Sandra Villaume

Drought stress tolerance in grapevine involves activation of polyamine oxidation contributing to improved immune response and low susceptibility to Botrytis cinerea

Environmental factors including drought stress may modulate plant immune responses and resistance to pathogens. However, the relationship between mechanisms of drought tolerance and resistance to pathogens remained unknown. In this study, the effects of drought stress on polyamine (PA) homeostasis and immune responses were investigated in two grapevine genotypes differing in their drought tolerance; Chardonnay (CHR), as sensitive and Meski (MSK), as tolerant. Under drought conditions, MSK plants showed the lowest leaf water loss and reduction of photosynthetic efficiency, and expressed a lower level of NCED2, a gene involved in abscisic acid biosynthesis, compared with CHR plants. The improved drought tolerance in MSK was also coincident with the highest change in free PAs and up-regulation of the genes encoding arginine decarboxylase (ADC), copper amine-oxidase (CuAO), and PA-oxidases (PAO) and their corresponding enzyme activities. MSK plants also accumulated the highest level of amino acids, including Arg, Glu, Gln, Pro, and GABA, emphasizing the participation of PA-related amino acid homeostasis in drought tolerance. Importantly, drought-tolerant plants also exhibited enhanced phytoalexin accumulation and up-regulation of PR genes, especially PR-2 and Chit4c, compared with the sensitive plants. This is consistent with a lower susceptibility of MSK than CHR to Botrytis cinerea. Data suggest a possible connection between water stress tolerance and immune response in grapevine. Pharmacological experiments revealed that under drought conditions CuAO and PAO pathways were involved in the regulation of photosynthetic efficiency, and also of immune response and resistance of grapevine to a subsequent pathogen attack. These results open new views to improve our understanding of crosstalk between drought tolerance mechanisms and immune response.

Kinetics of CO2 Fluxes Outgassing from Champagne Glasses in Tasting Conditions: The Role of Temperature

Measurements of CO(2) fluxes outgassing from a flute poured with a standard Champagne wine initially holding about 11 g L(-1) of dissolved CO(2) were presented, in tasting conditions, all along the first 10 min following the pouring process. Experiments were performed at three sets of temperature, namely, 4 degrees C, 12 degrees C, and 20 degrees C, respectively. It was demonstrated that the lower the champagne temperature, the lower CO(2) volume fluxes outgassing from the flute. Therefore, the lower the champagne temperature, the lower its progressive loss of dissolved CO(2) concentration with time, which constitutes the first analytical proof that low champagne temperatures prolong the drinks chill and helps retains its effervescence. A correlation was also proposed between CO(2) volume fluxes outgassing from the flute poured with champagne and its continuously decreasing dissolved CO(2) concentration. Finally, the contribution of effervescence to the global kinetics of CO(2) release was discussed and modeled by the use of results developed over recent years. The temperature dependence of the champagne viscosity was found to play a major role in the kinetics of CO(2) outgassing from a flute. On the basis of this bubbling model, the theoretical influence of champagne temperature on CO(2) volume fluxes outgassing from a flute was discussed and found to be in quite good accordance with our experimental results.

CO2 Volume Fluxes Outgassing from Champagne Glasses in Tasting Conditions: Flute versus Coupe

Measurements of CO(2) fluxes outgassing from glasses containing a standard Champagne wine initially holding about 11.5 g L(-1) of dissolved CO(2) were presented, in tasting conditions, during the first 10 min following the pouring process. Experiments were performed at room temperature, with a flute and a coupe, respectively. The progressive loss of dissolved CO(2) concentration with time was found to be significantly higher in the coupe than in the flute, which finally constitutes the first analytical proof that the flute prolongs the drinks chill and helps it to retain its effervescence in contrast with the coupe. Moreover, CO(2) volume fluxes outgassing from the coupe were found to be much higher in the coupe than in the flute in the early moments following pouring, whereas this tendency reverses from about 3 min after pouring. Correlations were proposed between CO(2) volume fluxes outgassing from the flute and the coupe and their continuously decreasing dissolved CO(2) concentration. The contribution of effervescence to the global kinetics of CO(2) release was discussed and modeled by use of results developed over recent years. Due to a much shallower liquid level in the coupe, bubbles collapsing at the free surface of the coupe were found to be significantly smaller than those collapsing at the free surface of the flute, and CO(2) volume fluxes released by collapsing bubbles only were found to be approximately 60% smaller in the coupe than in the flute. Finally, the contributions of gas discharge by invisible diffusion through the free surface areas of the flute and coupe were also approached and compared for each type of drinking vessel.

