Natural variation in yeast reveals multiple paths for acquiring higher stress resistance

Abstract

Background: Organisms frequently experience environmental stresses that occur in predictable patterns and combinations. For wild Saccharomyces cerevisiae yeast growing in natural environments, cells may experience high osmotic stress when they first enter broken fruit, followed by high ethanol levels during fermentation, and then finally high levels of oxidative stress resulting from respiration of ethanol. Yeast have adapted to these patterns by evolving sophisticated "cross protection" mechanisms, where mild 'primary' doses of one stress can enhance tolerance to severe doses of a different 'secondary' stress. For example, in many yeast strains, mild osmotic or mild ethanol stresses cross protect against severe oxidative stress, which likely reflects an anticipatory response important for high fitness in nature.

Results: During the course of genetic mapping studies aimed at understanding the mechanisms underlying natural variation in ethanol-induced cross protection against H₂O₂, we found that a key H₂O₂ scavenging enzyme, cytosolic catalase T (Ctt1p), was absolutely essential for cross protection in a wild oak strain. This suggested the absence of other compensatory mechanisms for acquiring H₂O₂ resistance in that strain background under those conditions. In this study, we found surprising heterogeneity across diverse yeast strains in whether CTT1 function was fully necessary for acquired H₂O₂ resistance. Some strains exhibited partial dispensability of CTT1 when ethanol and/or salt were used as mild stressors, suggesting that compensatory peroxidases may play a role in acquired stress resistance in certain genetic backgrounds. We leveraged global transcriptional responses to ethanol and salt stresses in strains with different levels of CTT1 dispensability, allowing us to identify possible regulators of these alternative peroxidases and acquired stress resistance in general.

Conclusions: Ultimately, this study highlights how superficially similar traits can have different underlying molecular foundations and provides a framework for understanding the diversity and regulation of stress defense mechanisms.

Keywords: Comparative genomics; Cross-stress protection; Natural variation; Regulation of gene expression; Stress biology; Yeast.

Fig.1

CTT1 is partially dispensable for salt-induced, but not ethanol-induced, cross protection against H₂O₂ in a wild oak strain. A Representative acquired H₂O₂ resistance assays for wild-type S288c (lab) and YPS163 (oak) strains and respective ctt1∆ mutants. Strains were exposed to a mild ‘primary’ stress pretreatment (5% ethanol or 0.4 M NaCl in culture media) or a mock control for 60 min, collected and resuspended in fresh media, exposed to a panel of 11 doses of severe ‘secondary’ doses of H₂O₂ for 2 h, and then spot plated to score viability. B A single survival score was calculated from the viability at all H₂O₂ doses (see Methods). Each plot shows the mean and standard deviation of three independent biological replicates. The replicates for some strain conditions all had the same tolerance score and thus zero standard deviation (see Additional file 6: Table S5 and Additional file 7: Table S6 for raw numerical data). Acquired H₂O₂ resistance was significantly higher in the YPS163 ctt1∆ mutant with NaCl as the pretreatment compared to the mock control (** P < 0.01, ordinal regression analysis on raw spot scores—see Methods), while the sensitizing effect of ethanol-pretreatment on S288c was eliminated in the S288c ctt1∆ mutant (** P < 0.01, ordinal regression analysis on raw spot scores)

Fig. 2

Diverse yeast strains show complex patterns of conditional CTT1 dependency. A Representative acquired H₂O₂ resistance assays of wild-type strains and their ctt1∆ mutant derivatives. B Percent acquired H₂O₂ resistance in ctt1∆ mutants relative to that of the wild type revealed three classes of conditional CTT1 dependency (based on a cutoff of 10% or higher residual acquisition, see Methods). A fourth class represents strains with poor acquired H₂O₂ resistance following ethanol pretreatment. Error bars denote the mean and standard deviation of biological duplicates, except for S288c and YPS163 which are in biological triplicate. C Hierarchical clustering of H₂O₂ survival scores for wild-type strains following either ethanol (E) or salt (N) pretreatments, or their ctt1∆ derivatives (∆E and ∆N). Strain labels are color coded according to their origin as indicated in the key on the right

