Little information is available about the precise mechanisms and determinants of freeze resistance in baker's yeast, Saccharomyces cerevisiae. Genomewide gene expression analysis and Northern analysis of different freeze-resistant and freeze-sensitive strains have now revealed a correlation between freeze resistance and the aquaporin genes AQY1 and AQY2. Deletion of these genes in a laboratory strain rendered yeast cells more sensitive to freezing, while overexpression of the respective genes, as well as heterologous expression of the human aquaporin gene hAQP1, improved freeze tolerance. These findings support a role for plasma membrane water transport activity in determination of freeze tolerance in yeast. This appears to be the first clear physiological function identified for microbial aquaporins. We suggest that a rapid, osmotically driven efflux of water during the freezing process reduces intracellular ice crystal formation and resulting cell damage. Aquaporin overexpression also improved maintenance of the viability of industrial yeast strains, both in cell suspensions and in small doughs stored frozen or submitted to freeze-thaw cycles. Furthermore, an aquaporin overexpression transformant could be selected based on its improved freeze-thaw resistance without the need for a selectable marker gene. Since aquaporin overexpression does not seem to affect the growth and fermentation characteristics of yeast, these results open new perspectives for the successful development of freezeresistant baker's yeast strains for use in frozen dough applications.Bread making is one of the oldest food-manufacturing processes and involves the fermentative capacity of the yeast Saccharomyces cerevisiae for the leavening of the dough. Special types of dough, such as sweet or sour dough, present specific challenges to the leavening activity of the yeast, and specific strains with better performance under such conditions have been selected. However, no appropriate strains of yeast are available yet for use in frozen doughs, an important recent development in the bakery industry (2, 33).The use of frozen doughs is steadily increasing in all industrialized countries because it offers great convenience, automation, and economy of scale. However, significant reduction of the leavening capacity during freeze storage is a serious drawback. Minimizing this loss requires specialized equipment for cold and rapid mixing of the dough which is not available to artisanal bakers. Moreover, these optimized production conditions still cannot completely overcome the drop in leavening activity during long-term storage.Conditions for production of baker's yeast have been optimized in the past decades and nowadays allow yeast with a very high stress resistance to be produced. Active dry yeast, for instance, is guaranteed to maintain its activity during shelf storage at room temperature for 2 years. However, the preparation of frozen doughs presents an unusual challenge. Although marketed baker's yeast is highly stress resistant, it rapidly lose...

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Revealing QTLs with a minor effect in complex traits remains difficult. Initial strategies had limited success because of interference by major QTLs and epistasis. New strategies focused on eliminating major QTLs in subsequent mapping experiments. Since genetic analysis of superior segregants from natural diploid strains usually also reveals QTLs linked to the inferior parent, we have extended this strategy for minor QTL identification by eliminating QTLs in both parent strains and repeating the QTL mapping with pooled-segregant whole-genome sequence analysis. We first mapped multiple QTLs responsible for high thermotolerance in a natural yeast strain, MUCL28177, compared to the laboratory strain, BY4742. Using single and bulk reciprocal hemizygosity analysis we identified MKT1 and PRP42 as causative genes in QTLs linked to the superior and inferior parent, respectively. We subsequently downgraded both parents by replacing their superior allele with the inferior allele of the other parent. QTL mapping using pooled-segregant whole-genome sequence analysis with the segregants from the cross of the downgraded parents, revealed several new QTLs. We validated the two most-strongly linked new QTLs by identifying NCS2 and SMD2 as causative genes linked to the superior downgraded parent and we found an allele-specific epistatic interaction between PRP42 and SMD2. Interestingly, the related function of PRP42 and SMD2 suggests an important role for RNA processing in high thermotolerance and underscores the relevance of analyzing minor QTLs. Our results show that identification of minor QTLs involved in complex traits can be successfully accomplished by crossing parent strains that have both been downgraded for a single QTL. This novel approach has the advantage of maintaining all relevant genetic diversity as well as enough phenotypic difference between the parent strains for the trait-of-interest and thus maximizes the chances of successfully identifying additional minor QTLs that are relevant for the phenotypic difference between the original parents.

