From grape berries to wine: population dynamics of cultivable yeasts associated to “Nero di Troia” autochthonous grape cultivar

The aim of this work was to study the biodiversity of yeasts isolated from the autochthonous grape variety called “Uva di Troia”, monitoring the natural diversity from the grape berries to wine during a vintage. Grapes were collected in vineyards from two different geographical areas and spontaneous alcoholic fermentations (AFs) were performed. Different restriction profiles of ITS–5.8S rDNA region, corresponding to Saccharomyces cerevisiae, Issatchenkia orientalis, Metschnikowia pulcherrima, Hanseniaspora uvarum, Candida zemplinina, Issatchenkia terricola, Kluyveromyces thermotolerans, Torulaspora delbrueckii, Metschnikowia chrysoperlae, Pichia fermentans, Hanseniaspora opuntiae and Hanseniaspora guilliermondii, were observed. The yeast occurrences varied significantly from both grape berries and grape juices, depending on the sampling location. Furthermore, samples collected at the end of AF revealed the great predominance of Saccharomyces cerevisiae, with a high intraspecific biodiversity. This is the first report on the population dynamics of ‘cultivable’ microbiota diversity of “Uva di Troia” cultivar from the grape to the corresponding wine (“Nero di Troia”), and more general for Southern Italian oenological productions, allowing us to provide the basis for an improved management of wine yeasts (with both non-Saccharomyces and Saccharomyces) for the production of typical wines with desired unique traits. A certain geographical-dependent variability has been reported, suggesting the need of local based formulation for autochthonous starter cultures, especially in the proportion of the different species/strains in the design of mixed microbial preparations.     

Reaction mechanism of grape catechol oxidase—a kinetic study

The initial velocity of the oxidation of 4-methylcatechol by grape catechol oxidase was determined. The kinetic analysis indicates that first there is random binding of an oxygen and a 4-methylcatechol molecule to the enzyme. Then one product molecule is released prior to the binding of second 4-methylcatechol molecule which is followed by the release of a second product molecule. The true K_m values were determined; they were found to be 0.5 mM for oxygen and 17 mM for 4-methylcatechol.     

Is vegetative area,photosynthesis, or grape C uploading involved in the climate change-related grape sugar/anthocyanin decoupling in Tempranillo?

Foreseen climate change is expected to impact on grape composition, both sugar and pigment content. We tested the hypothesis that interactions between main factors associated with climate change (elevated CO₂, elevated temperature, and water deficit) decouple sugars and anthocyanins, and explored the possible involvement of vegetative area, photosynthesis, and grape C uploading on the decoupling. Tempranillo grapevine fruit-bearing cuttings were exposed to CO₂ (700 vs. 400 ppm), temperature (ambient vs. +?4 °C), and irrigation levels (partial vs. full) in temperature-gradient greenhouses. In a search for mechanistic insights into the underlying processes, experiments 1 and 2 were designed to maximize photosynthesis and enlarge leaf area range among treatments, whereas plant growth was manipulated in order to deliberately down-regulate photosynthesis and control vegetative area in experiments 3 and 4. Towards this aim, treatments were applied either from fruit set to maturity with free vegetation and fully irrigated or at 5–8% of pot capacity (experiments 1 and 2), or from veraison to maturity with controlled vegetation and fully irrigated or at 40% of pot capacity (experiments 3 and 4). Modification of air ¹³C isotopic composition under elevated CO₂ enabled the further characterization of whole C fixation period and C partitioning to grapes. Increases of the grape sugars-to-anthocyanins ratio were highly and positively correlated with photosynthesis and grape ¹³C labeling, but not with vegetative area. Evidence is presented for photosynthesis, from fruit set to veraison, and grape C uploading, from veraison to maturity, as key processes involved in the establishment and development, respectively, of the grape sugars to anthocyanins decoupling.     