Fig. 3

Extensive variation in stress responsive gene expression across diverse yeast isolates. Hierarchical clustering of 4,262 genes with significant (FDR < 0.01) differential ethanol-responsive expression in at least one strain relative to the mean of all strains (A), and 3,918 genes with significant (FDR < 0.01) differential salt-responsive expression in at least one strain relative to the mean of all strains (B). Genes are shown as rows and log₂ fold changes for each strain’s stress response (left) or difference in stress response (right) are shown as columns. Red indicates induced and blue indicates repressed expression in response to stress, while violet indicates higher and brown indicates lower expression relative to the mean stress response of all strains. Significant regulatory enrichments are annotated to the left and functional enrichments are annotated to the right (Bonferroni-corrected P < 0.01). Full lists of regulatory and functional enrichments are in Additional file 3: Table S2

Fig. 4

Strain-specific variation in different classes of stress-activated expression changes. Total number of genes for each strain with significantly (FDR < 0.01) different (> 1.5-fold) induction patterns (A) or repression patterns (B) relative to the mean or ‘consensus’ response (see Methods). Additional file 11: Table S8 and Additional file12: Table S9 contain the genes for ethanol-responsive and salt-responsive categories, respectively

Fig. 5

msn2∆ msn4∆, skn7∆, and yap1∆ mutants have varied effects on intrinsic and acquired H₂O₂ resistance. Survival score plots representing the mean and standard deviation for biological triplicates for transcription factor mutant strains with either 5% ethanol (A) or 0.4 M NaCl (B) as the mild pretreatment. Because each transcription factor mutant was tested alongside a wild-type YPS606 and YPS606 ctt1∆ control including those shown in Additional file 2: Fig. S2, those bar graphs depict 24 replicates across all experiments. The replicates for NaCl-treated yap1∆ and skn7∆ mutants all had the same tolerance score and thus zero standard deviation (see Additional file 6: Table S5 and Additional file 7: Table S6 for raw numerical data). Asterisks represent significant differences in acquired resistance between denoted strains (*** P < 0.001 ordinal regression analysis on raw spot scores)

Fig. 6

Msn2/4, Skn7, and Yap1 regulate shared and unique targets for the YPS606 ethanol and NaCl responses. The Venn diagrams represent genes with significant defective induction for each transcription factor mutant’s (msn2∆msn4∆, skn7∆, or yap1∆) stress response (FDR < 0.01 and at least 1.5-fold reduced expression). Significant functional enrichments (Bonferroni-corrected P < 0.01) are shown below for all unique and shared sets of regulated genes

Fig. 7

Stress-responsive expression variation in genes involved in detoxifying reactive oxygen species. Hierarchical clustering of all yeast genes annotated as having peroxidase activity in response to either ethanol (A) or NaCl (B). Genes are shown as rows and log₂ fold changes for each strain’s stress response (left) or difference in stress response (right) are shown as columns. Red indicates induced and blue indicates repressed expression in response to stress, while violet indicates higher and brown indicates lower expression relative to the mean stress response of all strains

Fig. 8

CTT1-dispensable acquired H₂O₂ resistance requires glutathione biosynthesis. Survival score plots indicating the mean and standard deviation for biological triplicates for depicted strains with either 5% ethanol (A) or 0.4 M NaCl (B) as the mild pretreatment. Error bars denote the standard deviation. The replicates for NaCl-treated YPS606, YPS606 gsh1∆, M22, and M22 gsh1∆ strains all had the same tolerance score and thus zero standard deviation (see Additional file 6: Table S5 and Additional file 7: Table S6 for raw numerical data). Asterisks represent significant differences in acquired resistance between denoted strains (* P < 0.05, ns = not significant; ordinal regression analysis on raw spot scores)