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。     

脱落酸激素诱导拟南芥幼苗中花青素的合成

脱落酸(abscisic acid,ABA)激素是一类重要的生长调节物质,参与调控植物的多种生理过程。花青素(anthocyanins)是植物次生代谢产生的类黄酮化合物,对植物的生长发育和逆境胁迫响应有重要作用。该文以拟南芥(Arabidopsis thaliana)为研究对象,探讨ABA信号对花青素生物合成的调控功能和作用机制。结果表明:外源施加ABA显著提高野生型幼苗茎尖中花青素的积累。相一致的是,ABA能诱导某些与花青素合成相关的转录因子及合成酶基因的表达。遗传学分析发现,ABA诱导花青素合成部分依赖于MBW复合体中的核心转录因子,如TTG1、TT8及MYB75等。初步机制研究揭示,ABA信号途径中的bZIP类转录因子ABI5能与TTG1、TT8及MYB75等相互作用形成蛋白复合物。综上结果认为,ABA信号诱导拟南芥幼苗中花青素的积累,并可能通过ABI5与MBW复合体协同作用调控花青素的合成。     

Abscisic Acid and Its Catabolites in Leaves and Berries of Chardonnay are Affected by Water Status

The biosynthetic pathway of abscisic acid (ABA) is well known. The aim of this study was to investigate the relationship among various ABA catabolites in leaves and berries of Chardonnay grapevines grown under various irrigation regimes. An irrigation trial was set up in one vineyard, located in Niagara-on-the-Lake, ON, Canada, consisting of seven treatments: control (non-irrigated), plus three water levels (100, 50, and 25 % of estimated crop evapotranspiration) combined with two irrigation imposition times (fruit set, veraison). No irrigation occurred prior to treatment imposition. ABA, phaseic acid (PA), dihydrophaseic acid (DPA), 7′-hydroxy-ABA, 8′-hydroxy-ABA, neophaseic acid, and ABA glucose ester (ABA-GE) were quantified in leaves and berries by HPLC–MS. ABA was likely catabolized by conjugation to form ABA-GE in treatments under high levels of water deficit, while in treatments with high water status, the oxidation pathway leading to DPA or PA predominated. Concentrations of ABA and its catabolites therefore reflected vine water status, whereby the specific ABA catabolic pathways in leaves and berries were determined by water status level. Hormonal profiles suggested a direct relationship between ABA and vine water status. The concentration of ABA in Chardonnay may explain why and how white cultivars adapt to drought stress versus red cultivars.

     

ABA Initiates Anthocyanin Production in Grape Cell Cultures

Abscisic acid (ABA) has a well-known positive impact on grape ripening, especially color development, but its role in the initiation of anthocyanin synthesis remains unclear. To elucidate this point, ABA treatment was applied to a simple Vitis vinifera model, consisting of Cabernet Sauvignon cell suspensions that do not spontaneously produce anthocyanins under laboratory conditions. Endogenous ABA levels, the expression of some genes in the upstream part of the anthocyanin pathway, and anthocyanin content were determined. Exogenous ABA treatment sharply increased cell ABA content and induced both structural and regulatory genes involved in anthocyanin production. These changes were promptly detected, as early as 6 h after ABA treatment, whereas anthocyanin production was observed only after 4 days in culture. These results demonstrate that ABA promotes anthocyanin synthesis in grape cell culture.     

Water deficit and exogenous ABA significantly affect grape and wine phenolic composition under in field and in-vitro conditions

Phenolic compounds are components responsible of the sensorial characteristics of red wine. Indeed, they present important properties for human health. In the present work, the effects of combined in field treatments (water-stress and exogenous ABA) on phenolic accumulation were evaluated for berries and wine. The responses were assessed by UV–Vis and CZE. A differential phenolic regulation was observed under the same conditions and good correlations were achieved for grape and wine. ABA appears to regulate the content of each phenols under study depending on plant water-status. Although the effects of water-stress and exogenous ABA were similar in magnitude, our results support evidence suggesting that both affect different metabolic pathways. ABA supply increased catechin and malvidin synthesis for both water statuses while the resveratrol was enhanced only for water-stress. Indeed, ABA reduced the quercetin content for both water statuses. In vitro assays were carried out to estimate the effects of combined treatments (temperature-exogenous ABA) on anthocyanin accumulation in berries and pulp. These tests demonstrated that the effect of ABA was dependent on the hormonal level (207% for 0.5 g L⁻¹ and 307% for 1 g L⁻¹ of ABA). Effect of temperature varied according to the phenological state. In pulps, we detected an increase of anthocyanin after ABA treatments.     

Abscisic acid is involved in phenolic compounds biosynthesis,mainly anthocyanins,in leaves of Aristotelia chilensis plants (Mol.) subjected to drought stress

Abscisic acid (ABA) regulates the physiological and biochemical mechanisms required to tolerate drought stress, which is considered as an important abiotic stress. It has been postulated that ABA might be involved in regulation of plant phenolic compounds biosynthesis, especially anthocyanins that accumulate in plants subjected to drought stress; however, the evidence for this postulate remains elusive. Therefore, we studied whether ABA is involved in phenolic compounds accumulation, especially anthocyanin biosynthesis, using drought stressed Aristotelia chilensis plants, an endemic berry in Chile. Our approach was to use fluridone, an ABA biosynthesis inhibitor, and then subsequent ABA applications to young and fully‐expanded leaves of drought stressed A. chilensis plants during 24, 48 and 72 h of the experiment. Plants were harvested and leaves were collected separately to determine the biochemical status. We observed that fluridone treatments significantly decreased ABA concentrations and total anthocyanin (TA) concentrations in stressed plants, including both young and fully‐expanded leaves. TA concentrations following fluridone treatment were reduced around fivefold, reaching control plant levels. ABA application restored ABA levels as well as TA concentrations in stressed plant at 48 h of the experiment. We also observed that TA concentrations followed the same pattern as ABA concentrations in the ABA treated plants. Quantitative real‐time PCR revealed that AcUFGT gene expression decreased in fully‐expanded leaves of stressed plants treated with fluridone, while a subsequent ABA application increased AcUFGT expression. Taken together, our results suggest that ABA is involved in the regulation of anthocyanin biosynthesis under drought stress.     

Organ-specific transcription of putative flavonol synthase genes of grapevine and effects of plant hormones and shading on flavonol biosynthesis in grape berry skins

In order to investigate the control mechanism of flavonol biosynthesis of grapevine, we obtained five genomic sequences (FLS1 to FLS5) of putative flavonol synthase genes from Vitis vinifera cv. Cabernet Sauvignon. The mRNA of five FLSs accumulated in flower buds and flowers, while the mRNA of FLS2, FLS4, and FLS5 accumulated in small berry skins and then decreased toward veraison. At the ripening stage, the mRNA of only FLS4 and FLS5 accumulated again. This change in mRNA accumulation did not contradict the flavonol accumulation in the berry skins. Shading of the berries completely inhibited the increase in flavonol content and mRNA accumulation of FLS4, but did not affect the mRNA accumulation of FLS5. The effects of light and plant hormones on flavonol accumulation were different from those on anthocyanin accumulation. Thus flavonol biosynthesis appears to be under a different control system from that of anthocyanin biosynthesis.     

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.     

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.