Figure 1. SCH9 deletion and GCN4 overexpression modulate growth and metabolite production under winemaking conditions. (A) Cell viability in natural grape juice fermentations of the wild-type C9 strain, mutants sch9Δ and gcn4 Δ, and the GCN4 overexpressing strain, measured as cfu/mL. (B) Survival curve of the aforementioned strains under winemaking conditions. Cell viability at day 8 of panel A was taken as 100% survival. (C) Sugar consumption during fermentations. Ethanol (D), glycerol (E) and acetic acid (F) production at the end of fermentations. Experiments were done in triplicate. The mean and standard deviation are provided. *P < 0.05, **P < 0.01, ***P < 0.005, unpaired t-test, two tailed. From: Sch9p kinase and the Gcn4p transcription factor regulate glycerol production during winemaking FEMS Yeast Res. 2016;17(1). doi:10.1093/femsyr/fow106 FEMS Yeast Res | © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com
Figure 2. Deletion of aquaporin Aqy2p impairs fermentation Figure 2. Deletion of aquaporin Aqy2p impairs fermentation. (A) Viability count of GUT2 and PEX12 deletion strains during fermentations in synthetic must MS75. (B) Same as (A) with aquaporin AQY1 and AQY2 deletion strains. (C) Sugar consumption of the above-mentioned fermentations. (D) Growth curve in YPD of AQY1 and AQY2 deletion strains. Experiments were done in triplicate and a representative curve is shown. (E) Final glycerol production during fermentations. Experiments were done in triplicate. The mean and standard deviation are provided. From: Sch9p kinase and the Gcn4p transcription factor regulate glycerol production during winemaking FEMS Yeast Res. 2016;17(1). doi:10.1093/femsyr/fow106 FEMS Yeast Res | © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com
Figure 3. SCH9 deletion increases the Gpd1p protein levels during winemaking. (A) Wild-type and sch9Δ strains that contained the GFP-tagged version of the Gpd1p protein were grown in synthetic must, and cells were analysed by fluorescence microscopy at 1, 4 and 7 days of growth. (B) The same analysis for a Pex12-GFP strain. (C) Western blot using an anti-GFP antibody of the extracts from the strains described in panel A. The control was a non-tagged wild-type strain. From: Sch9p kinase and the Gcn4p transcription factor regulate glycerol production during winemaking FEMS Yeast Res. 2016;17(1). doi:10.1093/femsyr/fow106 FEMS Yeast Res | © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com
Figure 4. Sch9p is required for stress tolerance in the stationary phase. (A) Survival after the incubation of stationary cultures of mutants sch9Δ and gcn4Δ, and the GCN4 overexpressing strain on YPD at 46°C, 1 M NaCl and 10% ethanol for 1 h. Cells were diluted, plated on YPD plates, and counted before and after stress application. (B) Growth inhibition by hydrogen peroxide on the stationary grown cells by measuring the halo diameter on YPD plates. Experiments were done in triplicate. The mean and standard deviation are provided. From: Sch9p kinase and the Gcn4p transcription factor regulate glycerol production during winemaking FEMS Yeast Res. 2016;17(1). doi:10.1093/femsyr/fow106 FEMS Yeast Res | © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com
Figure 5. SCH9 deletion and GCN4P overexpression had a strong impact on metabolism. (A) PCA of the five samples of the wild-type, SCH9 deletion and GCN4 overexpression strains grown to the stationary phase is synthetic must. (B) Metabolites in the central metabolism (glycolysis and TCA) affected by both genetic manipulations. Green (downward arrow) indicates downregulation and red (upward arrow) indicates upregulation, while grey (cross) indicate the metabolites that remain unchanged. From: Sch9p kinase and the Gcn4p transcription factor regulate glycerol production during winemaking FEMS Yeast Res. 2016;17(1). doi:10.1093/femsyr/fow106 FEMS Yeast Res | © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com
Figure 6. Both SCH9 deletion and GCN4 overexpression control carbon metabolism. (A) Box analysis of the metabolites related to glycerol production in the wild-type, SCH9 deletion and GCN4 overexpression strains. (B) Same for the metabolites devoted to polysaccharide production and trehalose. (C) Glycogen plate assay. Patches of strains of interest were plated and incubated for 2 days. Glycogen was detected by I<sub>2</sub> vapours by only placing I<sub>2</sub> on the plate until colour developed. From: Sch9p kinase and the Gcn4p transcription factor regulate glycerol production during winemaking FEMS Yeast Res. 2016;17(1). doi:10.1093/femsyr/fow106 FEMS Yeast Res | © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com