Reciprocity between abscisic acid and ethylene at the onset of berry ripening and after harvest

Background  

The ripening of grape berry is generally regulated by abscisic acid (ABA), and has no relationship with ethylene function. However, functional interaction and synergism between ABA and ethylene during the beginning of grape berry ripening (véraison) has been found recently.     

A carotenoid cleavage dioxygenase from Vitis vinifera L.: functional characterization and expression during grape berry development in relation to C13-norisoprenoid accumulation

A potential Carotenoid Cleavage Dioxygenase (CCD) gene was identified among a Vitis vinifera L. EST collection and a full-length cDNA (VvCCD1) was isolated. Recombinant expression of VvCCD1 confirmed that the gene encoded a functional CCD. Experimental evidence was obtained that VvCCD1 cleaves zeaxanthin symmetrically yielding 3-hydroxy-beta-ionone, a C(13)-norisoprenoidic compound, and a C(14)-dialdehyde. Expression of the gene was studied by real-time PCR at different developmental stages of grape berries from Muscat of Alexandria and Shiraz cultivars. A significant induction of the gene expression approaching véraison was observed in both cultivars. In parallel, the C(13)-norisoprenoid level increased from véraison to maturity in both cultivars.     

cDNA microarray analysis of developing grape (Vitis vinifera cv. Shiraz) berry skin

Microarray analysis of Vitis vinifera cv. Shiraz developing berries has revealed the expression patterns of several categories of genes. Microarray slides were constructed from 4,608 PCR-amplified cDNA clones derived from a ripening grape berry cDNA library. The mRNA expression levels of the genes represented by these cDNAs were measured in flowers, week 2 post-flowering whole berries, week 5, week 8, week 10 (véraison, green berries), week 12 and week 13 berry skin. In addition, a comparison of RNA expression in pigmented and unpigmented berry skin at véraison (week 10) was undertaken. Image and statistical analysis revealed four sets of genes with distinctive and similar expression profiles over the course of berry development. The first set was composed of genes which had maximum RNA expression in flowers, followed by a steady decrease in expression. The most prominent group within this set were genes which have a role in photosynthesis. The second set of cDNAs was dominated by genes involved in flavonoid biosynthesis and had a peak of expression week 2 post-flowering. The data indicate co-ordinate regulation of flavonoid biosynthetic genes which code for the enzymes 4-coumarate-CoA ligase, chalcone synthase, chalcone isomerase, flavonone hydroxylase, anthocyanidin reductase and cytochrome b5. The third set of cDNAs exhibited maximum expression week 5 post-flowering, midway between flowering and véraison, a period of rapid berry growth. This set of cDNAs is dominated by genes which code for structural cell wall proteins. The fourth set of genes was dramatically up-regulated at véraison and remained up-regulated until 13 weeks post-flowering. This set of genes was composed of a diverse range of genes, a reflection of the complexity of ripening, most with no known function.     

A grape berry (Vitis vinifera L.) cation/proton antiporter is associated with berry ripening

We have cloned and characterized VvNHX1, a gene encoding a vacuolar cation/H(+) antiporter from Vitis vinifera cv. Cabernet Sauvignon. VvNHX1 belongs to the vacuolar NHX protein family and showed high similarity to other known vacuolar antiporters. The expression of VvNHX1 partially complements the salt- and hygromycin-sensitive phenotypes of an ena1-4 nhx1 yeast strain. Immunoblots of vacuoles of yeast expressing a VvNHX1, together with the expression of a VvNHX1-GFP (green fluorescent protein) chimera demonstrated that VvNHX1 localized to the vacuoles. VvNHX1 displayed low affinity K(+)/H(+) and Na(+)/H(+) exchange activities (12.8 and 40.2 mM, respectively). The high levels of expression of VvNHX1 during the véraison and post-véraison stages would indicate that the increase in vacuolar K(+) accumulation, mediated by VvNHX1, is needed for vacuolar expansion. This process, together with the rapid accumulation of reducing sugars, would drive water uptake to the berry and the concomitant berry size increase, typical of the post-véraison stage of growth.     

Proteomic analysis of grape berry cell cultures reveals that developmentally regulated ripening related processes can be studied using cultured cells

Background

This work describes a proteomics profiling method, optimized and applied to berry cell suspensions to evaluate organ-specific cultures as a platform to study grape berry ripening. Variations in berry ripening within a cluster(s) on a vine and in a vineyard are a major impediment towards complete understanding of the functional processes that control ripening, specifically when a characterized and homogenous sample is required. Berry cell suspensions could overcome some of these problems, but their suitability as a model system for berry development and ripening needs to be established first.

