合成相关基因的启动子序列,推测可通过调控这些基因的转录水平来调控葡萄果皮花青苷的积累。     

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.     

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.     

Flavonoid biosynthesis-related genes in grape skin are differentially regulated by temperature and light conditions

Temperature and light are important environmental factors that affect flavonoid biosynthesis in grape berry skin. However, the interrelationships between temperature and light effects on flavonoid biosynthesis have not been fully elucidated at the molecular level. Here, we investigated the effects of temperature and light conditions on the biosynthesis of flavonoids (anthocyanins and flavonols) and the expression levels of related genes in an in vitro environmental experiment using detached grape berries. Sufficient anthocyanin accumulation in the grape skin was observed under a low temperature (15?°C) plus light treatment, whereas high temperature (35?°C) or dark treatment severely suppressed anthocyanin accumulation. This indicates that the accumulation of anthocyanins is dependent on both low temperature and light. qRT-PCR analysis showed that the responses of three MYB-related genes (VlMYBA1-3, VlMYBA1-2, and VlMYBA2) to temperature and light differed greatly even though the products of all three genes had the ability to regulate anthocyanin biosynthesis pathway genes. Furthermore, the expression levels of other MYB-related genes and many flavonoid biosynthesis pathway genes were regulated independently by temperature and light. We also found that temperature and light conditions affected the anthocyanin composition in the skin through the regulation of flavonoid biosynthesis pathway genes. Our results suggest that low temperature and light have a synergistic effect on the expression of genes in the flavonoid biosynthesis pathway. These findings provide new information about the relationships between environmental factors and flavonoid accumulation in grape berry skin.     

A 2,4-D-inducible glutathione S-transferase from soybean (Glycine max). Purification, characterisation and induction

Glutathione S-transferases (GSTs; EC 2.5.1.18) have recently been proposed to form one large group among the auxin-induced proteins. However. the properties and regulation of such auxin-responsive GSTs in the plant still await detailed investigation. In this study, a 2,4-dichloro-phenoxyacetic acid (2,4-D)-inducible GST isozyme from soybean ( Glycine max [L.] Merr. cv. Williams) was purified to near homogeneity by anion-exchange and affinity chromatography on S-hexylglutathione agarose. The native enzyme had a molecular mass of 49 kDa, as determined by gel filtration, and consisted of 26-kDa subunits. The purified GST conjugated glutathione to 1-chloro-2,4-dinitrobenzene and to the herbicide metolachlor, but not to the other GST substrates atrazine. fluorodifen or trans-cinnamic acid. The N-termmal amino acid sequence shared significant homology with the deduced polypeptide sequences of two 2,4-D-inducible genes from tobacco, par A and CNT107 . The levels of the 26-kDa GST subunit protein in soybean hypocotyls were analysed by immunoblotting. At micromolar concentrations, 2,4-D induced a transient increase in net accumulation of GST, whereas indole-3-acetic acid or I-naphthaleneacetic acid did not increase the GST levels. Known inhibitors of polar auxin transport, including 2.3.5-tri-iodobenzoic acid. N-I-naphthylphthalamic acid and analogues thereof, differed widely in their ability to elicit GST protein accumulation. It is concluded that the induction of soybean GST by 2,4-D and by some of the auxin transport inhibitors is not related to auxin activity or to changes in the endogenous auxin levels.     

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.     

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.     

TRANSPARENT TESTA 19 is involved in the accumulation of both anthocyanins and proanthocyanidins in Arabidopsis

Flavonoid compounds such as anthocyanins and proanthocyanidins (PAs; so-called condensed tannins) have a multitude of functions in plants. They must be transported from the site of synthesis in the cytosol to their final destination, the vacuoles. Three models have been proposed for sequestering anthocyanins in vacuoles, but the transport machinery for PAs is poorly understood. Novel Arabidopsis mutants, transparent testa 19 (tt19), which were induced by ion beam irradiation, showed a great reduction of anthocyanin pigments in the vegetative parts as well as brown pigments in the seed coat. The TT19 gene was isolated by chromosome walking and a candidate gene approach, and was shown to be a member of the Arabidopsis glutathione S-transferase (GST) gene family. Heterologous expression of a putative ortholog, petunia anthocyanin 9 (AN9), in tt19 complemented the anthocyanin accumulation but not the brown pigmentation in the seed coat. This suggests that the TT19 gene is required for vacuolar uptake of anthocyanins into vacuoles, but that it has also a function different from that of AN9. The depositional pattern of PA precursors in the mutant was different from that in the wild type. These results indicate that TT19 participates in the PA pathway as well as the anthocyanin pathway of Arabidopsis. As involvement of GST in the PA pathway was previously considered unlikely, the function of TT19 in the PA pathway is also discussed in the context of the putative transporter for PA precursors.     

