Yeast strains origin, availability are described in Table 1. Two different strains of each species were studied. For S. cerevisiae, T73 model wine strain (Querol et al., 1994; Lopes et al., 2010) and the commercial wine strain Fermol Cryophile FCry (AEB Group); selected as adapted to low temperature (Gamero et al., 2013) were chosen. The 108 and Chr 16.2 strains isolated from natural environment were used as representatives of S. paradoxus. For S. uvarum, the 12600 and BMV58 strains isolated from wine in Spain were studied. BMV58 was commercialized (Lallemand Inc) because of its high glycerol production and good fermentative properties (patent ES2330709 B1). For S. kudriavzevii species, IFO1802 (type strain), and the CR85 wild strain isolated in Spain (Dušková et al., 2015b) were used. The S. cerevisiae BY4741Δhog1Δstl1 (Pérez-Torrado et al., 2009) was used as a laboratory strain for the expression of STL1 genes and comparison of the function of their products under hyperosmotic-stress conditions.

Yeast cells were maintained and grown in YPD medium (2% glucose, 2% Bacto peptone, and 1% Yeast extract) or SC-Ura medium (YNB 0.67%, glucose 2%, Drop-out –Ura 1.92 g/l (Formedium)) at 28°C for the S. cerevisiae and S. paradoxus species and 25°C for S. kudriavzevii and S. uvarum species.

The wine fermentations were performed in 250 ml bottles filled with 200 ml of MS300 synthetic must (100 g/L glucose, 100 g/L fructose, 6 g/L citric acid, 6 g/L malic acid, mineral salts, vitamins, anaerobic growth factors, 300 mg/L assimilable nitrogen) simulating standard grape juice (Bely et al., 2003) at 12°C with agitation (150 rpm) in triplicate. Overnight precultures were inoculated at 5.0 × 10⁶ cells/ml density determined by measuring OD₆₀₀. To study the expression of genes related to glycerol balance under hyperosmotic stress, the cells from exponentially growing precultures were washed with water and transferred to YP (2% Bacto peptone and 1% yeast extract) with 2% glucose or 2% mannitol as a source of carbon, to the same medium supplemented with 1 M sorbitol (hyperosmotic stress), which is not assimilable for any of the species studied, or to H₂O (hypoosmotic stress). This experiment was performed in 2 l flasks filled with 400 ml of media in triplicate at 25°C and 150 rpm.

The tolerance to hyperosmotic stress was evaluated by drop tests. Yeasts were grown overnight in YPD or SC-ura medium, then cultures were diluted to OD₆₀₀ = 0.2 and cells were allowed to grow in the same media until OD₆₀₀ = 1. Then, serial dilutions of cells were transferred to plates with YPD; YPD + 0.8 M NaCl; YPD + 1.25 M KCl, incubated at 12 and 25°C and evaluated each day. The growth of Saccharomyces species was also compared in plates with SC containing 2 M sorbitol or 2 M KCl and supplemented or not with 1 mM glycerol. To investigate the functional differences of Stl1, the growth of BY4741Δhog1Δstl1 cells transformed with appropriate plasmids was monitored on plates with SC-ura containing 0.7 M sorbitol, which or 0.3 M KCl and with or without 10 mM glycerol. Experiments were performed in triplicate, representative results are shown.

For the intracellular glycerol measurements, cells were grown in 250 ml flasks with SC-ura with 10% of glucose at 28°C with agitation (150 rpm) in triplicate until the glucose concentration achieved < 2g/l.

Plasmids expressing the S. cerevisiae T73, S. bayanus BMV58, and S. kudriavzevii IFO1802 STL1 genes under NHA1 gene promoter were constructed by exchanging the NHA1 coding sequence in pNHA1-985 (YEp352 derivative, Kinclová et al., 2001) by homologous recombination. All constructions were confirmed by diagnostic PCR and sequencing. The primers, listed in Table 2, were designed based on data from Saccharomyces Genome Database (Cherry et al., 2012) and used to amplify the DNA fragments (from genomic DNAs) with suitable flanking regions for homologous recombination and confirmation.

The extracellular glycerol concentrations and residual sugars (glucose and fructose) were determined in must and medium samples by HPLC (Thermo Fisher Scientific, Waltham, MA) equipped with a refraction index detector. The column employed was a HyperREZTM XP Carbohydrate H+ 8 μm (Thermo Fisher Scientific) and the conditions used in the analysis were as follows: eluent, 1.5 mM H₂SO₄; flux, 0.6 ml/min; and oven temperature, 50°C. The samples were diluted, filtered through a 0.22-μm nylon filter (Symta, Madrid, Spain) and injected in duplicate.