Methodology/Principal Findings

In this study we report on the proteomic evaluation of the cytosolic proteins obtained from synchronized cell suspension cultures that were established from callus lines originating from green, véraison and ripe Vitis vinifera berry explants. The proteins were separated using liquid phase IEF in a Microrotofor cell and SDS PAGE. This method proved superior to gel-based 2DE. Principal component analysis confirmed that biological and technical repeats grouped tightly and importantly, showed that the proteomes of berry cultures originating from the different growth/ripening stages were distinct. A total of twenty six common bands were selected after band matching between different growth stages and twenty two of these bands were positively identified. Thirty two % of the identified proteins are currently annotated as hypothetical. The differential expression profile of the identified proteins, when compared with published literature on grape berry ripening, suggested common trends in terms of relative abundance in the different developmental stages between real berries and cell suspensions.

Conclusions

The advantages of having suspension cultures that accurately mimic specific developmental stages are profound and could significantly contribute to the study of the intricate regulatory and signaling networks responsible for berry development and ripening.     

Damage Patterns caused by Lygocoris spinolae (Hemiptera: Miridae) on ‘Campbell Early’ Grapes

The phenology and damage patterns of Lygocoris spinolae (Meyer-Dür) on ‘Campbell Early’ grape were examined in different grape development stage of inflorescence formation, blooming, and berry set. Nymphs fed on newly unfolding leaves of shoot tip before inflorescence formation. As the inflorescences were clearly visible, nymphs on leaves moved to flower clusters. Sting spots with brown color occurred at sucking sites of leaves, then expanded to hole as the leaves developed, and resulted in leaf malformation. The nymphs on flower clusters sucked developing florets, which induced the drying of florets followed by defoliation. After flower caps had fallen, L. spinolae fed on young berries causing blackening of berry skin around the sting. The blackening of berry skin changed to corky-scarred tissue as the berry developed. L. spinolae feeding significantly reduced the number of berries per cluster in all grape stages. A few berries became corky-scarred or shot berries when fed at inflorescence formation period. Feeding at blooming caused shot berries with low rate of corky-scarred berry. Most of the berries were corky-scarred or became shot berries when fed at berry set. Feeding by adults caused the same damage patterns as nymphs. Heavy damages were observed where L. spinolae were introduced after inflorescence formation: reduced number of berries per cluster by 35.0 to 99.1% at blooming and by 80.0% at berry setting depending on L. spinolae densities.     

Cloning and Expression of a Hexose Transporter Gene Expressed during the Ripening of Grape Berry

The ripening of grape (Vitis vinifera L.) is characterized by massive sugar import into the berries. The events triggering this process and the pathways of assimilate transport are still poorly known. A genomic clone Vvht1 (Vitis vinifera hexose transporter1) and the corresponding cDNA encoding a hexose transporter whose expression is induced during berry ripening have been isolated. Vvht1 is expressed mainly in the berries, with a first peak of expression at anthesis, and a second peak about 5 weeks after véraison (a viniculture term for the inception of ripening). Vvht is strictly conserved between two grape cultivars (Pinot Noir and Ugni-Blanc). The organization of the Vvht1 genomic sequence is homologous to that of the Arabidopsis hexose transporter, but differs strongly from that of the Chlorella kessleri hexose transporter genes. The Vvht1 promoter sequence contains several potential regulating cis elements, including ethylene-, abscisic acid-, and sugar-responsive boxes. Comparison of the Vvht1 promoter with the promoter of grape alcohol dehydrogenase, which is expressed at the same time during ripening, also allowed the identification of a 15-bp consensus sequence, which suggests a possible co-regulation of the expression of these genes. The expression of Vvht1 during ripening indicates that sucrose is at least partially cleaved before uptake into the flesh cells.     