The routine production and storage of frozen doughs are still problematic. Although commercial baker's yeast is highly resistant to environmental stress conditions, it rapidly loses stress resistance during dough preparation due to the initiation of fermentation. As a result, the yeast loses gassing power significantly during storage of frozen doughs. We obtained freeze-tolerant mutants of polyploid industrial strains following screening for survival in doughs prepared with UV-mutagenized yeast and subjected to 200 freeze-thaw cycles. Two strains in the S47 background with a normal growth rate and the best freeze tolerance under laboratory conditions were selected for production in a 20-liter pilot fermentor. Before frozen storage, the AT25 mutant produced on the 20-liter pilot scale had a 10% higher gassing power capacity than the S47 strain, while the opposite was observed for cells produced under laboratory conditions. AT25 also retained more freeze tolerance during the initiation of fermentation in liquid cultures and more gassing power during storage of frozen doughs. Other industrially important properties (yield, growth rate, nitrogen assimilation, and phosphorus content) were very similar. AT25 had only half of the DNA content of S47, and its cell size was much smaller. Several diploid segregants of S47 had freeze tolerances similar to that of AT25 but inferior performance for other properties, while an AT25-derived tetraploid, TAT25, showed only slightly improved freeze tolerance compared to S47. When AT25 was cultured in a 20,000-liter fermentor under industrial conditions, it retained its superior performance and thus appears to be promising for use in frozen dough production. Our results also show that a diploid strain can perform at least as well as a tetraploid strain for commercial baker's yeast production and usage.

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Previous observations that aquaporin overexpression increases the freeze tolerance of baker's yeast (Saccharomyces cerevisiae) without negatively affecting the growth or fermentation characteristics held promise for the development of commercial baker's yeast strains used in frozen dough applications. In this study we found that overexpression of the aquaporin-encoding genes AQY1-1 and AQY2-1 improves the freeze tolerance of industrial strain AT25, but only in small doughs under laboratory conditions and not in large doughs under industrial conditions. We found that the difference in the freezing rate is apparently responsible for the difference in the results. We tested six different cooling rates and found that at high cooling rates aquaporin overexpression significantly improved the survival of yeast cells, while at low cooling rates there was no significant effect. Differences in the cultivation conditions and in the thawing rate did not influence the freeze tolerance under the conditions tested. Survival after freezing is determined mainly by two factors, cellular dehydration and intracellular ice crystal formation, which depend in an inverse manner on the cooling velocity. In accordance with this so-called two-factor hypothesis of freezing injury, we suggest that water permeability is limiting, and therefore that aquaporin function is advantageous, only under rapid freezing conditions. If this hypothesis is correct, then aquaporin overexpression is not expected to affect the leavening capacity of yeast cells in large, industrial frozen doughs, which do not freeze rapidly. Our results imply that aquaporinoverexpressing strains have less potential for use in frozen doughs than originally thought.Although much correlative evidence is available, the determinants of freeze resistance in Saccharomyces cerevisiae are largely unknown (for a review see reference 34). The injury sustained during freezing and thawing is caused by a combination of multiple types of stress imposed on the cells, such as changes in temperature, water content, water state, pH, and free radical, ion, and solute concentrations. The concomitant loss of survival is not attributable to any one form of injury and depends on the cooling and warming rates, the final temperature, the duration of freezing, and the suspending medium (23,24).When a cell suspension is cooled to a temperature of 0°C or less, both the suspending medium and the cells initially supercool. Extracellular ice crystal formation precedes intracellular freezing and is determined by the freezing point of the suspending medium and the presence of ice-nucleating agents. Based only on osmolarity, the freezing point of the cytoplasm is predicted to be approximately Ϫ1°C. Nevertheless, the cell interior typically remains unfrozen until the temperature is Ϫ10 to Ϫ15°C. This intracellular supercooling may be due to prevention of growth of ice into the cell interior by the cell membrane and due to the lack of nucleators of supercooled water within the cell (23, 24).Following external fr...

