Proteomic analysis of grape berry skin responding to sunlight exclusion

The most obvious effect of sunlight exclusion from grape clusters is the inhibition of anthocyanin biosynthesis in the berry skin so that no color develops. Two-dimensional gel electrophoresis coupled with mass spectrometry was used to characterize the proteins isolated from berry skins that developed under sunlight exclusion versus those from sunlight-exposed berries. Among more than 1500 spots resolved in stained gels, the accumulation patterns of 96 spots differed significantly between sunlight-excluded berry skin and that of sunlight-exposed control berries. Seventy-two proteins, including 35 down-regulated and 37 up-regulated proteins, were identified and categorized. Proteins involved in photosynthesis and secondary metabolism, especially UDP-glucose:flavonoid 3-O-glucosyltransferase (UFGT), the key step for anthocyanin biosynthesis in grape berry skin, were accumulated less in the absence of sunlight. Several isoforms of heat shock proteins were also down-regulated. The proteins that were over-accumulated in sunlight-excluded berry skin were more often related to energy production, glycolysis, the tricarboxylic-acid cycle, protein synthesis and biogenesis of cellular components. Their putative role is discussed in terms of their relevance to sunlight exclusion processes.     

A DIGE-based quantitative proteomic analysis of grape berry flesh development and ripening reveals key events in sugar and organic acid metabolism

Grapevine (Vitis vinifera L.) is an economically important fruit crop. Quality-determining grape components, such as sugars, acids, flavours, anthocyanins, tannins, etc., are accumulated during the different grape berry development stages. Thus, correlating the proteomic profiles with the biochemical and physiological changes occurring in grape is of paramount importance to advance the understanding of the berry development and ripening processes. Here, the developmental analysis of V. vinifera cv. Muscat Hamburg berries is reported at protein level, from fruit set to full ripening. A top-down proteomic approach based on differential in-gel electrophoresis (DIGE) followed by tandem mass spectrometry led to identification and quantification of 156 and 61 differentially expressed proteins in green and ripening phases, respectively. Two key points in development, with respect to changes in protein level, were detected: end of green development and beginning of ripening. The profiles of carbohydrate metabolism enzymes were consistent with a net conversion of sucrose to malate during green development. Pyrophosphate-dependent phosphofructokinase is likely to play a key role to allow an unrestricted carbon flow. The well-known change of imported sucrose fate at the beginning of ripening from accumulation of organic acid (malate) to hexoses (glucose and fructose) was well correlated with a switch in abundance between sucrose synthase and soluble acid invertase. The role of the identified proteins is discussed in relation to their biological function, grape berry development, and to quality traits. Another DIGE experiment comparing fully ripe berries from two vintages showed very few spots changing, thus indicating that protein changes detected throughout development are specific.     

Proteomic Analysis of Sauvignon Blanc Grape Skin,Pulp and Seed and Relative Quantification of Pathogenesis-Related Proteins

Thaumatin-like proteins (TLPs) and chitinases are the main constituents of so-called protein hazes which can form in finished white wine and which is a great concern of winemakers. These soluble pathogenesis-related (PR) proteins are extracted from grape berries. However, their distribution in different grape tissues is not well documented. In this study, proteins were first separately extracted from the skin, pulp and seed of Sauvignon Blanc grapes, followed by trypsin digestion and analysis by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Proteins identified included 75 proteins from Sauvignon Blanc grape skin, 63 from grape pulp and 35 from grape seed, mostly functionally classified as associated with metabolism and energy. Some were present exclusively in specific grape tissues; for example, proteins involved in photosynthesis were only detected in grape skin and proteins found in alcoholic fermentation were only detected in grape pulp. Moreover, proteins identified in grape seed were less diverse than those identified in grape skin and pulp. TLPs and chitinases were identified in both Sauvignon Blanc grape skin and pulp, but not in the seed. To relatively quantify the PR proteins, the protein extracts of grape tissues were seperated by HPLC first and then analysed by SDS-PAGE. The results showed that the protein fractions eluted at 9.3 min and 19.2 min under the chromatographic conditions of this study confirmed that these corresponded to TLPs and chitinases seperately. Thus, the relative quantification of TLPs and chitinases in protein extracts was carried out by comparing the area of corresponding peaks against the area of a thamautin standard. The results presented in this study clearly demonstrated the distribution of haze-forming PR proteins in grape berries, and the relative quantification of TLPs and chitinases could be applied in fast tracking of changes in PR proteins during grape growth and determination of PR proteins in berries at harvest.     

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.     

