ROS accumulation and oxidative damage to cell structures in Saccharomyces cerevisiae wine strains during fermentation of high-sugar-containing medium

Biochimica et Biophysica Acta 1780 (2008) 892–898

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Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b b a g e n

ROS accumulation and oxidative damage to cell structures in Saccharomyces cerevisiae wine strains during fermentation of high-sugar-containing medium Sara Landolfo a, Huguette Politi a, Daniele Angelozzi a, Ilaria Mannazzu a,b,⁎ a b

Dipartimento di Scienze degli Alimenti, Università Politecnica delle Marche, Via Brecce Bianche, 60131, Ancona, Italy Dipartimento di Scienze Ambientali, Agrarie e Biotecnologie Agroalimentari, Università degli Studi di Sassari, Viale Italia 39, 07100 Sassari, Italy

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Article history: Received 28 August 2007 Received in revised form 11 March 2008 Accepted 12 March 2008 Available online 18 March 2008 Keywords: Fermentative stress Wine yeast ROS Membrane permeability Protein catabolism Trehalose Viability

a b s t r a c t To further elucidate the impact of fermentative stress on Saccharomyces cerevisiae wine strains, we have here evaluated markers of oxidative stress, oxidative damage and antioxidant response in four oenological strains of S. cerevisiae, relating these to membrane integrity, ethanol production and cell viability during fermentation in high-sugar-containing medium. The cells were sampled at different fermentation stages and analysed by flow cytometry to evaluate membrane integrity and accumulation of reactive oxygen species (ROS). At the same time, catalase and superoxide dismutase activities, trehalose accumulation, and protein carbonylation and degradation were measured. The results indicate that the stress conditions occurring during hypoxic fermentation in high-sugar-containing medium result in the production of ROS and trigger an antioxidant response. This involves superoxide dismutase and trehalose for the protection of cell structures from oxidative damage, and protein catabolism for the removal of damaged proteins. Cell viability, membrane integrity and ethanol production depend on the extent of oxidative damage to cellular components. This is, in turn, related to the ‘fitness’ of each strain, which depends on the contribution of individual cells to ROS accumulation and scavenging. These findings highlight that the differences in individual cell resistances to ROS contribute to the persistence of wine strains during growth under unfavourable culture conditions, and they provide further insights into our understanding of yeast behaviour during industrial fermentation. © 2008 Elsevier B.V. All rights reserved.

1. Introduction Reactive oxygen species (ROS) are normal by-products of cell metabolism. However, when their production prevails over the cellular defence systems, this can result in damage to nucleic acids, proteins and lipids, and to other cellular components. The mitochondrial respiratory chain is the main source of ROS in aerobically growing cells [1]. For this reason, the production of ROS has been traditionally associated with respiring cells and has not been considered significant under fermentative conditions, during which classical oxidative phosphorylation rapidly drops to less than significant levels [2] due to the reduction in the levels of cytochromes, oxidases and F1-ATPase [3,4]. However, experimental evidence indicates that ROS production occurs also in fermenting yeasts. Salmon et al. [5] noted that notwithstanding the presence of repressive glucose concentrations, during the first stages of fermentation Saccharomyces cerevisiae carries out partial and limited synthesis of mitochondrial cytochromes and maintains the ability to consume oxygen through mitochondria-related activities. In the absence of oxygen, S. cerevisiae activates NAD(P)H-dependent pathways, such as cytochrome P450 systems, which produce significant levels of H2O2, UO−2 and UOH [6].