The importance of aquaporin expression in water permeability in Saccharomyces cerevisiae was assessed by measuring the osmotic water permeability coefficient (P f ) and the activation energies (E a ) from both hypo-and hypertonic experiments performed with whole protoplasts from four strains differing in aquaporin level of expression: parental, double-deleted and overexpressing AQY1 or AQY2. Double-deleted (lower P f ) and AQY1-overexpressing strains (higher P f ) presented linear Arrhenius plots with E a consistent with fluxes mainly through the lipids [16?3 kcal mol "1 (68?2 kJ mol )], respectively. The Arrhenius plots for the parental (swelling experiments) and overexpressing AQY2 strains (swelling and shrinking experiments) were not linear, presenting a break point with a change in slope around 23 6C. The E a values for these strains, calculated for temperatures ranging from 7 to 23 6C, were lower [9?5 kcal mol "1 (39?7 kJ mol The permeabilities for each strain relative to the deletion strain show that an increase in permeability due to the presence of aquaporins was more relevant at low temperatures. Following our results, we propose that water channels play an important role for osmotic adjustment of yeast cells at low temperature.

Aquaporins are members of the major intrinsic protein superfamily of integral membrane proteins which enable the transport of water, glycerol, and other solutes across membranes in various organisms. In microorganisms, the physiological role of aquaporins is not yet defined. We found a clear correlation between expression of the Candida albicans aquaporin-encoding gene AQY1 and freeze tolerance. A connection with the function for the aquaporin in the natural environment of C. albicans is, however, not obvious.Aquaporins are members of the major intrinsic protein (MIP) superfamily of integral membrane proteins (4, 23). They mediate water transport across cell membranes in numerous species (7). Mammalian and plant aquaporins have important functions in water homeostasis and osmoregulation (1,20), but the physiological function of microbial aquaporins remains a matter of debate (9,16,17,18,26,27). Transport processes that involve the uptake or efflux of water mediated by water channel proteins are nevertheless believed to be of major importance for microorganisms, particularly under conditions where the passive diffusion of water through the lipid bilayer is insufficient.In Saccharomyces cerevisiae, two aquaporin-encoding genes have been identified and characterized, AQY1 and AQY2 (6,11,21). Aquaporin expression is strongly correlated with freeze tolerance (28). Deletion of both aquaporin genes from a laboratory strain makes the cells more sensitive to freezing, and overexpression of the genes improves freeze tolerance (28). These findings are consistent with a role for plasma membrane water transport activity in determining freeze tolerance in S. cerevisiae.A functional water channel is known in Candida albicans (12), but a aquaporin null strain does not have a pronounced phenotype (12). Whereas an S. cerevisiae aquaporin null strain displays a small but significant decrease in sensitivity to osmotic shock (6), deletion of C. albicans AQY1 resulted in a far less pronounced phenotype (12). The minor effects of aquaporin deletion on cell surface hydrophobicity, flocculation, cell aggregation, and invasive growth observed in S. cerevisiae (11) were not noticeable in C. albicans (12).In this study, we determined whether the aquaporin-encoding gene AQY1 is a determinant of C. albicans freeze tolerance, as previously demonstrated for S. cerevisiae AQY1 and AQY2 (28). We verified the freeze-sensitive C. albicans AQY1 deletion phenotype, which is remedied by reintroduction of the gene. These findings strengthen the correlation between aquaporin function and freeze tolerance in microorganisms.We compared the freeze tolerance of heterozygous AQY1 deletion strain JC0186 (aqy1⌬/AQY1) (12) and homozygous AQY1 deletion strain JC0188 (aqy1⌬/aqy1⌬) (12). Both strains were grown overnight in 5 ml YPD (1% wt/vol yeast extract, 2% wt/vol Bactopeptone, 2% glucose) or uracil-deficient minimal medium (6.67 g/liter yeast nitrogen base without amino acids [Bio101, Qbiogene, France], 0.77 g/liter complete supplement mixture minus uracil [...