Proteomic analysis of β-1,3-glucanase in grape berry tissues

Grape berries are considered recalcitrant materials in proteomic analysis, because berry tissues contain large amounts of secondary metabolites, especially phenolic compounds, which severely interfere with protein extraction and electrophoresis separation. We report hereby a PVPP/TCA-based protein extraction protocol for grape berries. Phenolic compounds in berry extracts were removed with repeated PVPP cleanups, and proteins were recovered with TCA precipitation. Protein resolution in 2-D gels was gradually improved with the increase of PVPP cleanup steps. By the protocol, about 760 protein spots of berry tissues were clearly resolved in 2-D gels with CBB staining. This protocol was also used to analyze β-1,3-glucanase (EC 3.2.1.39) in berry tissues. An anti-synthetic peptide antibody was prepared against 15 amino acid sequence residing on the surface of β-1,3-glucanase molecule. It detected two major spots in 2-D blots of berry extracts. The spots were identified by MALDI-TOF analysis as β-1,3-glucanase. The present study validates that β-1,3-glucanase is present in higher abundance in berry skins than in pulps, and in red berries than in white berries. Therefore, β-1,3-glucanase displays a tissue-specific expression. The preferential accumulation of β-1,3-glucanase in skins may be relevant to berry ripening.     

Berry morphology and composition in irrigated and non-irrigated grapevine (Vitis vinifera L.)

The present study was carried out in a 5-year-old vineyard (Vitis vinifera L., cv. Aglianico) located in Southern Italy. Half of the plants (IRR) were fully irrigated, whereas the other half were not irrigated (NIRR). In both of the treatments, plant water status, gas exchange, photosynthetic efficiency and productive performance were determined. The arid conditions resulted in significant decreases in stem water potential in NIRR (minimum values of -1.34 and -1.52 MPa in IRR and NIRR, respectively). The values of yield per plant, cluster weight and total berry weight were significantly higher in IRR. Grape berries were separated into four weight classes, and morphometric and microscopic analyses were carried out to measure and calculate berry skin characteristics. Irrigation determined a marked shift toward heavier (+23% in the class ≥ 1.25 g) and bigger (336.35 mm3 vs 299.15 mm3) berries, and induced significant changes in other morphometric berry parameters. No differences among berry weight classes and irrigation treatments were observed for berry skin thickness. In all of the berry weight classes, total anthocyanins extracted from berry skins were significantly higher in NIRR than in IRR (12301.53 and 9585.52 mg kg?1 fresh berry skin, respectively), and appeared to be positively related to berry weight, whereas total flavonols were not significantly different between the two treatments. Qualitative changes in the levels of single anthocyanin and flavonol compounds were detected between IRR and NIRR. In addition, iron, copper and zinc, whose high concentration can negatively affect wine quality, were significantly higher in the IRR treatment. The results highlighted that the absence of irrigation did not determine decreases in grape quality. Such data can be of primary importance in environments where water availability is by far the most important limiting factor for plant growth.     

An in vivo experimental system to study sugar phloem unloading in ripening grape berries during water deficiency stress

An in vivo experimental system-called the 'berry-cup' technique-was developed to study sugar phloem unloading and the accumulation of sugar in ripening grape berries. The berry-cup system consists of a single peeled grape berry immersed in a buffer solution in a cup prepared from a polypropylene syringe. A small cross-incision (2 mm in length) is made on the stylar remnant of a berry during its ripening phase, the skin of the berry then being easily peeled off, exposing the dorsal vascular bundles without damaging either these or the pulp tissue of the berry. The sites of sugar phloem unloading are thus made directly accessible and may be regulated by the buffer solution. In addition, the unloaded photoassimilates are easily transported into the buffer solution in the berry-cup. With the berry-cup technique, it takes 60 min to purge the sugar already present in the apoplast, after which the amount of sugar in the buffer solution is a direct measure of the sugar unloading from the grape berry phloem. The optimum times for sampling were 20 or 30 min, depending on the type of experiment. Sugar phloem unloading was significantly inhibited by the inclusion of either 7.5 mm NaF or 2.5 mm PCMB in the buffer solution. This study indicates that sugar phloem unloading in ripening grape berries is via the apoplastic network and that the process requires the input of energy. The system was shown to be an appropriate experimental system with which to study sugar phloem unloading in ripening grape berries, and was applied successfully to the study of berry sugar unloaded from grapevines subjected to water stress. The results showed that water deficiency inhibits sugar unloading in grape berries.     

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.     

Polysaccharides from grape berry cell walls. Part I: tissue distribution and structural characterization of the pectic polysaccharides

Buffer-soluble arabinogalactan-proteins (AGPs) and pectins from grape berry skin and pulp tissues have been isolated and their structure has been partly determined. Pectic polysaccharides from the cell wall material were solubilized by treating pulp and skin cell walls with homogeneous glycosyl hydrolases. Homogalacturonans, rhamnogalacturonans I (RG-I), and rhamnogalacturonan II (RG-II) of each tissue have been fractionated by high resolution size exclusion chromatography and their relative distribution and major structural features have been determined. It has been shown that pulp tissue contains two-fold more buffer-soluble AGPs and pectins than skin tissue and we have determined that 75% of the grape berry walls originates from the skin tissue. There is three-fold more RG-I and RG-II in skin tissue than in pulp tissue and three-fold more RG-I than RG-II in the grape berry cell walls.

The results of this study have shown that the grape polysaccharide content of a wine is related to the type of tissue used for wine making and to the solubility of the grape polysaccharides and their resistance to fragmentation by grape and yeast glycanases.     

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.     