⁎ Corresponding author. DiSAABA, Università degli Studi di Sassari, Viale Italia 39, 07100 Sassari, Italy. Tel.: +39 079 229314; fax: +39 079 229287. E-mail address: [email protected] (I. Mannazzu). 0304-4165/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2008.03.008

Moreover, S. cerevisiae undergoes transient oxidative stress upon exposure to anoxia [7]. It has also been shown that during must fermentation wine yeasts are subject to the effects of nutritional and environmental stress factors [8], the synergistic activities of which can result in ROS production and in generation of a stress condition [9]. In agreement with this, wine yeasts show correlations between fermentative behaviour and stress resistance [10–13]. Moreover, during the first hours of fermentation, wine strains of S. cerevisiae increase transcription levels of stress-response genes [14,15] and induce expression of proteins involved in the response to oxidative stress [16]. Costa et al. [17] have shown that in the presence of ROS, molecular chaperones, such as Hsp60, and glycolytic enzymes are subject to protein carbonylation. This leads to the impairment of both protein folding and membrane translocation [17] and to a reduction in the expression of glycolytic enzymes [18]. Under these conditions NADPH production occurs via the pentose phosphate pathway [19] and cells undergo a reduction in their ethanol production. Thus, the ROS scavenging ability is involved in the maintenance of the fermentative ability of yeast strains used in industrial processes. However, to the best of our knowledge, the kinetics of ROS accumulation and scavenging and the effects exerted by ROS on sensitive cellular targets, such as proteins and cell membranes, have not been characterized during progression through a long-lasting batch fermentation process.

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To determine the effects of such stress factors upon wine strains during fermentation of high-sugar-containing media, we have here evaluated some biomarkers of oxidative stress, oxidative damage and antioxidant response in four oenological strains of S. cerevisiae, and we have related these to membrane integrity, ethanol production and cell viability during fermentation of high-sugar-containing medium. 2. Materials and methods 2.1. Strains and culture conditions Three commercial oenological strains of S. cerevisiae were used: L2056, EC1118 and Lalvin M69 (Lallemand Montreal, Canada), along with M25, a wine strain belonging to the Culture Collection of DiSAABA (University of Sassari, Italy). The yeast strains were pre-cultured aerobically in YEPD (2% glucose,1% yeast extract, 2% peptone) and 5 × 105 cells ml− 1 were inoculated into 100-ml flasks containing 75 ml SJ medium (0.2% YNB without amino acids, 0.7% ammonium sulphate, 12% glucose, 12% fructose) pH 4.3. Each flask was equipped with a glass capillary stopper, a flask for each sampling time was inoculated and all flasks were incubated statically at 20 °C for 20 days. Oxoid strips saturated with resazurin (Anaerobic Indicator BR0055B, Oxoid Ltd) were fixed to the glass capillary stoppers in the head space of the flask. The colour change from pink to white indicates the lack of oxygen in the head space of each flask. Fermentation trials were carried out in triplicate and the cells underwent the analyses described below. Yeast growth was determined by total cell counting in a haemocytometer and viable plate counting.

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0.24 g l− 1 KH2PO4, pH 7.4) to a final concentration of 1–2 × 106 cells ml− 1 (OD600 0.08– 0.20). The cell suspensions were stained according to the following procedures: (i) Propidium iodide (PI): 10 μl PI stock solution (10 mg ml− 1) in PBS was added to 1 ml of cell suspension just prior to the analysis. Fluorescence was detected in fluorescence channel 3. (ii) Dihydroethidium (DHE): 5 μl DHE stock solution (10 mg ml− 1 in DMSO, stored at − 20 °C) was added to 1 ml of cell suspension, and the samples were incubated at room temperature in the dark for 10 min before analysis. DHE emission was detected in fluorescence channel 3. At least 1.5 × 104 cells were analysed for each experiment. Data were visualized using the WinMDI flow cytometry software (Joseph Trotter, Salk Institute for Biological Studies, La Jolla, CA, USA). 2.4. Preparation of yeast crude extract Yeast crude extracts for antioxidant enzyme assays were prepared in 0.1 M Tris (pH 7.6) and following a mechanical lysis protocol [21]. Crude extracts for Western blotting of carbonylated and ubiquitinated proteins were prepared in potassium phosphate buffer (pH 7.0) containing a mixture of protease inhibitors (30 μg ml−1 pepstatin, 30 μg ml−1 leupeptin, 6 μg ml−1 antipain and 6 mM EDTA) [22] and according to the same protocol [21]. Crude extracts for the proteinase A activity assay were prepared in 0.1 M Tris–HCl (pH 7.6) by vigorous shacking of the cell suspension in the presence of glass beads for 3 min. Total protein content was assayed according to Bradford [23]. 2.5. Measurement of enzyme activities

