## 3 Results and Discussion
### Adaptive Evolution-Mediated Changes in Glycerol Concentrations
Therefore, the next question that we asked was whether an enhanced tolerance of high osmotic stress as well as the increased levels of glycerol synthesis exhibited by the evolved strain might be linked to any changes in osmoresisponse gene expression. To answer this question, all investigated samples were collected after the end of the evolutionary experiment and were analyzed for their glycerol biosynthesis-related gene expressions. Among the osmoresisponse genes that are responsible for glycerol production and whose expressions are partially Hog1 dependent are two paralogs of NAD+dependent glycerol-3-phosphate dehydrogenase (GPDs), that is _GPD1_ and _GPD2_[40; 41; 42], as well as two paralogs of specific phosphatases (GPPs), that is _GPP1_ and _GPP2_[41]. The action of GPDs leads to the reduction of the glycolytic intermediate di-hydroxyl-acetone phosphate (DHAP) to glycerol-3-phosphate (G3P) [42; 43; 44], which in turn is dephosphorylated by GPPs [41]. However, semi-quantitative RT-PCR analysis showed only  
Figure 3: **(A)** Concentrations of glycerol [g/L] after the end of fermentation. Grape juice containing 18% sugars (10.2 g/L glucose and 7.7 g/L fructose) or 30% sugars (15.82 g/L glucose; 13.55 g/L fructose) were used as fermentation media. Data are presented as the means \(\pm\) SD; \(n\) = 3. Significant differences are marked by \(\star\) (at \(p\) < 0.05). 1 gen. –1 st generation (ancestral strains); YPD-YPD-evolved derivatives (control); KCl_a–1.25 M KCl-evolved derivatives; KCl_b–1.5 M KCl-evolved derivatives. **(B)** Semi-quantitative PCR analysis of _Hog1_, _GPD1_, _GPD2_, _GPP1_, and _GPP2_ expression levels determined for ancestral and evolved yeast strains 1, 2, and 3. Actin was used as an internal control. Representative gels are shown. **1**, **2**, and **3**–ancestral strains 1, 2, and 3, respectively; 1_YPD, 2_YPD, 3_YPD-YPD-evolved derivatives of strains 1, 2, and 3 (control), respectively; 1_KCl_a, 2_KCl_a, and 3_KCl_a-1.25 M KCl-evolved derivatives of strains 1, 2, and 3, respectively; 1_KCl_b, 2_KCl_b, and 3_KCl_b-1.5 M KCl-evolved derivatives of strains 1, 2, and 3, respectively. slight increases in _GPD1_, _GPP1_, and _GPP2_ expression levels in the case of the KCl-evolved strain 3 (3_KCl_a strain), when compared to its ancestral strain (Figure 3B). Nevertheless, it has been discussed whether all four genes played equally important roles in osmoadaptation. For example, [PERSON] et al. [41], who revised data concerning the osmoadaptation mechanisms of yeast cells, reported that such a successful adaptation is conferred by the upregulation of only _GPD1_ and _GPP2_[41]. In support of this, there are data showing that expression of their paralogs, _GPD2_[45] and _GPP1_[43], respectively, are also influenced by other stresses, such as hypoxia [43; 45]. In addition, [PERSON] et al. [46] showed that a major role in glycerol production is played by GPD1, which is only partially controlled by the HOG1 pathway, while the role of _GPD2_ during anaerobic fermentation was found to be of little significance [46]. Therefore, the enhanced levels of _GPD1_ and _GPP2_ as well as _GPP1_ transcripts noticed in the 3_YPD_KCl_a (Figure 3B) might result not only from hyperosmolarity but also from hypoxic stress, which is associated with the fermentation process. In addition, such an observation might confirm the data obtained by [PERSON] et al. [47], who showed that yeast coped with stressful changing environments by cross-protection, which is a situation when one environmental stressor protects cells against a second stressor [48]. Indeed, recent studies on _S. cerevisiae_ adaptations have shown enhanced stress tolerance after long-term exposure to glycerol [49] even though glycerol does not cause osmotic stress [19]. One of these mechanisms that has been recognized as a physiological adaptation by yeast cells to cope with changing environments is an increase in genome-scale gene expression for short durations [47], which can also be mediated by duplicated genes [49].