Foaming properties of various Champagne wines depending on several parameters: Grape variety, aging, protein and CO2 content

A comparison of the foaming parameters of various Champagne wines was undergone with two well distinct methods: (i) a classical gas-sparging method providing standardized but artificial effervescence conditions (the so-called Mosalux), and (ii) a computer assisted viewing equipment (CAVE), much closer to the real champagne tasting conditions. The latter one is the only apparatus which enables a thorough descriptive analysis of foam behavior, during the pouring process of a sparkling wine, and from the end of its pouring. Various Champagne wines elaborated from two grape varieties (Chardonnay and Pinot Meunier) and having experienced different aging-periods (15 months and 5 years) were analyzed and compared to a model sparkling wine, elaborated from a model base wine (devoid of grape colloids). The CO(2) and protein content was also investigated to discuss the foaming behavior of these wines. A significant loss of the CO(2) content during aging was observed and might be the reason for the worse foaming properties of the old champagnes, as determined with CAVE. It is worth noting that contradictory foaming parameters were obtained through the Mosalux method, which is indeed more intrusive than the CAVE, and finally far from the real champagne tasting conditions, since it requires filtration and champagne degassing prior experiment.

On the Losses of Dissolved CO2 during Champagne Serving

Pouring champagne into a glass is far from being consequenceless with regard to its dissolved CO(2) concentration. Measurements of losses of dissolved CO(2) during champagne serving were done from a bottled Champagne wine initially holding 11.4 +/- 0.1 g L(-1) of dissolved CO(2). Measurements were done at three champagne temperatures (i.e., 4, 12, and 18 degrees C) and for two different ways of serving (i.e., a champagne-like and a beer-like way of serving). The beer-like way of serving champagne was found to impact its concentration of dissolved CO(2) significantly less. Moreover, the higher the champagne temperature is, the higher its loss of dissolved CO(2) during the pouring process, which finally constitutes the first analytical proof that low temperatures prolong the drinks chill and helps it to retain its effervescence during the pouring process. The diffusion coefficient of CO(2) molecules in champagne and champagne viscosity (both strongly temperature-dependent) are suspected to be the two main parameters responsible for such differences. Besides, a recently developed dynamic-tracking technique using IR thermography was also used in order to visualize the cloud of gaseous CO(2) which flows down from champagne during the pouring process, thus visually confirming the strong influence of champagne temperature on its loss of dissolved CO(2).

One step purification of the grape vacuolar invertase

Invertase is a major protein of grape juice and wine. Accordingly, in order to study the biochemical and structural characteristics of this protein and for a better understanding of its physico-chemical properties, large amounts of the pure protein are needed. A simple method for the purification of the grape vacuolar invertase in a preparative-scale is described in this work. The grape protein was isolated and purified from must by ultrafiltration and anion exchange chromatography. The identification and purity determination of the grape invertase fraction were assessed by SDS-PAGE, and were then confirmed using nanoLC-chip-MS/MS analysis. The laboratory fractionation procedure presented in this work generated large quantities of pure grape vacuolar invertase from must.

Osmotic stress-induced polyamine oxidation mediates defence responses and reduces stress-enhanced grapevine susceptibility to Botrytis cinerea

Abiotic factors inducing osmotic stress can influence the plant immune response and resistance to pathogen infections. In this study, the effect of polyethylene glycol (PEG)- and sucrose-induced osmotic stress on polyamine (PA) homeostasis and the basal immune response in grapevine plantlets before and after Botrytis cinerea infection was determined. Pharmacological approaches were also addressed to assess the contribution of osmotic stress-induced PA oxidation to the regulation of defence responses and the susceptibility of grapevine to B. cinerea. Following osmotic stress or pathogen infection, PA homeostasis was linked to enhanced activity of diamine oxidases (CuAO) and PA oxidases (PAO) and the production of 1,3-diaminopropane. These responses paralleled the accumulation of the main stilbenic phytoalexins, resveratrol and ε-viniferin and upregulation of gene transcripts including STS (a stilbene synthase), PR-2 (a β-1,3-glucanase), PR3-4c (acidic chitinase IV), and PR-5 (a thaumatin-like protein), as well as NCED2 involved in abscisic acid biosynthesis. It was also demonstrated that leaves pre-exposed to osmotic stress and later inoculated with B. cinerea showed enhanced PA accumulation and attenuation of CuAO and PAO activities. This was consistent with the impaired production of phytoalexins and transcript levels of defence- and stress-related genes following infection, and the enhanced susceptibility to B. cinerea. Pharmacological experiments revealed that, under osmotic stress conditions, CuAO and PAO were involved in PA homeostasis and in the regulation of defence responses. Specific inhibition of CuAO and PAO in osmotically stressed leaves strongly attenuated the induction of defence responses triggered by B. cinerea infection and enhanced susceptibility to the pathogen. Taken together, this study reveals a contribution of PA catabolism to the resistance state through modulation of immune response in grapevine following osmotic stress and/or after B. cinerea infection.

