Proton Nuclear Magnetic Resonance-Spectroscopic Discrimination of Wines Reflects Genetic Homology of Several Different Grape (V. vinifera L.) Cultivars

Background and Aims

Proton nuclear magnetic resonance spectroscopy coupled multivariate analysis (¹H NMR-PCA/PLS-DA) is an important tool for the discrimination of wine products. Although ¹H NMR has been shown to discriminate wines of different cultivars, a grape genetic component of the discrimination has been inferred only from discrimination of cultivars of undefined genetic homology and in the presence of many confounding environmental factors. We aimed to confirm the influence of grape genotypes in the absence of those factors.

Methods and Results

We applied ¹H NMR-PCA/PLS-DA and hierarchical cluster analysis (HCA) to wines from five, variously genetically-related grapevine (V. vinifera) cultivars; all grown similarly on the same site and vinified similarly. We also compared the semi-quantitative profiles of the discriminant metabolites of each cultivar with previously reported chemical analyses. The cultivars were clearly distinguishable and there was a general correlation between their grouping and their genetic homology as revealed by recent genomic studies. Between cultivars, the relative amounts of several of the cultivar-related discriminant metabolites conformed closely with reported chemical analyses.

Conclusions

Differences in grape-derived metabolites associated with genetic differences alone are a major source of ¹H NMR-based discrimination of wines and ¹H NMR has the capacity to discriminate between very closely related cultivars.

Significance of the Study

The study confirms that genetic variation among grape cultivars alone can account for the discrimination of wine by ¹H NMR-PCA/PLS and indicates that ¹H NMR spectra of wine of single grape cultivars may in future be used in tandem with hierarchical cluster analysis to elucidate genetic lineages and metabolomic relations of grapevine cultivars. In the absence of genetic information, for example, where predecessor varieties are no longer extant, this may be a particularly useful approach.     

Transport and accumulation of flavonoids in grapevine (Vitis vinifera L.)

Flavonoids are a group of secondary metabolites widely distributed in plants that represent a huge portion of the soluble phenolics present in grapevine (Vitis vinifera L.). These compounds play different physiological roles and are often involved in protection against biotic and abiotic stress. Even if the flavonoid biosynthetic pathways have been largely characterized, the mechanisms of their transport and accumulation in cell wall and vacuole are still not completely understood. This review analyses the known mechanisms of flavonoid uptake and accumulation in grapevine, with reference to the transport models and membrane carrier proteins described in other plant species. The effect of different environmental factors on flavonoid biosynthesis and transporters is also discussed.Key words: ABC proteins, active transport, bilitranslocase, biotic and abiotic stress, flavonoid, secondary metabolites     

Phenolic and heterocyclic metabolite profiles of the grapevine pathogen Eutypa lata

The ascomycete Eutypa lata is the causative agent of eutypa dieback in grapevines, a serious economic problem in major wine grape producing areas. In order to develop a predictive, non-destructive assay for early detection of fungal infection, the phenolic metabolite profiles of 11 strains of E. lata grown on four different artificial growth media were analyzed by HPLC and their variability compared with growth on Cabernet Sauvignon grapevine wood and wood extracts. Six compounds were generally produced in significant amounts, namely eutypinol, eulatachromene, and eutypine and its benzofuran cyclization product, together with siccayne and eulatinol. The two most widely distributed and abundant metabolites were eutypinol and eulatachromene, which were present in 8 of the strains grown on grapewood aqueous extract fortified with sucrose. Metabolite production on grapevine extract was greatly enhanced relative to the artificial media, indicating that this native substrate provides optimal conditions and a more representative profile of the metabolites produced in the natural disease state. The primary metabolites were tested in a grapeleaf disc bioassay to establish their relative toxicity. Neither eutypinol nor siccayne were phytotoxic; eulatachromene, eulatinol, eutypine, and the benzofuran exhibited necrotic effects in the bioassay. The results indicate that eutypa dieback may be caused by several E. lata metabolites rather than a single compound.     

