Martine Pradal

Molecular characterization and expression analysis of the Rab GTPase family in Vitis vinifera reveal the specific expression of a VvRabA protein

As a first step to investigate whether Rab GTPases are involved in grape berry development, the Vitis vinifera EST and gene databases were searched for members of the VvRab family. The grapevine genome was found to contain 26 VvRabs that could be distributed into all of the eight groups described in the literature for model plants. Genetic mapping was successfully performed; VvRabs were mostly located on independent chromosomes, apart from eight that were located on the as yet unassigned portions of the genome clustered in the ChrUn Random chromosome. Conserved and divergent regions between VvRab protein sequences were identified. Transcript expression of 11 VvRabs was analysed by real-time quantitative RT-PCR. Except for VvRabA5b, transcript expression was detected, in general, in all the organs investigated, but with different patterns. In grape berries, VvRab transcripts were expressed at all stages of fruit development, with different profiles, except in the case of members of the A family which displayed generally similar patterns. The response to growth regulators in cell cultures was generally specific to each VvRab, with a differential pattern of expression for ethylene, auxin, and abscisic acid according to the VvRab. Interestingly, and unexpectedly considering transcript expression, western blotting using a monoclonal antibody raised against AtRabA5c (ARA4) showed a specific expression in the exocarp of ripe grape berries, in all seven red and white berry varieties tested. By contrast, no expression was detected in any of the other organs or tissues investigated. This paper contains the first description of Rab GTPases in V. vinifera. The involvement of a specific VvRab in grape berry late development and the potential role of this Rab GTPase are discussed in relation to literature data.

Impact of Nutrient Imbalance on Wine Alcoholic Fermentations: Nitrogen Excess Enhances Yeast Cell Death in Lipid-Limited Must

We evaluated the consequences of nutritional imbalances, particularly lipid/nitrogen imbalances, on wine yeast survival during alcoholic fermentation. We report that lipid limitation (ergosterol limitation in our model) led to a rapid loss of viability during the stationary phase of fermentation and that the cell death rate is strongly modulated by nitrogen availability and nature. Yeast survival was reduced in the presence of excess nitrogen in lipid-limited fermentations. The rapidly dying yeast cells in fermentations in high nitrogen and lipid-limited conditions displayed a lower storage of the carbohydrates trehalose and glycogen than observed in nitrogen-limited cells. We studied the cell stress response using HSP12 promoter-driven GFP expression as a marker, and found that lipid limitation triggered a weaker stress response than nitrogen limitation. We used a SCH9-deleted strain to assess the involvement of nitrogen signalling pathways in the triggering of cell death. Deletion of SCH9 increased yeast viability in the presence of excess nitrogen, indicating that a signalling pathway acting through Sch9p is involved in this nitrogen-triggered cell death. We also show that various nitrogen sources, but not histidine or proline, provoked cell death. Our various findings indicate that lipid limitation does not elicit a transcriptional programme that leads to a stress response protecting yeast cells and that nitrogen excess triggers cell death by modulating this stress response, but not through HSP12. These results reveal a possibly negative role of nitrogen in fermentation, with reported effects referring to ergosterol limitation conditions. These effects should be taken into account in the management of alcoholic fermentations.

A ‘fragile cell’ sub‐population revealed during cytometric assessment of Saccharomyces cerevisiae viability in lipid‐limited alcoholic fermentation

Aims:u2002 To show that in anaerobic fermentation with limiting lipid nutrients, cell preparation impacts the viability assessment of yeast cells, and to identify the factors involved.

Relief from nitrogen starvation triggers transient destabilization of glycolytic mRNAs in Saccharomyces cerevisiae cells

Rapid and transient down-regulation of glycolytic mRNAs was observed after replenishment of nitrogen-starved yeast. Glucose sensing, protein elongation, nitrogen metabolism, and TOR signaling are factors affecting glycolytic mRNA stability via carbon/nitrogen cross-talk. Destabilization occurs by the general mRNA decay pathway.

Relief from nitrogen starvation entails quick unexpected down-regulation of glycolytic/lipid metabolism genes in enological Saccharomyces cerevisiae