New Insights into the Ecological Interaction Between Grape Berry Microorganisms and Drosophila Flies During the Development of Sour Rot

In this work, we studied the ecological interactions between grape berry microorganisms and Drosophila sp. flies involved in sour rot disease during grape ripening. After veráison the total microbial counts of grape berries affected by sour rot increased from about 2 log CFU/g of berries to more than 7 log CFU/g. Berry damage provoked a clear shift in yeast diversity from basidiomycetes to ascomycetous fermentative species. The latter were mostly Pichia terricola, Hanseniaspora uvarum, Candida zemplinina, and Zygoascus hellenicus. However, these species were not able to produce the metabolites characteristic of sour rot (gluconic and acetic acids) in inoculated berries. On the contrary, the acetic acid bacteria Gluconacetobacter saccharivorans produced high levels of these acids, mainly when berries were incubated in the presence of the insect Drosophila sp. Sour rot was not observed when grape bunches were physically separated from insects, even when berries were artificially injured. The wounds made in berry skin healed in the absence of insects, thus preventing the development of sour rot. Therefore, in the vineyard, the induction of sour rot depends on the contamination of wounded berries by a microbial consortium--yeasts and acetic acid bacteria--transported by drosophilid insects which disseminate sour rot among damaged berries. In the absence of these insects, plant defense mechanisms are effective and lead to skin healing, preventing disease spread. Thus, we showed that Drosophila sp. act as a vector for microorganisms associated with grape sour rot disease.     

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 Leafhopper-Transmissible DNA Virus with Novel Evolutionary Lineage in the Family Geminiviridae Implicated in Grapevine Redleaf Disease by Next-Generation Sequencing

A graft-transmissible disease displaying red veins, red blotches and total reddening of leaves in red-berried wine grape (Vitis vinifera L.) cultivars was observed in commercial vineyards. Next-generation sequencing technology was used to identify etiological agent(s) associated with this emerging disease, designated as grapevine redleaf disease (GRD). High quality RNA extracted from leaves of grape cultivars Merlot and Cabernet Franc with and without GRD symptoms was used to prepare cDNA libraries. Assembly of highly informative sequence reads generated from Illumina sequencing of cDNA libraries, followed by bioinformatic analyses of sequence contigs resulted in specific identification of taxonomically disparate viruses and viroids in samples with and without GRD symptoms. A single-stranded DNA virus, tentatively named Grapevine redleaf-associated virus (GRLaV), and Grapevine fanleaf virus were detected only in grapevines showing GRD symptoms. In contrast, Grapevine rupestris stem pitting-associated virus, Hop stunt viroid, Grapevine yellow speckle viroid 1, Citrus exocortis viroid and Citrus exocortis Yucatan viroid were present in both symptomatic and non-symptomatic grapevines. GRLaV was transmitted by the Virginia creeper leafhopper (Erythroneura ziczac Walsh) from grapevine-to-grapevine under greenhouse conditions. Molecular and phylogenetic analyses indicated that GRLaV, almost identical to recently reported Grapevine Cabernet Franc-associated virus from New York and Grapevine red blotch-associated virus from California, represents an evolutionarily distinct lineage in the family Geminiviridae with genome characteristics distinct from other leafhopper-transmitted geminiviruses. GRD significantly reduced fruit yield and affected berry quality parameters demonstrating negative impacts of the disease. Higher quantities of carbohydrates were present in symptomatic leaves suggesting their possible role in the expression of redleaf symptoms.     

Impact of leaf removal as a source of stresses on grapevine yields,chemical characteristics,and anthocyanin content in the grapevine variety Babica

Leaf removal (LR) treatments improve the photosynthetic capacity of the remaining leaves and induce flavonoid synthesis as a stress response in the common grapevine (Vitis vinifera L.). However, excessive exposure of grape berries to UV-B radiation as a result of cultural practices in the Mediterranean climate may have negative effects on berry composition. This 2-year study determined the effects of defoliation on the autochthonous red grape variety ‘Babica’ in a Mediterranean climate (wine-growing region Dalmatia, Croatia). Six leaves were removed before flowering (FLR) and at the end of véraison (the onset of grape ripening; VerLR) and were compared to the untreated control. Yield parameters, sugar content, grape must pH, total polyphenols (TP), total anthocyanin (TA) content, and individual anthocyanin compounds were measured in grape skin extracts and wines. However, the greater mean daily temperature during the vegetation period and lesser rainfall before harvest in 2018 increased yield per vine, average cluster weights, density, and total acidity, compared to 2017. Both defoliation treatments significantly reduced TP in grape extracts, but these differences were not observed in wine. Compared to the control (NLR), VerLR treatment significantly reduced TA in grape skin extracts and wine. Significantly lesser TP concentrations, in grape skin extracts and wine, as well as TA were noticed during the 2017 season. VerLR treatment reduced the concentration of nine individual anthocyanins compared to the control in grape skin extracts, while this effect was not observed in wine. Season year was a statistically significant source of variability of the individual anthocyanin contents in wine. Under specific environmental conditions LR can decrease polyphenols, especially anthocyanins, and negatively impact grape and wine quality.