To determine intracellular glycerol content, 5 OD₆₀₀ units were harvested by filtration and quickly washed with 5 ml of water and transferred to a tube containing 1 ml of cold water. After no more than 20 s after sampling, the yeast suspension was boiled for 10 min, cooled on ice, and centrifuged at 15,300 × g for 10 min (4°C). The supernatant was collected, filtered and directly analyzed by HPLC. A second sample (5 OD₆₀₀ units) was harvested by filtration in cellulose membrane, 25 mm pore size 0.45 μm (MF-Milipore) previously dried in the microwave at 350W for 20 min. and weighed. To determine dry weight, the cells in the membrane were carefully washed with 1 ml of water and dried under the same conditions. The values obtained are expressed as μg of glycerol per mg of yeast cells. Experiments were performed in triplicate.

For each culture, 10–20 ml sample was taken at different times. The cells were quickly harvested by centrifugation, washed and frozen in liquid N_2.Then, frozen cells were lysed and homogenized by FastPrep-24 (MP Biomedicals) in LETS buffer (10 mm Tris pH 7.4, 10 mM lithium-EDTA, 100 mM lithium chloride, 1% lithium lauryl sulfate) with acid-washed glass beads (0.4 mm diameter; Sigma-Aldrich) for 30 s six times alternating with ice incubation. Total RNA was extracted and purified using the phenol:chloroform method with minor modifications (Combina et al., 2012). Then the RNA was converted to cDNA and the expression of GPD1, GPD2, STL1, and FPS1 genes was quantified by qRT-PCR (quantitative real-time PCR). The cDNA strand was constructed using 2 μg of RNA mixed with 0.8 mM dNTP's, 80 pmol Oligo (dT) in 13 μl. The mixture was heated to 65°C for 5 min and quenched on ice for 1 min. 5 mM dithiothreitol (DTT), 50 U of RNase inhibitor (Invitrogen), 1 × First Strand Buffer (Invitrogen) and 200 U Superscript III (Invitrogen) were added to the 20 μl mixture, which was incubated at 50°C for 60 min and the reaction was inactivated after 15 min at 70°C. qRT-PCR was performed with gene-specific primers (200 nM) designed for each specie (Table 2) from sequences consensus between the different strains in a 10 μl reaction, using the Light Cycler FastStart DNA MasterPLUS SYBR green (Roche Applied Science, Germany) in a LightCycler® 2.0 System (Roche Applied Science, Germany). All samples were processed for melting curve analysis, amplification efficiency and DNA concentration determination. A mixture of all samples and serial dilutions (10⁻¹ to 10⁻⁵) was used as standard curve. Two different constitutive reference genes were used (ACT1 and RDN18-1) to normalize the amount of mRNA and ensure accuracy, correct interpretation, and repeatability (Starovoytova et al., 2013). The results were normalized by using the normalization factor obtained from geNorm VBA applet (Vandesompele et al., 2002).

The behavior of S. cerevisiae and other Saccharomyces species interesting for industrial applications was evaluated in response to wine fermentation relevant stresses. We selected hyperosmotic (NaCl 0.8 M and KCl 1.25 M) and a combination of hyperosmotic and cold stresses (12°C), two frequent suboptimal conditions during winemaking. We performed a drop test with two strains of each species (S. paradoxus, S. cerevisiae, S. kudriavzevii, and S. uvarum) on complete media and compared the growth in the above mentioned conditions (Figure 1). The results revealed that the used stresses have a very different effect on yeast growth depending not only on the species but even on the strain. The stress with KCl 1.25 M is the condition that has less effect on the yeast growth capacity, and the NaCl 0.8 M plus 12°C the most severe stress. The conditions NaCl 0.8 M hyperosmotic stress and KCl 1.25 M at 12°C hyperosmotic-cold stress generated intermediate growth capacity levels. The results showed clearly that the strains can cope better with a higher osmotic stress (KCl 1.25 M) than with the sodium toxicity (NaCl 0.8 M). In hyperosmotic stress mediated by NaCl 0.8 M we observed that S. uvarum strains are the ones presenting the highest tolerance to hyperosmotic and a similar observation can be made in the most severe condition (NaCl 0.8 M plus 12°C). The other species showed similar behavior although S. kudriavzevii strains showed low growth levels in cold stress condition, especially IFO1802 strain. S. cerevisiae and S paradoxus strains showed similar growth levels but strain 108 in NaCl 0.8 M at 25°C and strain Chr16.2 in KCl 1.25 M at 12°C presented lower growth levels than S. cerevisiae strains.

