Involvement of ethylene signalling in a non-climacteric fruit: new elements regarding the regulation of ADH expression in grapevine

Although grape berries have been classified as non climacteric fruits, ongoing studies on grape ethylene signalling lead to challenge the role of ethylene in their ripening. One of the significant molecular changes in berries is the up regulation of ADH (alcohol dehydrogenase, EC. 1.1.1.1) enzyme activity at the inception of fruit ripening and of VvADH2 transcript levels. This paper shows that the ethylene signal transduction pathway could be involved in the control of VvADH2 expression in grapevine berries and in cell suspensions. The induction of VvADH2 transcription, either in berries at the inception of ripening or in cell suspensions, was found to be partly inhibited by 1 methylcyclopropene (1 MCP), an inhibitor of ethylene receptors. Treatment of cell suspensions with 2 chloroethylphosphonic acid (2-CEPA), an ethylene releasing compound, also resulted in a significant increase of ADH activity and VvADH2 transcription under anaerobiosis, showing that concomitant ethylene and anaerobic treatments in cell suspensions could result in changes of VvADH2 expression. All these results, associated with the presence in the VvADH2 promoter of regulatory elements for ethylene and anaerobic response, suggest that ethylene transduction pathway and anaerobic stress could be in part involved in the regulation of VvADH2 expression in ripening berries and cell suspensions. These data open new aspects of the expression control of a ripening-related gene in a non climacteric fruit

Yeast cell death caused by nutrient desequilibrium during alcoholic fermentation is impacted by nitrogen sources

Nutrients availability is a key factor for controlling wine alcoholic fermentation. Among them, nitrogen has been identified as an essential parameter, controlling both the fermentation rate and the durationof the fermentation. However, nitrogen is not sufficient to ensure a correct fermentation and other nutrients such as vitamins and lipids, present in lower quantities, are required. Furthermore, we showed in a previous study that an excess of nitrogen combined with a depletion in certain micronutrients can lead to cell death and sluggish or stuck fermentation. In this study, we provide evidence of the mechanism controlling cell death and we show that all the nitrogen sources are not equivalent in the initiation of this phenomenon.Fermentations limited in oleic acid, pantothenic acid and nicotinic acid showed yeast cell death linked to a high nitrogen content. In each case, lowering the nitrogen level restored yeast viability. We evidenced that yeast cell lack of a correct stress response to those micronutrient starvations in presence of high levels of nitrogen. A transcriptional analysis showed a correct stress response suggesting that the lack of resistance originates from a post-transcriptional control mechanism. We then provide evidence that the nitrogen Tor/Sch9 signaling pathway is involved in triggering cell death.Yeast cell viability was then monitored and compared during fermentation starting at different nitrogen levels, with the addition of different nitrogen sources (19 amino acids and NH4+) and two different timing of NH4+ addition. We observed that cell death was triggered with different intensities.Yeast cell death associated to disequilibrium between micronutrients and nitrogen has been evidenced and its implication on fermentations highlighted. We showed a strong impact of both the nature of the nitrogen source and time of addition on yeast cell death and fermentation outcome

Genetic bases of nitrogen requirement in wine yeast assessed trhrough QTL analysis

In grape must, nitrogen content is ofteninsufficient for the completion of alcoholic fermentation by yeast. For Saccharomyces cerevisiae, response to nitrogen deficiency is strain-dependent, some strains being able to complete fermentation despite nitrogen deficiency whereas others are not and result in sluggish or stuck fermentation. Thus, it is of high interest to study the mechanisms behind those different responses and exploit them to improve yeast strain for wine fermentation when nitrogen content is low. Previous study highlighted different genomic regions involved in nitrogen requirement through BSA (Bulk Segregant Analysis), and the contributions of three genes: MDS3, GCN1, and ARG81 have been shown (1). However, many other large genomic regions were also defined for which we could not find evident candidate genes. In addition, BSA did not provide any information on possible interactions between loci. In order to explore further the genetic bases of nitrogen requirement, we applied a QTL analysis to the fermentation rate in nitrogen deficient medium, on a population of 131 individually genotyped segregants obtained from the same cross as (1). The dense genetic map available for the segregant population (3727 markers) enabled us to perform single and multiple map QTL and thus define genomic regions which could be implied in low nitrogen requirement. In order to further validate the impact of candidate genes on the phenotype, alleles were “swapped” by CRISPR-Cas9 technique and phenotype was evaluated in comparison with haploid parent strains.Several regions with high LOD scores were identified, some above the significance threshold, and others below, among which the regions containing the genes identified by (1), probably in relation with the multigenic character of the trait. In the region with the highest LOD score, two candidate genes in relation with nitrogen metabolism (namely, Target of Rapamycin (TOR) pathwayand lifespan regulation) were identified. In addition, in order to reveal possible interaction between genes, strains carrying different combinations of GCN1 and MDS3 parental alleles (implied in TOR pathway) have been evaluated. These constructions confirm their role on the fermentation rate in low-nitrogen conditions and indicate dependence on the genetic background. These results confirm the complexity of mechanisms involved in nitrogen requirement during alcoholic fermentation and will permit to optimise wine yeast strain selection in response to winemaking industry demands

