The molecular mechanisms that enable yeast cells to detect and transmit cold signals and their physiological significance in the adaptive response to low temperatures are unknown. Here, we have demonstrated that the MAPK Hog1p is specifically activated in response to cold. Phosphorylation of Hog1p was dependent on Pbs2p, the MAPK kinase (MAPKK) of the high osmolarity glycerol (HOG) pathway, and Ssk1p, the response regulator of the two-component system Sln1p-Ypd1p. However, Sho1p was not required. Interestingly, phosphorylation of Hog1p was stimulated at 30°C in cells exposed to the membrane rigidifier agent dimethyl sulfoxide. Moreover, Hog1p activation occurred specifically through the Sln1 branch. This suggests that Sln1p monitors changes in membrane fluidity caused by cold. Quite remarkably, activation of Hog1p at low temperatures affected the transcriptional response to cold shock. Indeed, the absence of Hog1p impaired the cold-instigated expression of genes for trehalose-and glycerol-synthesizing enzymes and small chaperones. Moreover, a downward transfer to 12 or 4°C stimulated the overproduction of glycerol in a Hog1p-dependent manner. However, hog1⌬ mutant cells showed no growth defects at 12°C as compared with the wild type. On the contrary, deletion of HOG1 or GPD1 decreased tolerance to freezing of wildtype cells preincubated at a low temperature, whereas no differences could be detected in cells shifted directly from 30 to ؊20°C. Thus, exposure to low temperatures triggered a Hog1p-dependent accumulation of glycerol, which is essential for freeze protection.Variations in the surrounding temperature are probably the most common stress for all living organisms. In particular, a downshift in temperature leads to a reduction in membrane fluidity, impaired protein biosynthesis, and stabilization of secondary structures of DNA and RNA (1). Consequently, the adaptive response to cold shock in most organisms, studied so far, includes a change in the lipid composition of membranes and the remodeling of the transcriptional and translational machinery. These changes are mainly triggered by a drastic variation in the gene expression program, which leads to both survival and adaptation to low temperatures.Unlike other stress conditions, the biochemical mechanisms by which eukaryotic cells sense changes and respond to a downshift in temperature are poorly understood. Using the cyanobacteria Synechocystis as an experimental model, Murata and Los (2) suggested that a phase transition of the plasma membrane upon cold shock would trigger a conformational change of a putative cold sensor, this being the primary event in the transduction of the temperature signal. According to this hypothesis, a membrane-bound histidine kinase, Hik33, has been identified as a cold sensor in this organism, which is able to detect changes in the physical state of the membrane and regulate the expression of most cold-induced genes (3, 4). Other putative cold sensors, such as DesK in Bacillus subtilis (5) and transient receptor potential (T...