Impact of diurnal temperature variation on grape berry development, proanthocyanidin accumulation, and the expression of flavonoid pathway genes

Little is known about the impact of temperature on proanthocyanidin (PA) accumulation in grape skins, despite its significance in berry composition and wine quality. Field-grown grapes (cv. Merlot) were cooled during the day or heated at night by +/-8 °C, from fruit set to véraison in three seasons, to determine the effect of temperature on PA accumulation. Total PA content per berry varied only in one year, when PA content was highest in heated berries (1.46 mg berry(-1)) and lowest in cooled berries (0.97 mg berry(-1)). In two years, cooling berries resulted in a significant increase in the proportion of (-)-epigallocatechin as an extension subunit. In the third year, rates of berry development, PA accumulation, and the expression levels of several genes involved in flavonoid biosynthesis were assessed. Heating and cooling berries altered the initial rates of PA accumulation, which was correlated strongly with the expression of core genes in the flavonoid pathway. Both heating and cooling altered the rate of berry growth and coloration, and the expression of several structural genes within the flavonoid pathway.     

Proteomic analysis of berry-sizing effect of GA3 on seedless Vitis vinifera L

Gibberellin (GA) is widely used in the table grape and raisin industries to enlarge the berries of seedless varieties. However, the mechanism underlying its berry‐sizing effect is poorly understood. In this study, clusters of Centennial Seedless (Vitis vinifera L.) were treated with 30 ppm GA₃ on day 12 after flowering, and berries were sampled at development stages I, II and III for proteomic analysis. Among the 1479 proteins detected on 2‐DE maps, 19, 70 and 69 spots in stages I, II and III, respectively, showed an at least twofold difference in volume between treatments and controls. Of these, 125 proteins were successfully identified and assigned to eight functional groups, chief among them are metabolism and energy, stress response, expression regulation and cytoskeleton proteins. Stress‐response proteins were predominantly down‐regulated in GA₃‐treated berries in stages I and II, and significantly up‐regulated in stage III. Up‐regulation of cytoskeleton, cell‐wall modification and other important proteins was found in the two latter stages of berry development. Our proteomic results and subsequent validation revealed, for the first time, the role of redox homeostasis in GA₃‐induced berry enlargement and markedly remodeled cellular protein expression in treated berries.     

Expression and In Situ Localization of Two Major PR Proteins of Grapevine Berries during Development and after UV-C Exposition

In grapevine Vitis vinifera L. cv Pinot noir, the Pathogenesis-Related (PR) proteins CHI4D and TL3 are among the most abundant extractable PR proteins of ripe berries and accumulate during berry ripening from véraison until full maturation. Evidence was supplied in favor of the involvement of these two protein families in plant defense mechanisms and plant development. In order to better understand CHI4D and TL3 function in grapevine, we analyzed their temporal and spatial pattern of expression during maturation and after an abiotic stress (UV-C) by in situ hybridization (ISH) and immunohistolocalization. In ripening berries, CHI4D and TL3 genes were mainly expressed in the exocarp and around vascular bundles of the mesocarp. In UV-C exposed berries, CHI4D and TL3 gene expression was strongly induced before véraison. Corresponding proteins localized in the exocarp and, to a lesser extent, around vascular bundles of the mesocarp. The spatial and temporal accumulation of the two PR proteins during berry maturation and after an abiotic stress is discussed in relation to their putative roles in plant defense.     

Role of ABA and Gibberellin A<Subscript>3</Subscript> on gene expression pattern of sugar transporters and invertases in <Emphasis Type="Italic">Vitis vinifera</Emphasis> cv. Malbec during berry ripening

Sugars are key constituents that affect quality of grape berries, and consequently the grape metabolic profile relevant to wine’s industry. However, enzymes and transporter genes expression involved in sugar transport at different phenological stages are scarcely studied. In addition, little is known about the role of the plant hormones ABA and Gibberellin (GA₃) as endogenous regulators, over the expression pattern of the sugars transporters genes in grapevine. The aim of this study was to analyze the expression pattern of the most relevant sugar transporters and invertases in leaves and berries of grapevine plants cv. Malbec during berry ripening stages and its shift after ABA and GA₃ sprays. In leaves, VvHT1 was the sugar transporter highly expressed, whereas VvHT6 was the most abundant in berries throughout berry ripening. Moreover, VvSUC12 and VvSUC27 were expressed at veraison greater in leaves than in berries, suggesting an active phloem loading at the onset of ripening. Applications of ABA and GA₃ enhanced the expression of VvSUC12 and VvSUC27 in pre-veraison leaves. Furthermore, hormones increased the expression of VvHT2, VvHT3 and VvHT6 in berries at different stages of ripening favoring sugar unloading from phloem. In conclusion, ABA and GA₃ are involved in the long-distance sugar transport from leaves to berries in Vitis vinifera L. cv. Malbec, and their exogenous application could be a suitable strategy to improve the process.