Importance and role of lipids in wine yeast fermentation

This review summarizes the current knowledge on the importance and role of lipids in wine yeast fermentation. Lipids play an important role in membrane structure, adaptation to stress, or as signaling molecules. They are also essential nutrients whose availability can vary depending on winemaking technology, with major effects on yeast alcoholic fermentation. Moreover, lipid supplementation can greatly stimulate the formation of yeast volatile metabolites

A simple FCM method to avoid misinterpretation in Saccharomyces cerevisiae cell cycle assessment between G0 and Sub-G1

Extensively developed for medical and clinical applications, flow cytometry is now being used for diverse applications in food microbiology. Most uses of flow cytometry for yeast cells are derived from methods for mammalian cells, but yeast cells can present specificities that must be taken into account for rigorous analysis of the data output to avoid any misinterpretation. We report an analysis of Saccharomyces cerevisiae cell cycle progression that highlights possible errors. The cell cycle was analyzed using an intercalating fluorochrome to assess cell DNA content. In analyses of yeast cultures, the presence of a sub-G1 peak in the fluorescent signal is often interpreted as a loss of DNA due to its fragmentation associated with apoptosis. However, the cell wall and its stucture may interfere with the fluorescent signal recorded. These observations indicate that misinterpretation of yeast DNA profiles is possible in analyses based on some of the most common probes: cells in G0 appeared to have a lower DNA content and may have been mistaken as a sub-G1 population. However, careful selection of the fluorochrome for DNA quantification allowed a direct discrimination between G0 and G1 yeast cell cycle steps, without additional labeling. We present and discuss results obtained with five current fluorochromes. These observations led us to recommend to use SYTOX Green for cycle analysis of living cells and SYBR Green I for the identification of the apoptosis sub-G1 population identification or the DNA ploidy application

Carbonic Maceration Wines: Characteristics and Winemaking Process

International audienceInvented by Michel Flanzy in 1934, carbonic maceration involves placing the intact grape clusters into a closed tank with a carbon dioxide-rich atmosphere. The berries subsequently undergo an intracellular fermentation without yeast intervention. Complex changes occur during this process which entail the transformation of a small amount of sugar into alcohol (1.5-2% alcohol), the reduction of malic acid content by about half, and the generation of secondary products. Compared with wines produced by conventional techniques, carbonic maceration produces wines of distinctive character of superior quality possessing a harmonious balance. It can be used to generate a wide range of wines (red as well as rosé), to be drunk young or aged. The process is composed of four steps: vatting of intact berries, "maceration-fermentation," pumping off, and pressing, followed by a second fermentation phase. Exchanges and interactions occur between grape berries, the gaseous atmosphere, and the must present at the bottom of the tank during the first step of the carbonic maceration winemaking process. Yeast fermentation starts at this stage, in the liquid phase, and continues throughout the second step as well, with the malolactic fermentation. The specific conditions required for a good handling of carbonic maceration are presented

Clonage et caractérisation de l'expression des gènes CAD et CCR chez le pommier

National audienc

Sampling size for determination of adenine nucleotide contents in mature grape berries: application to reference samples analysis

International audienc

Responses of Saccharomyces cerevisiae to nitrogen starvation in wine alcoholic fermentation.

UMR SPO - Equipe MICROBIONitrogen is an important nutrient in alcoholic fermentation because its starvation affects both fermentation kinetics and the formation of yeast metabolites. In most alcoholic fermentations, yeasts have to ferment in nitrogen-starved conditions, which requires modifications of cell functions to maintain a high sugar flux and enable cell survival for long periods in stressful conditions. In this review, we present an overview of our current understanding of the responses of the wine yeast Saccharomyces cerevisiae to variations of nitrogen availability. Adaptation to nitrogen starvation involves changes in the activity of signaling pathways such as target of rapamycin (TOR) and nitrogen catabolite repression (NCR), which are important for the remodeling of gene expression and the establishment of stress responses. Upon starvation, protein degradation pathways involving autophagy and the proteasome play a major role in nitrogen recycling and the adjustment of cellular activity. Recent progress in the understanding of the role of these mechanisms should enable advances in fermentation management and the design of novel targets for the selection or improvement of yeast strain

Analysis of Vitis vinifera alcohol dehydrogenase promoter regulation

International audienc