2.2. Analytical determinations of fermented SJ medium

Catalase (CAT) activity was determined as described by Luck [24] and expressed as U (mg protein)− 1. Superoxide dismutase (SOD) activity was measured as described by Oyanagui [25] and expressed as U (mg protein)− 1. Protease A activity was evaluated as indicated by Jones [26] and expressed as µg Tyr min− 1 (mg protein)− 1.

The 10139106035 and 10176290035 enzymatic kits (R-Biopharm BoehringerMannheim, Germany) were used for the determination of the residual glucose and fructose contents and for the production of ethanol, respectively, at the time points indicated during fermentation.

2.6. Western blotting of carbonylated and ubiquitinated proteins

2.3. Flow cytometry A Coulter Epics XL (Beckman Coulter, Inc. Fullerton, CA, USA) equipped with a 15-mW air-cooled argon-ion laser (emission, 488 nm) was used, with five sensors for the detection of forward and side light scatter, green (525 nm, channel 1), yellow (575 nm, channel 2), and orange–red (620 nm, channel 3) fluorescence. Size calibration was performed using the flow cytometry size-calibration kit (Molecular Probes, Inc. Eugene, OR, USA). Yeast cells were harvested at the indicated times. As ethanol toxicity is correlated with the production of ROS [20], ethanol-treated cells (70%, 40 min) were included as the positive control for oxidation. The samples were then washed and re-suspended in PBS (8.0 g l− 1 NaCl, 0.20 g l− 1 KCl, 1.44 g l− 1 Na2HPO4,

Protein carbonyls were detected using a 2, 4-dinitrophenylhydrazine (DNPH)binding method, as described by Levine et al. [27]. Aliquots of crude extracts containing 15 μg protein were derivatized with 20 mM DNPH in 10% trifluoroacetic acid (Sigma Aldrich Chemie GmbH, Steinheim, Germany) and loaded onto polyacrylamide gels (10%) [28]. After electrophoresis the proteins were transferred to PVDF membranes and probed with anti-DNP IgG (Sigma Aldrich Inc., St Louis, MO, USA) at a 1:5000 dilution as the first antibody, and goat anti-rabbit IgG linked to alkaline phosphatase (Sigma Aldrich Inc., St Louis, MO, USA) at a 1:5000 dilution as the second antibody. Positive controls of protein carbonylation were obtained by treating yeast cells growing exponentially in liquid YEPD with 70 μM plumbagine (Sigma Aldrich Inc., St Louis, MO, USA) for 60 min as indicated by Cabiscol et al. [29]. Ubiquitinated proteins were detected as follows: after electrophoresis 15 µg of proteins were transferred onto PVDF membranes and probed with anti-ubiquitin

Fig. 1. Evaluation of intracellular ROS content. Cells were sampled 1, 3, 7 and 20 days after inoculation, treated with DHE, and analysed by flow cytometry for ROS content. Abscissa: fluorescence intensity as logarithmic scale (increases from left to right); ordinate: number of events (cells). EtOH: cells treated with ethanol, as positive control for oxidation. Data are from a single experiment, representative of three independent experiments.

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antibody (U5379, Sigma) diluted 1:100 as the first antibody, and rabbit anti-IgG (Sigma Aldrich Inc., St Louis, MO, USA) diluted 1:2000 as the second antibody. The integrity of the crude extracts and the amount of proteins loaded in each lane were checked on SDS PAGE stained with Coomassie Blue. Immunodetection was performed by the BCIP/NBT staining method (Sigma Aldrich Inc., St Louis, MO, USA). 2.7. Trehalose evaluation Trehalose was evaluated as described by Parrou et al. [30]. The glucose produced by trehalase hydrolytic activity was measured with the Glucose Oxidase (GO) Assay kit (Sigma Aldrich, Inc., St Louis, MO, USA). 2.8. Data analysis All experiments were carried out in triplicate from independent pre-cultures. Statistical analyses of the data were performed using ANOVA followed by Tukey Kramer HSD test (all pair comparison) using the JMP version 3.1.5 software (SAS Institute Inc.). Linear regression analyses were performed and the coefficients of determination (R2) were used to evaluate the correlations between ethanol production and percentages of cells with membranes permeable to PI.