Losses of Dissolved CO2 Through the Cork Stopper during Champagne Aging: Toward a Multiparameter Modeling

Measurements of dissolved CO(2) concentrations from Champagne bottles initially holding the same CO(2) level after having been elaborated (close to 11.5 g L(-1)), but having experienced different periods of aging after having been corked with natural cork stoppers, were done. Losses of dissolved CO(2) close to 3.5 g L(-1) experienced by the oldest Champagne samples aged for about 75 months were reported. This very significant loss of dissolved CO(2) was logically interpreted as a consequence of the continuous diffusion of gaseous CO(2) through the pores of the cork stopper. By combining the diffusion principle through a porous medium with Henrys law (which links the solubility of a gas species in a liquid medium with its partial pressure in the vapor phase), a multiparameter model was built that provides the dissolved CO(2) content found in Champagne during its whole aging period. Both Champagne temperature and bottle volume were found to be key parameters with regard to the kinetics of CO(2) losses through the cork.

Quantification of chitinase and thaumatin-like proteins in grape juices and wines.

Chitinases and thaumatin-like proteins are important grape proteins as they have a great influence on wine quality. The quantification of these proteins in grape juices and wines, along with their purification, is therefore crucial to study their intrinsic characteristics and the exact role they play in wines. The main isoforms of these two proteins from Chardonnay grape juice were thus purified by liquid chromatography. Two fast protein liquid chromatography (FLPC) steps allowed the fractionation and purification of the juice proteins, using cation exchange and hydrophobic interaction media. A further high-performance liquid chromatography (HPLC) step was used to achieve higher purity levels. Fraction assessment was achieved by mass spectrometry. Fraction purity was determined by HPLC to detect the presence of protein contaminants, and by nuclear magnetic resonance (NMR) spectroscopy to detect the presence of organic contaminants. Once pure fractions of lyophilized chitinase and thaumatin-like protein were obtained, ultra-HPLC (UHPLC) and enzyme-linked immunosorbent assay (ELISA) calibration curves were constructed. The quantification of these proteins in different grape juice and wine samples was thus achieved for the first time with both techniques through comparison with the purified protein calibration curve. UHPLC and ELISA showed very consistent results (less than 16% deviation for both proteins) and either could be considered to provide an accurate and reliable quantification of proteins in the oenology field.

Review of preparative and analytical procedures for the study of proteins in grape juice and wine.

Proteins have a great influence on wine quality as they exhibit a various range of properties. In fact, they are involved among others in white wine turbidity, organoleptic characteristics and foam formation in sparkling wines. These compounds could also be of major interest for varietal differentiation, regarding wine authentication and traceability issues. To provide a better understanding of the role played by these biomolecules in wine processing and explore their potential applications, there is a manifest need for the quantification and characterization of each individual one in terms of sequence, structure and intrinsic and functional properties. We thus present an overview of preparative and analytical methods for the study of proteins in grape juices and wines, from routine techniques to dedicated methodologies. They include sample preparation with chromatographic methods for the purification and identification of proteins, quantification protocols and characterization procedures such as electrophoretic techniques, immunological methods, sequencing, mass spectrometry, physico-chemical and structural analyses, and so on. We expose advantages and limits of each technique and focus on the different but complementary information they can provide. Despite the past years advances in the field proteins identification, the elucidation of the full protein profile for grape juices and wines remains strenuous. Their interactions with other wine compounds make the challenge even harder. We therefore emphasize the requirement of the techniques to be refined and suggest the developments to be expected.