α-glucosidase from grape berries: Partial purification and characterization

-Glucosidase (-D-glucoside glucohydrolase EC 3.2.1.20) was purified approximately 30-fold from grape berries (Vitis vinifera var. Riesling). Besides maltose the enzyme preparation hydrolyzes to a lesser extent maltotriose, isomaltose, and starch. It has a pH optimum of 5.1 and a molecular weight of about 100,000. Tris, glycerol, several mono-and disaccharides were tested as inhibitors. The kinetic behavior of ribose, fructose, cellobiose, sucrose, turanose, methylglucopyranoside, Tris, and glycerol was fully investigated. The inhibition studies suggest a Ping-Pong mechanism, with the second substrate concentration being constant, that can be treated as a Uni Bi system. The purified enzyme is stable when stored frozen at-20° C. The grape-berry -glucosidase may exist as multiple forms (pI 7.2 and 8.2 respectively), and it does not require ions for its activity.This work was supported by the consiglio Nazionale delle Ricerche, Roma, Italy     

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.     

Light quality affectsin Vitro growth of grape ‘Teleki 5BB’

We investigated the effects of various qualities of light-emitting diode (LED) light sources on the growth and carbohydrate accumulation of grape rootstock ‘Teleki 5BB’ culturedin vitro. Shoot fresh and dry weights and net photosynthetic rates were increased when plants were exposed to fluorescent lighting (control), red light, or a mixture of blue plus red, but were unaffected by blueonly radiation. Shoot elongation was significantly stimulated by red light whereas the combination of blue and red light was associated with the shortest shoots. However, the number of nodes did not differ among these treatments. Under monochromic blue or red light, sugar content and starch accumulation increased under the mixed-radiation treatment.     

Effects of PEG-induced water stress onin vitro hardening of ‘Valiant’ grape

Polyethylene glycol was added to the rooting medium ofmicropropagated grape shoots to induce water stress. At the end of the rooting stage, plantlets treated with 2% polyethylene glycol were compared with untreated control plantlets and greenhouse-grown plants. Leaves of treated plantlets had the highest deposition of epicuticular wax, followed by those of the greenhouse and control. Stomatal index did not vary among treatments. However, differences in leaf epidermal cell configuration were observed among treatments. The morphological changes of treated plantlets, including substantial deposition of epicuticular wax and modified leaf surface anatomy were associated with increasedex vitro survival after four weeks in the greenhouse.     

Leaf anatomy and water loss of in vitro PEG-treated ‘Valiant’ grape

Polyethylene glycol was used to induce water stress of micropropagated Valiant grape. Reduced growth and slow rooting were observed in treated plantlets with 2, 4 and 6% polyethylene glycol as compared to control plantlets with no polyethylene glycol in the rooting medium. At high concentrations of 4 and 6%, leaves exhibited wilting and necrosis. At the 2% level, plantlets recovered and grew satisfactorily. Detached leaves of treated plantlets with 2% polyethylene glycol lost less water than controls when exposed to low humidity for 4 hours. Leaf anatomy of plantlets treated with 2% polyethylene glycol, control (in vitro plantlets) and greenhouse-grown plants were compared under light microscopy. Leaves from control plantlets contained larger mesophyll cells, lacked normal palisade layer formation, had greater intercellular pore spaces and fewer chloroplasts. Leaves from polyethylene glycol-treated plantlets, however, had smaller mesophyll cells, a more defined palisade layer, reduced intercellular pore space and the greatest number of chloroplasts. These results suggest that an osmoticum such as polyethylene glycol may be used to induce more normal leaf anatomy and reduced water loss in micropropagated Valiant grapes.Abbreviations BA 6-benzylaminopurine - FAA formalin-acetol-alcohol - MS Murashige & Skoog (1962) medium - MW molecular weight - NAA napthaleneacetic acid - PEG polyethylene glycol - TBA tertiary butyl alcohol     

The dynamics of grape leafhopper Empoasca vitis Göthe populations in southern Switzerland and the implications for habitat management