Unsaturated fatty acids play an essential role in the biophysical characteristics of cell membranes and determine the proper function of membrane-attached proteins. Thus, the ability of cells to alter the degree of unsaturation in their membranes is an important factor in cellular acclimatization to environmental conditions. Many eukaryotic organisms can synthesize dienoic fatty acids, but Saccharomyces cerevisiae can introduce only a single double bond at the ⌬ 9 position. We expressed two sunflower (Helianthus annuus) oleate ⌬ 12 desaturases encoded by FAD2-1 and FAD2-3 in yeast cells of the wild-type W303-1A strain (trp1) and analyzed their effects on growth and stress tolerance. Production of the heterologous desaturases increased the content of dienoic fatty acids, especially 18:2⌬ 9,12 , the unsaturation index, and the fluidity of the yeast membrane. The total fatty acid content remained constant, and the level of monounsaturated fatty acids decreased. Growth at 15°C was reduced in the FAD2 strains, probably due to tryptophan auxotrophy, since the trp1 (TRP1) transformants that produced the sunflower desaturases grew as well as the control strain did. Our results suggest that changes in the fluidity of the lipid bilayer affect tryptophan uptake and/or the correct targeting of tryptophan transporters. The expression of the sunflower desaturases, in either Trp ؉ or Trp ؊ strains, increased NaCl tolerance. Production of dienoic fatty acids increased the tolerance to freezing of wild-type cells preincubated at 30°C or 15°C. Thus, membrane fluidity is an essential determinant of stress resistance in S. cerevisiae, and engineering of membrane lipids has the potential to be a useful tool of increasing the tolerance to freezing in industrial strains.Tolerance to freezing is an essential trait influencing the viability and leavening capacity of baker's yeast in frozen dough (4, 42). The so-called frozen-dough technology has been widely accepted by consumers and bakers due to several advantages, which include supplying oven-fresh bakery products and improving labor conditions. However, no appropriate industrial strain with a high tolerance to freezing is available, and it is unlikely that the classical breeding program could significantly improve this trait. Freezing is a complex and multifaceted stress, in which different stressors and stress responses appear to play important roles. Cells exposed to subzero temperatures are injured by the formation of ice crystals and crystal growth during frozen storage (33). At a slow freezing rate, cells are exposed to hyperosmotic solutions and equilibrate by movement of water across the membranes (65). Finally, during the thawing process, cells can suffer biochemical damage by oxidative stress (16). It is not surprising, therefore, that tolerance to freezing involves several mechanisms working in concert.Biological membranes are the first barrier that separates cells from their environment and are a primary target for damage during environmental stress. Sudden changes in envi...

The parasitic mite Varroa destructor has a significant worldwide impact on bee colony health. In the absence of control measures, parasitized colonies invariably collapse within 3 years. The synthetic pyrethroids tau-fluvalinate and flumethrin have proven very effective at managing this mite within apiaries, but intensive control programs based mainly on one active ingredient have led to many reports of pyrethroid resistance. In Europe, a modification of leucine to valine at position 925 (L925V) of the V. destructor voltage-gated sodium channel was correlated with resistance, the mutation being found at high frequency exclusively in hives with a recent history of pyrethroid treatment. Here, we identify two novel mutations, L925M and L925I, in tau-fluvalinate resistant V. destructor collected at seven sites across Florida and Georgia in the Southeastern region of the USA. Using a multiplexed TaqMan® allelic discrimination assay, these mutations were found to be present in 98% of the mites surviving tau-fluvalinate treatment. The mutations were also found in 45% of the non-treated mites, suggesting a high potential for resistance evolution if selection pressure is applied. The results from a more extensive monitoring programme, using the Taqman® assay described here, would clearly help beekeepers with their decision making as to when to include or exclude pyrethroid control products and thereby facilitate more effective mite management programmes.

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SummaryA sudden overaccumulation of methylglyoxal (MG) induces, in Saccharomyces cerevisiae , the expression of MG-protective genes, including GPD1 , GLO1 and GRE 3. The response is partially dependent on the transcriptional factors Msn2p/Msn4p, but unrelated with the general stress response mechanism. Here, we show that the high-osmolarity glycerol (HOG)-pathway controls the genetic response to MG and determines the yeast growth capacity upon MG exposure. Strains lacking the MAPK Hog1p, the upstream component Ssk1p or the HOG-dependent nuclear factor Msn1p, showed a reduction in the mRNA accumulation of MG-responsive genes after MG addition. Moreover, hyperactivation of Hog1p by deletion of protein phosphatase PTP2 enhanced the response, while blocking the pathway by deletion of the MAPKK PBS 2 had a negative effect. In addition, the activity of Hog1p affected the basal level of GPD1 mRNA under non-inducing conditions. These effects had a great influence on MG resistance, as hog1 D D D D and other HOGpathway mutants with impaired MG-specific expression displayed MG sensitivity, whereas those with enhanced expression exhibited MG resistance as compared with the wild-type. However, MG does not trigger the overphosphorylation of Hog1p or its nuclear import in the parental strain. Moreover, dual phosphorylation of Hog1p appears to be dispensable in the triggering of the transcriptional response, although a phosphorylable form of Hog1p is fundamental for the transcriptional activity. Overall, our results suggest that the basal activity of the HOGpathway serves to amplify the expression of MGresponsive genes under non-inducing and inducing conditions, ensuring cell protection against this toxic glycolytic by-product.

