## 3 Results and Discussion
### KCl-ALE-Based Improvement of Wine Yeasts' Tolerance to Osmotic Stress and Glycogen Accumulation  
After the end of the ALE experiment, we assessed an effect of 2M KCl-induced osmotic stress on the growth phenotype of the analyzed strains by using a spot test assay, which is a standard method to evaluate the growth phenotype [35; 36; 37]. We showed that KCl-evolved derivatives of strains 2 and 3 (2_KCl_a, 2_KCl_b as well as 3_KCl_a, and 3_KCl_b, respectively) significantly increased their fitness to high osmotic pressure. Interestingly, this effect was also generational dependent because enhanced tolerance to high osmotic stress was also observed for two derivatives of strains 2 and 3 that were cultured for 250 generations under standard conditions (2_YPD and 3_YPD, respectively) (Figure 2A). In contrast to strains 2 and 3, evolved derivatives of strain 1 showed only slight increases in tolerance to high osmotic stress, when compared to the corresponding ancestral strain (Figure 2A)  
Figure 2: (**A**) Analysis of the effect of KCl-induced osmotic stress on the growth phenotype of the analyzed strains by spot test assays. Yeast cells were grown on solid YPD medium (control) or on YPD medium supplemented with 2 M KCl (osmotic stress conditions). Representative photographs are shown. (**B**) Iodine-staining of glycogen stones (indicated by reddish-brown coloration) of yeast colonies. Ancestral and evolved derivatives of strains 1, 2, and 3 were grown for 48, 72, and 96 h in standard solid YPD medium, YPD medium supplemented with 1.5 M KCl, or YPlu containing 30% glucose. The staining reactions of the colonies were recorded for 1 min after adding the iodine solution. Control-yeast colonies not treated with iodine solution. 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-125 M KCl-evolved derivatives of strains 1, 2, and 3, respectively; 1_KCL_b, 2_KCL_b, and 3_KCL_b-15 M KCl-evolved derivatives of strains 1, 2, and 3, respectively.  
Similarly, adaptive evolution under osmotic stress conditions had an impact on strains 2 and 3 with respect to the ability for glycogen accumulation. This effect was observed for 2_KCL_a, 3_KCL_a, and 3_KCL_b strains throughout the 96 h of growth under control conditions and in the presence of 1.5 M KCl as was indicated by the reddish-brown coloration of yeast colonies after iodine staining (Figure 2B). Although the impact of glycogen on the internal osmotic pressure of the cell is low, this macromolecule plays a crucial role in cells' survival during prolonged nutrient deprivation. Therefore, yeast cells with an increased ability for glycogen accumulation show enhanced fitness and vitality [20; 38; 39]. Indeed, cells of _Sacchaormyces_ accumulate storage carbohydrates very efficiently, as glycogen along with trehalose can reach 25% dry weight [8]. From the winemaking point of view, yeast cells overproducing glycogen are desirable, because they display improved performance upon inoculation into grape juice [32] and are more tolerant to different enological-related stresses. For example, it has been shown that yeast strains overexpressing _GSY2_, encoding glycogen synthase, exhibited enhanced viability under both laboratory growth and fermentation conditions in synthetic and natural musts. In particular, they showed increased resistance to glucose deprivation conditions [20]. It should be mentioned that we observed enhanced glycogen accumulation in all evolved derivatives of strain 2 under high glucose concentration conditions, when compared to its corresponding ancestral strain. Although there were no differences in glycogen accumulation over 48 h, significant increases in its content were observed at 72 and 96 h of growth (Figure 2B). Perhaps, changes in glycogen accumulation, mediated by adaptive evolution, might be explained by the fact that glycogen metabolism is strongly affected by a wide variety of cellular processes, including osmotic stress [39].