3. Results 3.1. ROS accumulation The four S. cerevisiae wine strains were incubated statically for 20 days in SJ medium, a synthetic medium that mimics the composition of grape must in terms of sugar and nitrogen contents. The intracellular levels of ROS were evaluated at different stages of the fermentation to determine the levels of stress symptoms. For this, the yeast cells were sampled 1, 3, 7 and 20 days after inoculation, corresponding to the mid-exponential phase, the early and late stationary phases, and the end of fermentation, respectively. These samples were stained with dihydroethidium (DHE) and analysed by flow cytometry. Once inside viable cells DHE can be oxidized to ethidium by ROS [31]. Ethidium intercalates with nucleic acids and emits red fluorescence [31,32]. Thus, flow cytometric analysis of DHE cell associated fluorescence provides an indirect measure of the intracellular levels of ROS [31,33,34]. The single uniform peaks produced by DHE stained cells on day 1 indicated that the four strains produced rather homogeneous populations regarding the intracellular levels of ROS. These levels were lower

Fig. 3. Percentages of cells permeable to PI. Cells were sampled 1, 3, 7 and 20 days after inoculation, stained with PI, and analysed by flow cytometry for percentages of PIstained cells. Data are means ± standard deviation of three independent experiments.

than those seen for the ethanol-treated cells used as the positive controls for oxidation, as indicated by the position of the peaks (further to the left for day 1 samples) on the X axis (Fig. 1). In the later samples, all of the strains showed subpopulations made of cells characterized by different mean fluorescence intensities as indicated by the coexistence of more than one peak in different position on the X axis. Those cells showing a fluorescence intensity comparable to the ethanol-treated control samples (Fig. 1; peaks further to the right) should represent cells that were highly oxidized, senescent and/or dead. Those characterized by lower mean fluorescence intensities (Fig. 1; peaks further to the left) contained lower intracellular levels of ROS. At day 20, as well as showing increases in the percentages of highly oxidized cells, all of the strains also differentiated subpopulations that were characterized by lower intracellular levels of ROS. Different degrees of this phenotypic heterogeneity were seen across the four S. cerevisiae wine strains examined. Considering that the intracellular levels of ROS depend on the balance between ROS production and scavenging, this phenotypic heterogeneity could depend on cell-to-cell variations in the cellular responses to the fermentative stress, as also suggested by Bishop et al. [35]. 3.2. Oxidative damage to cell structures, viability and fermentative ability To evaluate whether ROS accumulation was accompanied by oxidative damage to cell structures, Western blotting of carbonylated proteins was carried out [7,29,36]. The production of carbonyl groups is

Fig. 2. Oxidative damage to proteins. Cells were sampled 1, 3, 7 and 20 days after inoculation, and subjected to Western blotting for determination of protein carbonylation. YEPD: YEPD exponentially growing cells used as the negative control of carbonylation; YEPD + plumbagine: YEPD exponentially growing cells treated with 70 μM plumbagine for 60 min, used as the positive control of carbonylation. In L2056 ⁎ indicates increases in carbonyl groups in respect to day 7. Data are from a single experiment, representative of three independent experiments.

Fig. 4. Yeast viability during fermentation. Cells were sampled at the indicated times after inoculation and subjected to total and viable cell counts. Viability is expressed as percentages of viable versus total cells counted. Error bars represent standard deviations; where not seen, they lie within the symbol (n = 3).