Engineering grapevine for increased resistance to fungal pathogens without compromising wine stability

The vast majority of wine proteins have recently been identified as pathogenesis-related (PR) proteins. During the growing season, these proteins are expressed in developmentally dependent and inducible manners in grapevine leaves and grape berries, in which they are believed to play an important role in protection against fungal pathogens and possibly other stresses. Because of their inherent resistance to proteolytic attack and to the low pH values characteristic of wines, vinification can be seen as a "purification strategy" for grape PR proteins. The inevitable consequent accumulation of these proteins in wines becomes a technological nuisance because they adversely affect the clarity and stability of wines. Genetically modified vines underexpressing PR proteins would certainly lead to stable wines but would increase the plant susceptibility to fungal attack, and the actual trend seems to be in the opposite direction, that is overexpressing these proteins to obtain plants with enhanced resistance to pathogens--a trend that will probably augment problems associated with protein instability in the resulting wines.     

Transporters expressed during grape berry (Vitis vinifera L.) development are associated with an increase in berry size and berry potassium accumulation

Potassium accumulation is essential for grapevine (Vitis vinifera L.) growth and development, but excessive levels in berries at harvest may reduce wine quality particularly for red wines. In addition to decreasing the free acid levels, potassium also combines with tartaric acid to form largely insoluble potassium bitartrate. This precipitates during winemaking and storage, resulting in an increase in wine pH that is associated with negative impacts on wine colour, flavour, and microbiological stability. For these reasons, a better understanding of potassium transport and accumulation within the vine and berries is important for producing fruit with improved winemaking characteristics. Here two genes encoding KUP/KT/HAK-type potassium transporters that are expressed in grape berries are described. Their function as potassium transporters was demonstrated by complementation of an Escherichia coli mutant. The two transporters are expressed most highly in the berry skin during the first phase of berry development (pre-veraison), with similar patterns in two grapevine varieties. The timing and location of expression of these transporters are consistent with an involvement in potassium accumulation in grape berries.     

Interactions between Brettanomyces bruxellensis and other yeast species during the initial stages of winemaking

AIMS: Wine is the product of complex interactions between yeasts and bacteria in grape must. Amongst yeast populations, two groups can be distinguished. The first, named non-Saccharomyces (NS), colonizes, with many other micro-organisms, the surface of grape berries. In the past, NS yeasts were primarily considered as spoilage micro-organisms. However, recent studies have established a positive contribution of certain NS yeasts to wine quality. Amongst the group of NS yeasts, Brettanomyces bruxellensis, which is not prevalent on wine grapes, plays an important part in the evolution of wine aroma. Some of their secondary metabolites, namely volatile phenols, are responsible for wine spoilage. The other group contributing to wine aroma, which is also the main agent of alcoholic fermentation (AF), is composed of Saccharomyces species. The fermenting must is a complex microbial ecosystem where numerous yeast strains grow and die according to their adaptation to the medium. Yeast-yeast interactions occur during winemaking right from the onset of AF. The aim of this study was to describe the interactions between B. bruxellensis, other NS and Saccharomyces cerevisiae during laboratory and practical scale winemaking. METHODS AND RESULTS: Molecular methods such as internal transcribed spacer-restriction fragment length polymorphism and polymerase chain reaction and denaturing gradient gel electrophoresis were used in laboratory scale experiments and cellar observations. The influence of different oenological practices, like the level of sulphiting at harvest time, cold maceration preceding AF, addition of commercial active dry yeasts on B. bruxellensis and other yeast interactions and their evolution during the initial stages of winemaking have been studied. Brettanomyces bruxellensis was the most adapted NS yeast at the beginning of AF, and towards the end of AF it appeared to be more resistant than S. cerevisiae to the conditions of increased alcohol and sugar limitation. CONCLUSIONS: Among all NS yeast species, B. bruxellensis is better adapted than other wild yeasts to resist in must and during AF. Moreover, B. bruxellensis appeared to be more tolerant to ethanol stress than S. cerevisiae and after AF B. bruxellensis was the main yeast species in wine. SIGNIFICANCE AND IMPACT OF THE STUDY: Brettanomyces bruxellensis interacts with other yeast species and adapts to the wine medium as the dominant yeast species at the end of AF. Contamination of B. bruxellensis might take place at the beginning of malolactic fermentation, which is a critical stage in winemaking.     