Since hyperosmotic and also cold stress responses are unequivocally related to glycerol accumulation we wanted to determine glycerol levels during hyperosmotic-cold stress in wine fermentations. Thus we performed wine fermentations in synthetic must with the studied Saccharomyces species and strains, and we measured intra- and extracellular amount of glycerol during the first hours and days of the fermentation. In the results presented in Figure 2 we observed two steps regarding glycerol accumulation in S. cerevisiae strains. In the first step, glycerol starts to accumulate inside the cell (Figure 2B) immediately after inoculating into the cold-hyperosmotic condition, reaching a maximal value after 24 h. Also, minimal glycerol levels are accumulated in extracellular media in the beginning of our experiment (Figure 2A). In the next 2 days, intracellular glycerol is reduced and tends to recover its original levels whereas extracellular glycerol increases with the time. In the case of S. paradoxus and S. kudriavzevii, maximal intracellular glycerol accumulation, which are approximately half of those in S. cerevisiae strains, occurs in the first hours and levels are maintained during 48 h. Analyzing the intracellular glycerol level (Figure 2B), it is interesting to note that, comparing with the other species, S. cerevisiae strains accumulated the higher levels of glycerol between 4 and 48 h of incubation. The S. uvarum strains showed the lowest values of intracellular glycerol with a maximal level after 1 h in the case of BMV58 and after 48 h in the case of 12600. Regarding extracellular glycerol (Figure 2A), S. paradoxus presented similar levels and accumulation pattern as S. cerevisiae and S. uvarum and, in addition, S. kudriavzevii showed a similar pattern but higher accumulation levels (around five times more). Is interesting to emphasize that S. uvarum and S. kudriavzevii showed a higher extracellular glycerol accumulation rate compared to the other two species. Interestingly, no extracellular glycerol was observed at time 0 in any species. It should be noted that strains do not show significant growth after 1 or 4 h and maximal yeast biomass was observed at the 24 or 48 h time point except for the IFO1802 that show very low growth level in grape must (Supplementary Figure 1), in concordance with data observed in Figure 1 in osmotic and cold stress conditions.

To gain insights on the regulation of glycerol pools balance we studied variation in mRNA levels of key genes related to glycerol biosynthesis (GPD1 and GPD2), efflux (FPS1), and influx (STL1) in the same wine fermentation conditions described above and in the same strains and species. The results (Figure 3) clearly revealed different patterns and levels of gene expression among the species in all four genes studied. In the case of GPD1, all the strains showed a general pattern of induction after the first hour but with marked differences in the expression levels. S. kudriavzevii strains showed the highest mRNA levels, specially IFO1802 strain that presented elevated expression of GPD1 before stress and even more after 1 h of inoculation. For the GPD2 gene, which is mainly involved in redox balance, some of the strains presented an induction with maximal levels after four (S. uvarum strains, FCry, 108, and CR85) or 48 (T73) hours whereas other strains (Chr16.2 and IFO1802) seem to not activate this gene showing low mRNA levels. The FPS1 gene expression peaked after 1 h (108, CR85, S. cerevisiae, and S. uvarum strains) or 4 h (Fcry), with the S. cerevisiae and S. uvarum strains showing the highest levels. The IFO1802, Chr16.2, and S. kudriavzevii strains did not showed significant increase of mRNA levels compared to the inoculum. Finally, the SLT1 gene presented the most variable mRNA levels among the species showing highest values for the S. uvarum strains, especially BMV58, with a maximum after 1 h. Other species showed a moderate amount of mRNA with maximum levels after 1 h (S. kudriavzevii strains) or 4 h (Fcry). S. paradoxus strains showed very low SLT1 mRNA levels along the experiment. Yeast growth phase does not has a dramatic impact in activation of gene expression in this conditions since the most important inductions were observed at 1 and 4 h, were grow was not observed. The comparison between glycerol content and gene expression results emphasize the importance of GPD1 and STL1 in S. kudriavzevii and S. uvarum respectively, regarding their increased glycerol accumulation (Figure 2A). In the case of S. cerevisiae strains, increased GPD2 levels, especially in Fcry strain, could explain the high intracellular glycerol levels observed in Figure 2B. On the contrary, FPS1 increased expression does not reflect extracellular glycerol levels in S. cerevisiae probably due to the tight regulation of this channel by posttranslational mechanisms.