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Fig. 5. Ethanol production and sugar consumption during fermentation. Culture broth was sampled 1, 3, 7 and 20 days after inoculation and analysed for ethanol and residual sugars. Closed symbols sugar consumption; open symbols ethanol production. Error bars represent standard deviations; where not seen, they lie within the symbol (n = 3).

due to the oxidation of specific amino acids (arginine, histidine, lysine and proline) and to cleavage of the polypeptide chains (at aspartate, glutamate and proline) [17], and so the evaluation of carbonylated proteins was carried out on cell samples during fermentation. Similarly, cells growing exponentially in YEPD were used as the negative control of carbonylation, with the same after plumbagine treatment (70 μM; 1 h) for the positive control of carbonylation (Fig. 2). The pattern of carbonylated proteins was generally comparable in all of the strains, thus indicating common targets for ROS species (Fig. 2). Moreover, all strains showed a high content of carbonylated proteins at day 1, as compared to the negative control in YEPD. However, some differences were seen across the yeast wine strains regarding the kinetics of production and disposal of carbonyl groups. The M25 strain increased the amount of carbonylated proteins at days 3 and 7 according with the increment in ROS-associated fluorescence (peaks further to the right), at the corresponding days (Fig. 2). At day 20, protein carbonylation appeared not to correlate with the intracellular content of ROS of the vast majority of cells. This discrepancy was also seen in M69 and EC1118 in which the decrease in the amount of carbonylated proteins did not correlate with the observed increases in the percentages of cells containing high intracellular ROS levels (Fig. 1). L2056 strain showed decreases in the amounts of carbonylated proteins at days 3 and 7, with a slight increase at day 20. EC1118 showed the lowest level of carbonylated proteins after plumbagine treatment. This was even lower than that

Fig. 6. Trehalose accumulation during fermentation. Cells were sampled 1, 3, 7 and 20 days after inoculation and analysed for trehalose accumulation (expressed as μg glucose mg dry weight−1). Data are means ± standard deviation of three independent experiments.

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Fig. 7. SOD activity. Cells were sampled at the indicated times after inoculation and analysed for superoxide activity. Error bars represent standard deviations; where not seen, they lie within the symbol (n = 3).

found at day 1 of fermentation thus indicating that cells are subjected to highly stressful conditions after the inoculum in SJ. To determine whether this oxidative damage to proteins was accompanied by a loss of membrane selectivity, flow cytometric analysis was carried out on propidium iodide (PI)-stained cells sampled during fermentation in SJ medium. PI stains the nucleic acids in cells with defective membrane integrity, and for this reason it is widely used as a measure of membrane permeability [37,38]. The four S. cerevisiae wine strains examined maintained good levels of membrane integrity until day 7, and increased significantly the percentages of cells with permeable membranes at day 20 (Fig. 3). This was consistent with the decreases in the percentages of viable M69 and EC1118 cells at day 20 (Fig. 4). In particular, in these strains the percentages of PI-stained cells (49% and 53%, respectively) were similar to that of non-viable cells (43% and 55%, respectively). On the contrary, in the M25 and L2056 strains the percentages of viable cells were lower than expected on the basis of the flow cytometry analysis of PI-stained cells, possibly due to the generation of viable but not culturable cells [39]. At day 20 the four strains also showed significant differences in their fermentative abilities in terms of ethanol production and sugar consumption (Fig. 5). M25 and EC1118 strains were the lowest and the highest ethanol producers, respectively (P b 0.05). L2056 and M69 showed an intermediate behaviour even though with significant differences for both parameters (P b 0.05). Ethanol is a pro-oxidant that at concentrations above 15 g l− 1 leads to cell death by decreasing the membrane integrity [38]. Accordingly, cell membrane permeability showed a positive correlation with the final ethanol concentrations in the three commercial strains (R2 = 0.96) but not in the M25 strain,

Fig. 8. Proteinase A activity. Cells were sampled at the indicated times after inoculation and analysed for proteinase A activity. Error bars represent standard deviations; where not seen, they lie within the symbol (n = 3).