The life cycle of Empoasca vitis and its most important parasitoid Anagrus atomus in southern Switzerland can be divided into three phases. In phase I the adults of E. vitis move from overwintering sites, i.e. primarily conifers, over deciduous plants into the vineyards. The parasitoid A. atomus on the other hand, appears to overwinter in leafhopper eggs, mainly on roses and blackberries. It subsequently completes one generation in leafhopper eggs primarily on blackberries and hazelnuts, before it attacks E. vitis eggs in the vineyards. For phase II, a population model with time‐varying age structures and stochastic properties was constructed for E. vitis. For this purpose a time‐varying distributed delay model with attrition was constructed. Given the calibrated initial density of overwintering females, the model predicts an unacceptable number of E. vitis for the growing season. However, if egg parasitism of A. atomus and Stethynium triclavatum is introduced into the model as an external variable E. vitis densities are predicted which are economically irrelevant. Inphase III E. vitis adults leave the vineyards for the overwintering sites. The model shows the importance of the parasitoid A. atomus during phase II. A. atomus should be favoured by surrounding the vineyards with host plants carrying leafhoppers’ eggs. Consequently, habitat management measures for E. vitis control could be applied to the surroundings of the vineyards.     

Study on russet-related enzymatic activity and gene expression in ‘Shine Muscat’ grape treated with GA3 and CPPU

Physiological (metabolite analysis) and molecular (gene expression) approaches were used to understand the mechanism underlying russet formation in response to the application of GA₃ and CPPU (Forchlorfenuron) in a Japanese table grape cultivar ‘Shine Muscat’. Several different concentrations of GA₃ and GA₃?+?CPPU [25?mg?L^?1 GA₃ (A), 25?mg?L^?1 GA₃?+?5?mg?L^?1 CPPU (B), 25?mg?L^?1 GA₃?+?10?mg?L^?1 CPPU (C), and 25?mg?L^?1 GA₃?+?15?mg?L^?1 CPPU (D)] were applied to grape berry clusters at two weeks after flowering (WAF). No russet was observed on the berries treated with the ‘C’ combination. Lower levels of phenylalanine ammonia-lyase (PAL) activity was observed in the treated samples, relative to the untreated material. Reduced peroxide (POD) activity was also observed in response to different treatments, while the expression of Peroxidase 17 and Phenylalanine ammonia-lyase G1 genes mirrored lignin content. Increased activity of 4-coenzyme A ligase (4CL) may contribute to decreasing the level of russet and help to improve grape berry quality.     

Exploring the biodiversity of two groups of Oenococcus oeni isolated from grape musts and wines: Are they equally diverse?

One hundred and four Oenococcus oeni isolates were characterised by the carbohydrate fermentation (CH) profile and DNA fingerprinting. Forty-four isolates came from grape must, and 60 from wines sampled at the end of alcoholic fermentation or during malolactic fermentation. The grape must isolates fermented more CH than the wine isolates. In genotypical terms, no clear boundary between grape must and wine isolates was found. Diversities were deduced by considering the isolates of grape must and of wine separately and jointly. By considering only CH fermentation abilities, the group of grape must isolates gave higher diversity index (DI_CH) values than those isolated from wine; i.e., these isolates were metabolically more diverse. The contrary occurred when the DNA fingerprints were used to calculate DI_RAPD-VNTR: wine isolates were genotypically more diverse than grape must ones. With a polyphasic approach, which considered metabolic and genotypic data, the diversity index of both isolate groups (from grape must and wine) was the same, 0.993, which was slightly lower than that calculated from all the isolates (0.997).     

