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

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

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

Proteome changes in leaves from grapevine (Vitis vinifera L.) transformed for alcohol dehydrogenase activity

International audienceA proteomic approach has been used to study changes in leaf protein content from plants transformed for alcohol dehydrogenase (ADH) activity. Individual quantitative analysis of 190-436 spots separated by two-dimensional electrophoresis was performed, and spots displaying significant quantitative changes between control (C), sense (S), and antisense (R) transformants were selected using Student's t test. Of the 14 spots selected and further analyzed after trypsic digestion, 9 could be identified by MS analysis and 5 by LC-MS/MS. Identified proteins had mainly a chloroplastic origin: four rubisco large subunits, one rubisco binding protein, two glutamine synthetases, one elongation factor Tu, one ATP synthase beta subunit, and one plastidic aldolase. Proteins with other localization were also identified, such as a UDP-glucose pyrophosphorylase, a mitochondrial aminomethyltransferase, a linalool synthase, which comigrated with the protein identified as elongation factor Tu, an enolase comigrating with a glyceraldehyde 3-phosphate dehydrogenase, and a mixture of eight proteins among which were a dehydroascorbate reductase, a chalcone isomerase, and a rubisco activase. The results emphasize the changes in carbon metabolism-associated proteins linked to the alteration in ADH activity of grapevine transformant leave

La mortalité des levures en fermentation alcoolique:: le rôle clé des limitations en micronutriments et de l'azote

La mortalité des levures en fermentation alcoolique:. le rôle clé des limitations en micronutriments et de l'azot

Berry size variability in Vitis vinifera L

International audienc

Soil reflectance and colouring of grape. Vine red light excitation could be decisive for grape berry quality

Various conditions of vine « solarisation » with sun light reflecting clothes partially colored in blue, green or red, were performed in field conditions during 1998 and 1999 years.The experiments were conducted both on table grapes (Muscat de Hambourg , Italia and Danuta) and on wine grapes varieties (Carignane, Mourvèdre, Shiraz, Muscat à petits grains). The « solarisation » clothes were set up at the cluster closure stage and removed at the harvest time. Effect of various experimental conditions on changes in situ berry colour was examined. The chromatic parameters using the Lab and LCH conventional systems were weekly measured on representative berry samples, and levels of significance of the obtained differences were determined by a variance analysis. Differences in the colouring dynamics and in the values of the chromatic parameters at harvest were statistically significant, in particular in function of the importance of the reflecting area set up per vinestock and also in function of the color of the reflecting cloth used. The results were compared to those obtained from various other analyses : reflectance spectra and composition of berries at harvest, tastings of the table grapes. As a main result, the red colour with a maximum of reflectance at 680 nm appeared as the more effective in improving quality of the berries. These results demonstrate the importance of the reflectance properties of the neighbours of the vine, in particular those of the soil of the parcel, on the composition and the quality of the grape. Moreover, they underline that these properties should be taken in account to explain some misunderstood terroir effects. Finally, they strongly suggest that the excitation of vine photoreceptor systems, specially with the clear red light, could be decisive in the signaling and regulatory mechanisms leading to an optimal ripeness of the grape

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

Determination de l'ethanol endogene des baies de raisin par analyse de l'atmosphere des enceintes experimentales

National audienc

Synthesis of volatile compounds is enhanced in leaves of trangenic grapevine overexpressing ADH

National audienceNADP/NAD-dependent oxidoreductases are involved in the biosynthesis of volatiles, specially in the interconversion of volatile alcohols and aldehydes. Multiple examples exist in the literature regarding alcohol dehydrogenases for conversion of short-chain aldehydes to alcohol, some terpene alcohols to aldehydes and also for modifications in fruit aroma C6 compound profile by genetic engineering. In the present study, we investigated the effect of alcohol dehydrogenase overexpression on the volatile compounds in grapevine leaves. Compared to control, grapevine plants overexpressing ADH displayed no changes in free volatile compounds, but showed significantly enhanced amounts of glycosidically volatile compounds such as C13 norisoprenoids, monoterpenes and shikimates. Additionally the highest level of ABA was found in transformed grapevine leaves. The results presented here showed for the first time that some links could exist between ADH activity and the synthesis ofseveral glycosidically volatile compounds. C13 norisoprenoids and ABA synthesis involve a carotene dioxygenase tlle activity of which might be enhanced in transformed plants