Analysis of the Expression of Anthocyanin Pathway Genes in Developing Vitis vinifera L. cv Shiraz Grape Berries and the Implications for Pathway Regulation

Anthocyanin synthesis in Vitis vinifera L. cv Shiraz grape berries began 10 weeks postflowering and continued throughout berry ripening. Expression of seven genes of the anthocyanin biosynthetic pathway (phenylalanine ammonia lyase [PAL], chalcone synthase [CHS], chalcone isomerase [CHI], flavanone-3-hydroxylase [F3H], dihydroflavonol 4-reductase [DFR], leucoanthocyanidin dioxygen-ase [LDOX], and UDP glucose-flavonoid 3-o-glucosyl transferase [UFGT]) was determined. In flowers and grape berry skins, expression of all of the genes, except UFGT, was detected up to 4 weeks postflowering, followed by a reduction in this expression 6 to 8 weeks postflowering. Expression of CHS, CHI, F3H, DFR, LDOX, and UFGT then increased 10 weeks postflowering, coinciding with the onset of anthocyanin synthesis. In grape berry flesh, no PAL or UFGT expression was detected at any stage of development, but CHS, CHI, F3H, DFR, and LDOX were expressed up to 4 weeks postflowering. These results indicate that the onset of anthocyanin synthesis in ripening grape berry skins coincides with a coordinated increase in expression of a number of genes in the anthocyanin biosynthetic pathway, suggesting the involvement of regulatory genes. UFGT is regulated independently of the other genes, suggesting that in grapes the major control point in this pathway is later than that observed in maize, petunia, and snapdragon.     

Expression of anthocyanin biosynthesis pathway genes in red and white grapes

The expression of seven genes from the anthocyanin biosynthesis pathway was determined in different tissues of Shiraz grapevines. All of the tissues contained proanthocyanidins, but only the berry skin accumulated anthocyanins. In most tissues, all of the flavonoid genes except UDP glucose-flavonoid 3-o-glucosyl transferase (UFGT) were expressed, but UFGT expression was only detected in berry skin. Similar patterns of expression were observed in the skin of other red grapes. In white grapes, UFGT expression was not detected. White grape cultivars appear to lack anthocyanins because they lack UFGT, although they also had decreased expression of other flavonoid pathway genes.     

乙烯利处理对葡萄花色苷合成相关基因表达的影响

利用荧光定量PCR技术分析‘京优’葡萄果实成熟过程中,花色苷生物合成途径相关酶基因mRNA转录水平的变化以及乙烯利处理对果皮中花色苷含量和关键酶基因转录水平的影响。结果显示,葡萄果实发育进入着色期,花色苷合成过程中主要相关基因(CHSs、CHIs、F3Hs、F3′H、F3′5′H、DFR、LDOX、UFGT、OMT和GST)和转录因子(MybA1和MybA1-2)转录水平都显著提高,其中UFGT、GST、MybA1和CHSs、CHIs、F3Hs基因家族中的CHS3、CHI2、F3H2随着花色苷合成而大量转录;乙烯利处理能够增强花色苷合成相关基因的转录,使其转录时期前移和转录水平提高,其中对GST、UFGT和MybA1转录的促进作用最明显。相关性分析表明,花色苷合成与一些花色苷合成相关基因(CHS3、CHI2、F3H2、F3′5′H、UFGT、GST)和转录因子(MybA1)的转录水平呈显著或极显著正相关;与CHS1、CHS2、CHI1、F3H1、DFR、F3′H、LDOX和OMT转录水平的相关性均不显著。本研究结果为进一步阐明花色苷生物合成机理和花色苷类色素的生产应用提供一定的理论依据。     

Anthocyanin, antioxidant activity and stress-induced gene expression in high CO2-treated table grapes stored at low temperature

A pretreatment with 20kPa CO2+20 kPa O2+60 kPa N2 for 3 days proved effective in maintaining the fruit quality and controlling decay in table grapes (Vitis vinifera cv. Cardinal) stored at 0 degrees C. In the present work, we analyzed whether total anthocyanin content, the molecular mechanism implicated in their biosynthesis and antioxidant activity is related to the beneficial effect of this gaseous treatment. We isolated partial cDNAs that codified for enzymes implicated in the anthocyanin biosynthesis such as l-phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS), and an antioxidant enzyme such as ascorbate peroxidase (APX). Low temperatures induced an accumulation of total anthocyanin content in the skin of both treated and non-treated grapes, although levels were lower in CO2-treated fruit. By contrast, antioxidant activity decreased during storage at 0 degrees C in non-treated grapes but did not change in CO2-treated grapes. The up-regulation of anthocyanin biosynthesis gene expression and VcAPX mRNA observed in non-treated grape is not enhanced in CO2-treated grapes, which presented low total decay. These results point out the ability of CO2-treated grapes to prevent the generation of reactive oxygen species rather than their inactivation by means of induction of studied defense systems.     

Control of anthocyanin biosynthesis pathway gene expression by eutypine,a toxin from Eutypa lata,in grape cell tissue cultures

Eutypine, 4-hydroxy-3-(3-methyl-3-butene-1-ynyl) benzaldehyde, is a toxin produced by Eutypa lata, the causal agent of Eutypa dieback in grapevine. The effect of the toxin on anthocyanin synthesis has been investigated in Vitis vinifera cv. Gamay cell cultures. At concentrations higher than 200 micromol/L, eutypine reduced anthocyanin accumulation in cells. The reduction in anthocyanin accumulation was proportional to the eutypine concentrations and HPLC analysis showed that eutypine affected the levels of all anthocyanins. The effect of eutypine application on the expression of five genes of the anthocyanin biosynthesis pathway, including chalcone synthase (CHS), flavonone-3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), leucoanthocyanidin dioxygenase (LDOX), and UDP glucose-flavonoid 3-O-glucosyl transferase (UFGT) was determined. Expression of CHS, F3H, DFR and LDOXwas not affected by the addition of eutypine to grapevine cell cultures. In contrast, expression of the UFGT gene was dramatically inhibited by the toxin. These results suggest that in grapevine cell cultures, eutypine strongly affects anthocyanin accumulation by inhibiting UFGT gene expression. The mechanism of action of eutypine is discussed.     