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Fig. 9. Protein ubiquitination in EC1118. Cells were sampled 1, 3, 7 and 20 days after inoculation, and subjected to Western blotting for determination of protein ubiquitination. Data are from a single experiment, representative of two independent experiments.

which had the highest percentage of permeable cells even though it showed the lowest ethanol production. 3.3. ROS buffering abilities of the wine strains To determine whether the differences seen in the intracellular contents of ROS and carbonylated proteins were due to differences in the ROS buffering abilities of these wine strains, trehalose accumulation, CAT and SOD activities were evaluated. As expected, none of the strains accumulated trehalose during exponential growth (to day 1). At day 3, all of the strains, except M25, showed increases in trehalose content (Fig. 6). Over the following days, while the EC1118 and M69 strains showed further significant increases in their intracellular contents of trehalose (P b 0.05), the L2056 and M25 strains did not (Fig. 6). Interestingly, in EC1118, M25 and M69 the intracellular trehalose was inversely related to membrane permeability. In contrast, L2056 did not show any correlation between trehalose content and membrane integrity. None of the strains showed appreciable catalase (CAT) activities (data not shown), thus suggesting that other peroxidases are involved in the disposal of the H2O2 that will be derived from UO2− dismutation. Superoxide dismutase (SOD) activity was comparable in the L2056 and EC1118 strains, which showed increases in their levels of this antioxidant enzyme at day 7 (Fig. 7). The M25 cells showed SOD levels that were comparable to those of L2056 and EC1118 at the beginning of the fermentation process, but they then underwent a delayed increase in the activity of this antioxidant enzyme (Fig. 7). Finally, the M69 strain showed a more precocious induction of SOD activity, although it maintained low SOD levels during the whole process. 3.4. Protein degradation during fermentation Proteolytic degradation of damaged proteins is required to avoid the accumulation of misfunctioning cell components under oxidative stress [40]. To evaluate whether the observed variations in the content of carbonylated proteins could depend on protein degradation, the time courses of both proteinase A activity and protein ubiquitination were analysed. According to the results of Alexandre et al. [41] who observed an increase in Proteinase A activity during fermentation, all strains showed significant increases in proteolytic activity at day 20 (Fig. 8) but EC1118 and M25 harboured a proteolytic activity that was significantly higher than that of L2056 and M69. Moreover, in EC1118 vacuolar degradation of proteins was accompanied by a marked increase in protein ubiquitination (Fig. 9) while the other strains did not show significant variations in the ubiquitination pattern (data not shown). 4. Discussion These data indicate that stressful conditions occurring during hypoxic fermentation of a high-sugar-containing medium result in the production of ROS and trigger an antioxidant response that involves SOD, trehalose and protein catabolism. SOD is responsible for the