To study the regulation of key genes related to intracellular glycerol balance under standard lab conditions (Figure 4) we used a representative strains of each species (T73, Chr16.2, BMV58, and IFO1802) and measured mRNA levels of GPD1, STL1 and FPS1 after half, 1 and 2 h of transfer cells to a non-stress SC media (Figure 4A), hyperosmotic SC 1 M sorbitol (Figure 4B) or hypoosmotic (water) media (Figure 4C). In addition, another analog set of experiments were performed but using mannitol as a carbon source (Figures 4D–F), which is a non-fermentable carbon source that complicates the energy supply for cellular processes. No yeast growth was observed during this experiment (results not shown). We can observe that all strains, especially T73 and BMV58, activate GPD1 0.5-1 h after hyperosmotic stress (Figure 4B) but is not activated in non-stress conditions (Figure 4A) or hypoosmotic stress (Figure 4C). A similar situation but with higher mRNA levels is observed in presence of mannitol instead of glucose where hyperosmotic stress (Figure 4E) activates GPD1 gene, especially for BMV58 and IFO1802. In this case, hypoosmotic stress (Figure 4F) does activate the GPD1 gene in the case of T73 and BMV58. The STL1 gene reacts with a similar patter as GPD1 increasing mRNA levels in hyperosmotic stress (Figure 4B) but not upon hypoosmotic stress in the presence of glucose (Figure 4C). STL1 shows also a similar patter as GPD1 in presence of mannitol, increasing expression levels after hyperosmotic stress (Figure 4E), though to higher levels compared to glucose (Figures 4B,E). Interestingly, S. cerevisiae T73 strain shows very low STL1 levels in any conditions and no significant activation (Figure 4F). On the contrary, the FPS1 gene seems to be unresponsive to any condition in all the strains with except for the case of T73 growth in mannitol and hypoosmotic stress (Figure 4F). Similar levels are presented for all strains and conditions although BMV58 presented lower levels that the other strains. Altogether, it is the STL1 gene whose expression shows the highest level of variation in different conditions and among the species and strains.

Since STL1 gene presented important differences in mRNA levels in strains from different Saccharomyces species we wanted to study the possible functional differences of this glycerol importer. For that we first compared the growth of a representative strain of S. cerevisiae (T73), S. uvarum (BMV58), S. paradoxus (Chr16.2), and S. kudriavzevii (IFO1802) species in conditions where the activity of Stl1 is important (Figure 5A). A drop test with the four strains was performed in non-stress media (SC), in hyperosmotic stress media (SC with 2 M sorbitol or 2 M KCl) and in hyperosmotic stress media supplemented with a very low amount of glycerol (SC with 2 M sorbitol (or 2 M KCl) and 1 mM glycerol). In these conditions, if the cells are able to efficiently import glycerol to the cytosol they have a growth advantage when extracellular glycerol is present, i.e., before they synthesize the necessary amount to counterbalance the external osmotic pressure. The results show that cell growth is affected by hyperosmotic stress conditions proportionally to the osmotic pressure, i.e., more in the presence of 2 M KCl than in the presence of 2 M sorbitol. We can observe that BMV58 is the strain with the lowest and Chr16.2 the highest survival level in both hyperosmotic stress conditions. Interestingly, as shown in the Figure 5A, some strains, as IFO1802 and especially BMV58, benefit from the presence of glycerol in the medium more than others (e.g., T73 and Chr16.2). These results are indicative of different capacity to import glycerol in response to hyperosmotic stress among the studied strains. Is interesting to highlight that osmotolerances in minimal media can be different to complete media (see BMV58 in Figure 1 compared to Figure 5A). This reflects that strains disposition to cope to osmotic stress could be different since complete and minimal media induce very different gene expression programs (Gasch et al., 2000; Miura et al., 2008).

To confirm these Stl1 functional differences we cloned the different STL1 alleles from T73, BMV58, and IFO1802 strains in an S. cerevisiae multicopy plasmid behind a weak and constitutive promoter, and expressed them in a laboratory osmosensitive S. cerevisiae strain (BY4741Δslt1Δhog1). As a control, this strain was also transformed with the empty YEp352. Then, the growth of strains was tested in non-stress media (SC), in hyperosmotic-stress media (SC with 0.7 M sorbitol or 0.3 M KCl) and in hyperosmotic-stress media supplemented with extracellular glycerol (SC 0.7 M sorbitol or 0.3 M KCl, and 10 mM glycerol). The results (Figure 5B) showed that the strains with the BMV58 and IFO1802 SLT1 allele are clearly able to recover growth when they have extracellular glycerol in the presence of a hyperosmotic stress. However, the strain containing the T73 STL1 allele presented only a minor growth recovery when it can use extracellular glycerol in the presence of a hyperosmotic stress.

We also evaluated the different Slt1 functionality by measuring the intracellular glycerol accumulation of the S. cerevisiae strains expressing different STL1 genes after 1 and 2 days of growth in 10% glucose (Figure 5C) without any additional osmotic agents. The strain with IFO1802 Stl1 was able to recover the original intracellular glycerol levels by importing some of the diffused out glycerol. The strain with BMV58 Stl1 was able to recover more than 80% of the original intracellular glycerol levels. On the contrary, after 2 days the strain with the T73 Stl1 showed intracellular glycerol levels recovery no significantly different than a strain without Slt1. This results points in the same direction of the previous experiments and suggest a low functionality of T73 Stl1 compared with BMV58 and IFO1802.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.