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...     

Influence of Berry Heterogeneity on Phenolics and Antioxidant Activity of Grapes and Wines: A Primary Study of the New Winegrape Cultivar Meili (Vitis vinifera L.)

Wine grapes are usually harvested in vineyards when they ripen. However, not all of the berries in a vineyard ripen homogeneously because of different microclimates around the clusters and berries. In this study, the influence of berry heterogeneity on the phenolic content and antioxidant capacity of grapes and wines under a continental monsoon climate was evaluated for a new wine grape cultivar Meili (Vitis vinifera L.). The total phenolic, flavonoid, flavanol, and monomeric anthocyanin contents in the skin and wine significantly increased with grape density; however, there was no significant difference in the seeds between the two lower densities. The highest values of DPPH free radical-scavenging activity, cupric-reducing antioxidant capacity, and hydroxyl radical-scavenging activity in the skin, seed and wine were detected for the densest berries. The sum of individual phenolic compounds in skin, seed and wine increased with berry density, though no significant difference for skin was observed between the two higher density classes. Hence, the chemical components of Meili grapes and wines were positively associated with the berry density at harvest under the continental monsoon climate.     

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.     

Impact of fruit feeding on overwintering survival of the multicolored Asian lady beetle, and the ability of this insect and paper wasps to injure wine grape berries

The establishment of the multicolored Asian lady beetle, Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae), in North America has resulted in negative impacts on fruit production. We investigated the overwintering survival of H. axyridis after feeding on four diets: injured wine grape berries, 25% sucrose solution, water, and a control containing no food or water. After being exposed to these diets for 6 days, live individuals were transferred to clean plastic Petri dishes, and held at 5 ± 1 °C in growth chambers throughout the winter. Survival was recorded every month. Adult lady beetles collected during the overwintering flight in mid-October had higher survival rates than beetles collected from soybean fields in mid-August. These results suggest that an adaptation period prior to diapause increases the chances of lady beetle survival over the winter. In addition, injured wine grape berries, sugar, and water decreased beetle mortality during the overwintering months. Our results also showed that under similar conditions, females have higher survival during the winter than males. The importance of sugar and water on winter survival may drive H. axyridis adults to vineyards for feeding on wine grapes. Finally, we tested if adults of H. axyridis and the European paper wasp, Polistes dominulus Christ (Hymenoptera: Vespidae), were able to break the grape skin. Harmonia axyridis adults and paper wasps were not able to cause the primary injury to berries of Frontenac grapes under laboratory conditions. These results suggest that control of paper wasps in vineyards may not affect H. axyridis aggregations, and that H. axyridis feeding on wine grapes depends on previous injury to grape berries.     

Sugars induce anthocyanin accumulation and flavanone 3-hydroxylase expression in grape berries

Flavanone 3-hydroxylase (EC 1.14.11.9, F3H) plays a key role in anthocyanin biosynthesis, and sugars enhance anthocyanin accumulation and F3H expression in some other plants. However, information about the relationship between sugars, anthocyanin accumulation and F3H expression in grape berries has been little reported. Present experiment was done with sliced grape berry system. The optimum fruit developmental stage, sugar concentration, and incubation time in sugar induction anthocyanin accumulation and F3H expression were determined. Mannose and 2-deoxyglucose, glucose analogs known to be phosphorylated by hexokinase but are poorly metabolized, obviously induced the anthocyanin accumulation and F3H expression, whereas 3-O-methylglucose and 6-deoxyglucose, glucose analogs transported inside the cell but not substrates for hexokinase, did not induce them. Glucosamine and mannoheptulose, the specific inhibitors of hexokinase, blocked the activation induced by sugar on both anthocyanin accumulation and F3H expression.     

Expression analysis of UDP-glucose:flavonoid 3-O-glucosyltransferase (UFGT) gene in an interspecific hybrid grape between Vitis ficifolia var. ganebu and Vitis vinifera cv. Muscat of Alexandria

Kadainou R-1, an interspecific hybrid grape derived from red (Vitis ficifolia var. ganebu) and white (V. vinifera cv. Muscat of Alexandria) grapes, accumulates high concentrations of anthocyanin in the berry skin. Hence, the expression of uridine 5′-diphosphate (UDP)-glucose:flavonoid 3-O-glucosyltransferase (UFGT), the key enzyme of the anthocyanin pathway, was examined in the berry skin of Kadainou R-1. As information on gene sequences of V. ficifolia var. ganebu and other wild grape species was unavailable, we performed GeneChip hybridization using biotin-labeled genomic deoxyribonucleic acid (DNA) to investigate how the genomic sequences of V. vinifera varieties and that of V. ficifolia var. ganebu differ. The study showed a lower correlation coefficient between V. vinifera cultivars and V. ficifolia var. ganebu than that among V. vinifera cultivars. The sequences of the UFGT gene derived from both parents of the red and white cultivars were sequenced in Kadainou R1 and revealed that both were expressed irrespective of the fact that it was not expressed in the white grape (male parent).