disposal of UO−2 and the two forms of SOD expressed by eukaryotic cells (cytosolic, Cu, Zn SOD; mitochondrial, Mn SOD) are involved in the protection of cell structures from oxidative damage [17]. Trehalose functions as a ROS scavenger and it is known to protect proteins against oxidative damage and to lower lipid oxidation in vivo [42]. Protein catabolism is involved in the removal of damaged/misfunctioning proteins [40]. The two ROS scavengers appeared to be involved in counteracting the oxidative damage to proteins, even though with some differences across different strains. In particular, the EC1118 and L2056 strains, that differed significantly in their patterns of carbonylated proteins, showed comparable kinetics of SOD activity, but differed significantly in the intracellular levels of trehalose. In L2056, the reduction in carbonyl groups seen at day 7 was concomitant with an increase in SOD activity; the later decrease in SOD activity, combined with a low level of trehalose and a higher ROS-associated fluorescence, resulted in a slight increase in carbonyl groups. The M69 and EC1118 strains showed a similar trend in the disappearance of carbonyl groups, but differed markedly in the kinetics of SOD induction, while showing similar levels of trehalose. In M25 the onset of trehalose synthesis at day 7 was not accompanied by a reduction in the carbonyl groups, which, on the contrary, appeared to be due, at least in part, to a dramatic increase in SOD activity at day 20, notwithstanding the increase in ROS associated fluorescence. Thus, the differences observed across the different strains suggest that even though both SOD and trehalose are required to avoid oxidative damage to proteins, the production of at least one of the two scavengers may be sufficient to reduce the negative effects of ROS. In contrast, low levels of both scavengers resulted in protein carbonylation (see M25 at days 3 and 7). This is expected to have negative consequences on the fermentative ability of a yeast. Indeed, under conditions of oxidative stress, glycolytic enzymes such as pyruvate decarboxylase and glyceraldehyde-3-phosphate dehydrogenase are susceptible to carbonylation [29]. The decreases in the activities of the glycolytic enzymes through the favouring of the activation of the pentose phosphate pathway over the regeneration of NADPH result in a decrease in ethanol production [22]. Accordingly, the M25 and L2056 strains showed marked damage to their cellular proteins and produced lower amounts of ethanol as compared to the EC1118 and M69 strains. The three commercial strains showed positive correlations (R2 = 0.96) between membrane permeability and ethanol production. Cell membranes are one of the main targets of ethanol, which is known to cause the uncoupling of the electron transport chain from the ATPase and to increase ROS production, thus increasing membrane fluidity and permeability [43,44]. For these reasons, the ethanol stress response is mediated by a re-modulation of the cellular lipid composition [45] and an activation of an adequate antioxidant response [46]. Indeed, contrary to what observed for L2056, the M25 cells are not able to adapt their lipid composition during SJ fermentation [39]. However, the dramatic increase in membrane permeability shown by the M25 strain was presumably a consequence of the oxidative damage to cell structures and one of the causes of both the low ethanol production and the loss of cell viability. Flow cytometry has here proven to be a useful tool for the study of fermentative stress. By allowing the evaluation of ROS content at the single-cell level, this use of flow cytometry has shown that single cells within isogenic populations can undergo transitions to different redox states during the progression of fermentation and that they present a marked phenotypic heterogeneity. The differentiation of cells characterized by lower intracellular levels of ROS is presumably responsible for the generation of subpopulations that were better adapted to unfavourable growth conditions during long-lasting batch cultures [35,48]. Interestingly, the increase in the percentage of highly oxidized cells, besides being in agreement with the natural increase in the number of aged and dead cells in batch cultures [47], was not accompanied by an increase in the amount of carbonylated proteins. According to Dudek

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et al. [49] carbonylated proteins are preferred substrates for ubiquitin, and the ubiquitin proteasome pathway is involved in the degradation of damaged proteins. Other authors indicate that the degradation of carbonylated proteins is ubiquitin independent and occurs mainly through the involvement of vacuolar proteases [50]. In our experimental conditions only EC1118 showed significant variations in the pattern of ubiquitinated proteins, indicative of a possible involvement of the ubiquitin proteasome pathway in the removal of carbonylated proteins. In accordance with the findings of Shringarpure et al. [40] the remaining strains confirmed that ubiquitin conjugation is not necessary for the degradation of oxidized proteins. On the contrary, the increase of proteinase A activity shown by the four strains at day 20 confirmed a role of vacuolar proteases in the turnover of oxidized proteins and explained the discrepancy between the amount of carbonylated proteins and the ROS-associated fluorescence at day 20. In conclusion, with the present study we have shown that wine strains undergo oxidative stress during fermentation. Their cell viability, membrane integrity and ethanol production depend on the extent of the oxidative damage to cellular structures. This is, in turn, related to the fitness of each strain, which depends on the contribution of individual cells to ROS accumulation and scavenging. These findings highlight that the differences in individual cell resistances to ROS contribute to the persistence of wine strains during growth under unfavourable culture conditions, and they provide further insights into our understanding of yeast behaviour during industrial fermentation. Acknowledgements The work was partially supported by MURST PRIN Anno 2003 — Prot. N 2003077174. D.A. received a grant from Enologica Fenocchio s.n.c., Grottammare (AP). References

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