Wine biotechnology in South Africa: towards a systems approach to wine science

The wine industry in South Africa is over three centuries old and over the last decade has reemerged as a significant competitor in world wine markets. The Institute for Wine Biotechnology (IWBT) was established in partnership with the Department of Viticulture and Oenology at Stellenbosch University to foster basic fundamental research in the wine sciences leading to applications in the broader wine and grapevine industries. This review focuses on the different research programmes of the Institute (grapevine, yeast and bacteria biotechnology programmes, and chemical-analytical research), commercialisation activities (SunBio) and new initiatives to integrate the various research disciplines. An important focus of future research is the Wine Science Research Niche Area programme, which connects the different research thrusts of the IWBT and of several research partners in viticulture, oenology, food science and chemistry. This 'Functional Wine-omics' programme uses a systems biology approach to wine-related organisms. The data generated within the programme will be integrated with other data sets from viticulture, oenology, analytical chemistry and the sensory sciences through chemometrics and other statistical tools. The aim of the programme is to model aspects of the wine making process, from the vineyard to the finished product.     

Engineering the Saccharomyces cerevisiae isoprenoid pathway for de novo production of aromatic monoterpenes in wine

Grape musts contain a variety of terpenols that significantly affect wine aroma. The amounts of these metabolites depend on the grape variety, and many cultivars are non-aromatic. Yeasts like Saccharomyces cerevisiae cannot produce and excrete monoterpenes efficiently, mainly due to their lack of monoterpene synthases. By metabolic engineering we have modified the isoprenoid biosynthesis pathway in a wine yeast strain of S. cerevisiae expressing the Clarkia breweri S-linalool synthase gene. Under microvinification conditions, without compromising other desirable and useful fermentative traits, the recombinant yeast efficiently excreted linalool to levels exceeding the threshold of human perception. Bearing in mind the possibility of (co-)expressing other genes that encode enzymes leading to the production of various aroma compounds and the feasibility of controlling the levels of their expression, the potential of this achievement for future genetic manipulation of wine varietal aroma or for use in other alcoholic drinks seems very promising.     

Differential Behaviour of Mycelial Growth of Several Botrytis cinerea Strains on either Patchoulol- or Globulol-amended Media

Botrydial and dihidrobotrydial are two characteristic metabolites of the phytopathogenic fungus Botrytis cinerea , which are involved in the development of necrotic lesions on grapevine and tobacco. Patchoulol and globulol, two natural products which are analogues to precursors of botrydial and dihidrobotrydial, were tested on 10 B. cinerea strains which were isolated from different hosts and varied in aggressiveness on grapevine leaves. Mycelial growth of all strains was prevented when they were grown on either patchoulol- or globulol-amended malt agar media (200  μ g/ μ l). Each strain displayed a specific response pattern to those products, according to the high variability previously described for this species. Furthermore, strains were different from one another with regard to their level of aggressiveness against leaves detached from sherry grapevine vineyards.     

Development of a transient expression system in grapevine via agro-infiltration

Transient expression of genes using Agrobacterium is a powerful tool for the analysis of gene function in plants. We have developed this method for the analysis of genes involved in disease resistance in grapevine leaves. Our research showed that the quality of the plant material, the plant genotype used for agro-infiltration and the presence of additional virulence factors (carried on plasmid pCH32) in the Agrobacterium strain are all important factors for success of the procedure. After optimising these factors, we consistently achieve sufficient acceptable levels of expression of the markers beta-glucuronidase (GUS) and green fluorescent protein (GFP) using vacuum infiltration of grapevine leaves from plants grown in vitro. We used this procedure to investigate the proposed role of stilbenes in defense against grapevine downy mildew (Plasmopara viticola) by transiently overexpressing stilbene synthase in grapevine leaves, before infection with P. viticola. We found that agro-infiltration itself induces the synthesis of stilbenes in grapevine leaves, thus preventing us to test the effect of the overexpression of stilbene synthase in defense. However, our results revealed that agro-infiltration before P. viticola inoculation had an effect on the development of the infection. Further research is required to show whether stilbenes or some other factor are the causal agent restricting pathogen development. The method described here provides and excellent tool to exploit at the many grapevine genomic resources now available, and will contribute to a better understanding of many areas of grapevine biology.     