Effects of the supplementation of distillers’ grape residues on ruminal degradability,whole tract digestibility and nitrogen metabolism in sheep

ABSTRACT

Five ruminally and duodenally cannulated Kazakh male lambs (30 ± 2.75 kg) maintained singly in a metabolic cage were used in a 5 × 5 Latin square experiment to investigate the effect of supplementing a ration with five different levels of distillers’ grape residue (DGR) on ruminal degradability, whole tract digestibility and nitrogen (N) metabolism of growing lambs. The rations were isoenergetic and isonitrogenous and contained 0, 3.85, 7.70, 11.55 and 15.41% DGR (DM basis). Each experimental period lasted for 18 d: 10 d for adaptation to the dietary treatment and 8 d for faecal, urinary, ruminal and duodenal digesta sample collections. The outflow rate of ruminal digesta increased (p = 0.032) linearly with the increased level of dietary neutral detergent fibre content, caused by the supplementation of DGR. As a result, the effective degradability of dry matter and crude protein decreased significantly with the treatments. Although the dietary intake of N, duodenal flow of total N, and the endogenous N at the duodenum were not affected by experimental treatments, N fractions in the digesta were altered. Ruminal microbial N decreased (p < 0.01) linearly; in contrast, ruminal un-degradable protein increased linearly (p < 0.01) in response to the increased addition of DGR. Although there was no significant difference in faecal N among treatments, N retention was increased linearly (p = 0.014), owing to the remarkable reduction (p = 0.016) of urinary N excretion with an increasing level of DGR. The results indicate that the DGR has some potential benefits of increasing the supply of bypass protein and of improving the utilisation efficiency of N for sheep. Therefore, the supplementation of DGR in ruminant feeding is recommended at levels not exceeding 10% of the diet.     

Why are grape/fresh wine anthocyanins so simple and why is it that red wine color lasts so long?

Vitis vinifera red berries are characterized by anthocyanins whose chemical structures are among the simplest encountered in higher plants. On the contrary, many plants, including orchids, petunias, red cabbage, elderberries, potatoes for instance, have developed very complicated anthocyanins featuring side-chains at the available positions of the aglycone skeleton. Such pigments were shown to possess bio-physico-chemical properties not to be seen with the grape common anthocyanins. Among beverages (water, tea, beer, wine, coffee, juices, milk), red wine is the only one whose organoleptic properties improve with time and this is called ageing. The grape/fresh red wine pigments, after a few months, disappear from the wine giving birth to new pigments resulting from the wine spontaneous chemistry allowing it to remain red for many years. What are the wine pigments and why are they so stable is the purpose of this mini-review. The structural simplicity of grape anthocyanins and the long lasting colour of red wine is another French paradox; we call it French paradox II.     

Genetic linkage map of the interspecific grape rootstock cross Ramsey (Vitis champinii) × Riparia Gloire (Vitis riparia)

The first genetic linkage map of grape derived from rootstock parents was constructed using 188 progeny from a cross of Ramsey (Vitis champinii) × Riparia Gloire (V. riparia). Of 354 simple sequence repeat markers tested, 205 were polymorphic for at least one parent, and 57.6% were fully informative. Maps of Ramsey, Riparia Gloire, and the F1 population were created using JoinMap software, following a pseudotestcross strategy. The set of 205 SSRs allowed for the identification of all 19 Vitis linkage groups (2n=38), with a total combined map length of 1,304.7 cM, averaging 6.8 cM between markers. The maternal map consists of 172 markers aligned into 19 linkage groups (1,244.9 cM) while 126 markers on the paternal map cover 18 linkage groups (1,095.5 cM). The expected genome coverage is over 92%. Segregation distortion occurred in the Ramsey, Riparia Gloire, and consensus maps for 10, 13, and 16% of the markers, respectively. These distorted markers clustered primarily on the linkage groups 3, 5, 14 and 17. No genome-wide difference in recombination rate was observed between Ramsey and Riparia Gloire based on 315 common marker intervals. Fifty-four new Vitis-EST-derived SSR markers were mapped, and were distributed evenly across the genome on 16 of the 19 linkage groups. These dense linkage maps of two phenotypically diverse North American Vitis species are valuable tools for studying the genetics of many rootstock traits including nematode resistance, lime and salt tolerance, and ability to induce vigor.