Varietal Differences of Prokupac,Evita and Čokot Zemun Based on Their Anthocyanins Content in Grape Skin Extract

In this study, we have analyzed the anthocyanin composition of skin extracts of three red grape varieties Prokupac, Evita and Čokot Zemun in order to distinguish these cultivars based on their anthocyanin profile. Also, mechanical analysis of grape bunches and berries was performed. According to our results, seventeen anthocyanins were identified using LC/MS technique and quantitative differences were recorded using HPLC-DAD method. The highest content of total anthocyanins was obtained for Evita variety and the lowest one was recorded in Prokupac. Also, clear differences were observed in anthocyanins ratios. In comparison to Prokupac and Evita varieties, Čokot Zemun was characterized with a high content of coumaroyl derivatives of anthocyanin compounds, while high levels of acetylated derivatives were recorded in Prokupac. Data reported in this study represent a certain contribution to a database of mechanical properties and chemical composition of grape varieties originating from Balkan.     

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.     

Evolutionary and structural analyses of GDAP1, involved in Charcot-Marie-Tooth disease, characterize a novel class of glutathione transferase-related genes

Mutations in the Ganglioside-induced differentiation-associated protein-1 (GDAP1) gene cause autosomal recessive Charcot-Marie-Tooth disease type 4A. The protein encoded by GDAP1 shows clear similarity to glutathione transferases (also known as glutathione S-transferases or GSTs). The human genome contains a paralog of GDAP1 called GDAP1L1. Using comparative genomics, we show that orthologs of GDAP1 and GDAP1L1 are found in mammals, birds, amphibians, and fishes. Likely orthologs of those genes in invertebrates and a low but consistent similarity with some plant and eubacterial genes have also been found. We demonstrate that GDAP1 and GDAP1L1 do not belong to any of the known classes of GST genes. In addition to having distinctive sequences, GDAP1 and its relatives are also characterized by an extended region in GST domain II, absent in most other GSTs, and by a C-terminal end predicted to contain transmembrane domains. Mutations affecting any of those characteristic domains are known to cause Charcot-Marie-Tooth disease. These features define the GDAP1 class of GST-like proteins.     

Whole genome survey of the glutathione transferase family in the brown algal model Ectocarpus siliculosus

We report here an exhaustive analysis of the glutathione transferases (GSTs) in the model brown alga Ectocarpus siliculosus using available genomic resources. A genome survey revealed the presence of twelve cytosolic GSTs, belonging to the Sigma class, two pseudogenes, one GST of the Kappa class, and three microsomal GSTs of the MGST3 family of membrane associated protein involved in eicosanoid and glutathione metabolism. Gene structure and phylogenetic analyses demonstrated the partition of the Sigma GSTs into two clusters which have probably evolved by duplication events. Gene expression profiling was conducted after the addition of high concentrations of chemicals, such as H(2)O(2), herbicides, heavy metals, as well as fatty acid derivatives, in order to induce stress conditions and to monitor early response mechanisms. The results of these experiments suggested that E. siliculosus GST genes are recruited in different and specific conditions. In addition, heterologous expression in yeast of two E. siliculosus microsomal GST showed that these enzymes feature peroxidase rather than transferase activity. The potential involvement of E. siliculosus GST in the metabolism of oxygenated polyunsaturated fatty acids is discussed.     

In vivo grapevine anthocyanin transport involves vesicle-mediated trafficking and the contribution of anthoMATE transporters and GST

In cells, anthocyanin pigments are synthesized at the cytoplasmic surface of the endoplasmic reticulum, and are then transported and finally accumulated inside the vacuole. In Vitis vinifera (grapevine), two kinds of molecular actors are putatively associated with the vacuolar sequestration of anthocyanins: a glutathione-S-transferase (GST) and two MATE-type transporters, named anthoMATEs. However, the sequence of events by which anthocyanins are imported into the vacuole remains unclear. We used MYBA1 transformed hairy roots as a grapevine model tissue producing anthocyanins, and took advantage of the unique autofluorescence of anthocyanins to study their cellular trafficking. In these tissues, anthocyanins were not only visible in the largest vacuoles, but were also present at higher concentrations in several vesicles of different sizes. In the cell, small vesicles actively moved alongside the tonoplast, suggesting a vesicular trafficking to the vacuole. Subcellular localization assays revealed that anthoMATE transporters were closely related with these small vesicles, whereas GST was localized in the cytoplasm around the nucleus, suggesting an association with the endoplasmic reticulum. Furthermore, cells in hairy roots expressing anthoMATE antisense did not display small vesicles filled with anthocyanins, whereas in hairy roots expressing GST antisense, anthocyanins were accumulated in vesicles but not in the vacuole. This suggests that in grapevine, anthoMATE transporters and GST are involved in different anthocyanin transport mechanisms.