Nematicidal potential of Brassicaceae

Brassicaceae Burnett (syn. Cruciferae A. L. de Jussieu) include many important economic plants used as edibile or ornamental. They are commonly known as the “mustard” plant family due to the sharp, potent flavour of their main metabolites, the glucosinolates (GLSs) which contain sulfur. Glucosinolates coexist in vivo with glycosylated thioglucosidases, myrosinase(s), responsible of their hydrolysis with the production of bioactive cognate isothiocyanates (ITC). GLSs and ITCs function as defence bioactive metabolites against plant pathogens, insects and herbivores. The present review paper focus on GLSs role as bionematicides. The current knowledge on the efficacy of these phytochemicals against the most common phytoparasitic nematodes affecting crops of agriculture importance such as tomato, potato and grapevine is reported. Data from our ongoing research on the in vitro biocidal activity of glucosinolate extracts, and their main components, against the virus-vector nematode Xiphinema index Thorne & Allen and the carrot cyst nematode Heterodera carotae Jones are also described.     

Future scenarios for viticultural zoning in Europe: ensemble projections and uncertainties

Optimum climate conditions for grapevine growth are limited geographically and may be further challenged by a changing climate. Due to the importance of the winemaking sector in Europe, the assessment of future scenarios for European viticulture is of foremost relevance. A 16-member ensemble of model transient experiments (generated by the ENSEMBLES project) under a greenhouse gas emission scenario and for two future periods (2011–2040 and 2041–2070) is used in assessing climate change projections for six viticultural zoning indices. After model data calibration/validation using an observational gridded daily dataset, changes in their ensemble means and inter-annual variability are discussed, also taking into account the model uncertainties. Over southern Europe, the projected warming combined with severe dryness in the growing season is expected to have detrimental impacts on the grapevine development and wine quality, requiring measures to cope with heat and water stress. Furthermore, the expected warming and the maintenance of moderately wet growing seasons over most of the central European winemaking regions may require a selection of new grapevine varieties, as well as an enhancement of pest/disease control. New winemaking regions may arise over northern Europe and high altitude areas, when considering climatic factors only. An enhanced inter-annual variability is also projected over most of Europe. All these future changes pose new challenges for the European winemaking sector.     

Impact of <Emphasis Type="Italic">Quillaja saponaria</Emphasis> saponins on grapevine ecosystem organisms

The control of grapevine pathogens is a rising concern in Vitis vinifera culture. The current international trend is toward banning chemicals that are highly toxic to the environment and human workers, and adopting tighter regulations. We evaluated the impact of saponins on three kinds of organisms found in grapevine culture. The ectoparasitic nematode Xiphinema index, the parasitic fungus Botrytis cinerea and various yeast strains representative of the must fermentation population were incubated on synthetic media supplemented with variable concentrations of Quillaja saponaria saponins. Saponins induced reduction in the growth of B. cinerea and showed nematicide effects on X. index. The control of X. index and Botrytis cinerea is discussed in the context of the potential use of these chemicals as environmentally-friendly grapevine treatments. With Saccharomyces cerevisiae and other yeasts, saponins showed higher toxicity against S. cerevisiae strains isolated from wine or palm wine whereas laboratory strains or strains isolated from oak exhibited better resistance. This indicates that Q. saponaria saponins effects against yeast microflora should be assessed in the field before they can be considered an environmentally-safe new molecule against B. cinerea and X. index.     

The impact of endogenous estradiol metabolites on carcinogenesis

The available literature on estrogen metabolism and estrogen metabolites involved in carcinogenesis is reviewed. Endogenous estradiol metabolism leads to metabolic products that can have various, and, to some extent, contrary, biologic effects. Thus, there are numerous research findings on the stimulation and inhibition of cancer growth by estrogen metabolites. Furthermore, there are indications that, in certain types of cancer, the production of growth-stimulating estradiol metabolites is increased. There are also reports on substances that can influence estradiol metabolism. So far, only a few estradiol metabolites have been examined with respect to their influence on the development and growth of cancer. It is presumed that other metabolites can also intervene directly or indirectly in the cancer process, but there is a great lack of research in this area. An understanding of the actions of estradiol metabolites may open up new avenues for the therapy of malignant diseases. Although little is known about the biologic effects of most of the estradiol metabolites, the reported actions of certain estradiol metabolites already justify clinical investigations on their possible beneficial uses in tumor therapy.