Nitrogen composition of the grape must has an impact on yeast growth and fermentation kinetics as well as on the organoleptic properties of the final product. In some technological processes, such as white wine/rose winemaking, the yeast-assimilable nitrogen content is sometimes insufficient to cover yeast requirements, which can lead to slow or sluggish fermentations. Growth is nevertheless quickly restored upon relief from nutrient starvation, e.g. through the addition of ammonium nitrogen, allowing fermentation completion. The aim of this study was to determine how nitrogen repletion affected the genomic cell response of a Saccharomyces cerevisiae wine yeast strain, in particular within the first hour after nitrogen addition. We found almost 4800 genes induced or repressed, sometimes within minutes after nutrient changes. Some of the transcriptional responses to nitrogen depended on the TOR pathway, which controls positively ribosomal protein genes, amino acid and purine biosynthesis or amino acid permease genes and negatively stress-response genes, RTG specific TCA cycle genes and NCR sensitive genes. Some unexpected transcriptional responses concerned all the glycolytic genes, the starch and glucose metabolism and citrate-cycle related genes that were down-regulated, as well as genes from the lipid metabolism.Nitrogen composition of the grape must has an impact on yeast growth and fermentation kinetics as well as on the organoleptic properties of the final product. In some technological processes, such as white wine/rosé winemaking, the yeast-assimilable nitrogen content is sometimes insufficient to cover yeast requirements, which can lead to slow or sluggish fermentations. Growth is nevertheless quickly restored upon relief from nutrient starvation, e.g. through the addition of ammonium nitrogen, allowing fermentation completion. The aim of this study was to determine how nitrogen repletion affected the transcriptional response of a Saccharomyces cerevisiae wine yeast strain, in particular within the first hour after nitrogen addition. We found almost 4800 genes induced or repressed, sometimes within minutes after nutrient changes. Some of these responses to nitrogen depended on the TOR pathway, which controls positively ribosomal protein genes, amino acid and purine biosynthesis or amino acid permease genes and negatively stress-response genes, and genes related to the retrograde response (RTG) specific to the tricarboxylic acid (TCA) cycle and nitrogen catabolite repression (NCR). Some unexpected transcriptional responses concerned all the glycolytic genes, carbohydrate metabolism and TCA cycle-related genes that were down-regulated, as well as genes from the lipid metabolism.

Kinetic analysis of yeast-yeast interactions in oenological conditions

Fermentation by microorganisms is a key step in the production of traditional food products such as bread, cheese, beer and wine. In these fermentative ecosystems, microorganisms interact in various ways (competition, predation, commensalism and mutualism). Traditional wine fermentation is a complex microbial process performed by different yeast species which can be classified in two groups: Saccharomyces and non-Saccharomyces species. To better understand the different interactions occurring within wine fermentation, isolated cultures were compared to cultures involving one reference strain of S. cerevisiae species and one strain of five non-Saccharomyces yeast species out of five (Metschnikowia pulcherrima, Metschnikowia fructicola, Torulaspora delbrueckii, Hanseniaspora opuntiae and Hanseniaspora uvarum). In each case, we studied the population dynamics, the resources consumption and the production of metabolites of central carbon metabolism. This deep phenotyping of competition kinetics allowed us to identify the main mechanisms of interaction. T. delbrueckii and S. cerevisiae compete for resources with comparable fitness in our experimental conditions. S. cerevisiae and H. uvarum and H. opuntiae are also competing for resources although both Hanseniaspora strains are characterized by a strong mortality in isolated and mixed fermentations. M. pulcherrima and M. fructicola have a negative interaction with S. cerevisiae, provoking a decrease in viability in co-culture, probably caused by the synthesis of a killer toxin. Overall, this work highlights the interest of measuring the population and metabolites kinetics in order to understand yeast-yeast interactions. These results are a first step for ecological engineering and the intelligent design of optimal multi-starter consortiums using modeling tools.Fermentation by microorganisms is a key step in the production of traditional food products such as bread, cheese, beer and wine. In these fermentative ecosystems, microorganisms interact in various ways, namely competition, predation, commensalism and mutualism. Traditional wine fermentation is a complex microbial process performed by Saccharomyces and non-Saccharomyces (NS) yeast species. To better understand the different interactions occurring within wine fermentation, isolated yeast cultures were compared with mixed co-cultures of one reference strain of S. cerevisiae with one strain of four NS yeast species (Metschnikowia pulcherrima, M. fructicola, Hanseniaspora opuntiae and H. uvarum). In each case, we studied population dynamics, resource consumed and metabolites produced from central carbon metabolism. This phenotyping of competition kinetics allowed us to confirm the main mechanisms of interaction between strains of four NS species. S. cerevisiae competed with H. uvarum and H. opuntiae for resources although both Hanseniaspora species were characterized by a strong mortality either in isolated or mixed fermentations. M. pulcherrima and M. fructicola displayed a negative interaction with the S. cerevisiae strain tested, with a decrease in viability in co-culture, probably due to iron depletion via the production of pulcherriminic acid. Overall, this work highlights the importance of measuring specific cell populations in mixed cultures and their metabolite kinetics to understand yeast-yeast interactions. These results are a first step towards ecological engineering and the rational design of optimal multi-species starter consortia using modeling tools. In particular the originality of this paper is for the first times to highlight the joint-effect of different species population dynamics on glycerol production and also to discuss on the putative role of lipid uptake on the limitation of some non-conventional species growth although interaction processes.