We used mRNA differential display to assess yeast gene expression under cold or freeze shock stress conditions. We found both up-and down-regulation of genes, although repression was more common. We identified and sequenced several cold-induced genes exhibiting the largest differences. We confirmed, by Northern blotting, the specificity of the response for TPI1, which encodes triose-phosphate isomerase; ERG10, the gene for acetoacetyl coenzyme A thiolase; and IMH1, which encodes a protein implicated in protein transport. These genes also were induced under other stress conditions, suggesting that this cold response is mediated by a general stress mechanism. We determined the physiological significance of the cold-induced expression change of these genes in two baker's yeast strains with different sensitivities to freeze stress. The mRNA level of TPI1 and ERG10 genes was higher in freeze-stressed than in control samples of the tolerant strain. In contrast, both genes were repressed in frozen cells of the sensitive strain. Next, we examined the effects of ERG10 overexpression on cold and freeze-thaw tolerance. Growth of wild-type cells at 10°C was not affected by high ERG10 expression. However, YEpERG10 transformant cells exhibited increased freezing tolerance. Consistent with this, cells of an erg10 mutant strain showed a clear phenotype of cold and freeze sensitivity. These results give support to the idea that a cause-and-effect relationship between differentially expressed genes and cryoresistance exists in Saccharomyces cerevisiae and open up the possibility of design strategies to improve the freeze tolerance of baker's yeast.Baker's yeast (Saccharomyces cerevisiae) responds to various stresses during its propagation and industrial application (3, 30). For example, extreme environmental conditions arise during freezing, frozen storage, and thawing of bread dough, resulting in yeast cells with reduced viability and dough-leavening capacity (11,14). These effects have a great technological and economic impact because the yeast gassing rate is critical in baking. Consequently, the improvement of the freeze tolerance in baker's yeast is of significant commercial importance.The response of S. cerevisiae to cold shock stress has not been characterized in detail, but it is generally accepted that freeze-thaw tolerance correlates with cellular factors including growth phase (39), respiratory metabolism (22), lipid composition of the membrane (10), and accumulation of trehalose (23,27,37). Cultural conditions that result in yeast cells with these characteristics, especially high trehalose content, are commonly employed in the production of baker's yeast (3, 30), even though they provide stress resistance only in the absence of fermentable sugars (37). Thus, an additional mechanism(s) is thought to be triggered in response to sharp downshift changes in temperature and to be required for the maintenance of freeze tolerance.Regulatory systems that control the stress response in S. cerevisiae act primarily at the transcr...

The inoculation of active dry wine yeast (ADWY) is one of the most common practices in winemaking. This inoculation exposes the yeast cells to strong osmotic, acidic and thermal stresses, and adaptation to the new medium is crucial for successful fermentation. We have analysed the changes that occur in the ADWY protein profile in the first hours after inoculation under enological-like conditions at a low temperature. Protein changes mainly included enzymes of the nitrogen and carbon metabolism and proteins related to the cellular stress response. Most of the enzymes of the lower part of the glycolysis showed an increase in their concentration 4 and 24 h after inoculation, indicating an increase in glycolytic flux and in ATP production. However, the shift from respiration to fermentation was not immediate in the inoculation because some mitochondrial proteins involved in oxidative metabolism were induced in the first hours after inoculation. Inoculation in this fresh medium also reduced the cellular concentration of stress proteins produced during industrial production of the ADWY. The only exception was Cys3p, which might be involved in glutathione synthesis as a response to oxidative stress. A better understanding of the yeast stress response to rehydration and inoculation will lead to improvements in the handling efficiency of ADWY in winemaking and presumably to better control of fermentation startup.

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