Osmotic shock does not result in detectable ROS production. The upper panels show ROS visualized with dihydroethidium in untreated samples or in samples treated with 1 mM tert-butyl hydroperoxide (positive control), 0.5 M NaCl, or 1 M sorbitol. The lower panels show the same cells through the bright filter.

Yeast cells recover faster from osmotic shock under anaerobiosis. Heat production was used to monitor metabolic activity. Solid lines represent the aerobic culture, and dashed lines represent the anaerobic culture. The first peak in heat production represents fermentative growth, and the second, which is present only in the aerobic culture, represents ethanol respiration. Panel A shows the complete heat production profile of the two cultures, while B is a logarithmic display of the critical period indicated by a hatched bar on the x axis in panel A. Exponentially growing cells were subjected to an osmotic shock at time zero. The dashed and solid vertical lines indicate when the anaerobic and aerobic cultures, respectively, resumed exponential heat production, which occurred after approximately 40 min in the anaerobic culture and after 90 min in the aerobic culture. Arrows indicate sampling points for global expression analysis.

The correlation between the aerobic and anaerobic responses to osmotic stress is very high. The figure illustrates the behavior of the genes included in this study and is a numerical representation of the clustering results (see Fig. 4). Expression of 105 genes was significantly downregulated upon osmotic shock under aerobic conditions. Expression of 101 (96%) of those was clearly downregulated also under anaerobic conditions, but only 11 (10%) of those passed the filtering criteria (row 1). Likewise, expression of 82 genes was significantly upregulated by osmotic shock under aerobic conditions. Expression of 77 (94%) of those was clearly upregulated also under anaerobic conditions, but only 42 (51%) passed the filtering criteria (row 2). Moreover, all 19 genes whose expression was significantly downregulated upon osmotic shock under anaerobic conditions were also downregulated under aerobic conditions (column 1). In contrast, only 52 (75%) of the 69 genes whose expression was significantly upregulated under anaerobic conditions were also upregulated aerobically (column 2).

Two-dimensional visualization of the correlation between aerobic and anaerobic expression. The upper (x) axis holds the cluster trees based on data obtained from the anaerobic culture. Clusters are labeled with roman numerals. The left (y) axis holds the cluster trees based on data obtained from the aerobic culture. These clusters are labeled with arabic numerals. Each gene occurs once on each axis, and ▪ marks the intersection between the positions on the two axes. The figure contains three different but connected plots that are separated by the dark grey lines. The upper left part contains the genes whose expression was significantly altered under both conditions (A versus C), the upper right part contains those whose expression was significantly altered under aerobic conditions only (A versus D), and the lower left contain those genes whose expression was significantly altered under anaerobic conditions only (B versus C). Cluster trees A and C are subdivided by general behavior, i.e., downregulation (group 1/I) or upregulation (group 2/II), by the lighter grey lines. These groups are further divided at a correlation of <0.75 by the thin black lines, grouping genes with closely related behavior. The grouping, rather than the physical distance, indicates similarity in behavior. Cluster trees B and D are subdivided by general behavior, i.e., downregulation (group 3/III), upregulation (group 5/V), and no or an ambiguous effect (groups 4/IV and 6). Figure 3 provides a numerical and simpler view. Further information can be found in Fig. S1 and S2 in the supplemental material. The color panels along the x and y axes show the induction profiles under anaerobic and aerobic conditions, respectively. Red indicates stimulated expression and green indicates diminished expression, compared to the average of the prestress time points. The time points are −30, −5, 10, 15, 20, 30, 40, and 60 min, starting from the periphery.

Expression profiles of selected genes. (A) Examples of expression profiles of genes discussed in this study. (B) Expression profiles of the 12 genes implicated in the oxidative stress response. The x axis displays time in minutes, and the y axis displays mRNA levels in arbitrary units. Closed squares represent aerobic expression patterns, and open squares represent anaerobic expression patterns.

Expression of genes encoding enzymes of the ergosterol biosynthetic pathway. The genes are listed in the order in which their products function in ergosterol biosynthesis. Erg10 to Erg20 constitute the first part of the pathway, and Erg9 to Erg4 constitute the second part. The left panels display the ratio between aerobic conditions and anaerobic conditions. Red indicates higher expression under aerobic conditions, and green indicates higher expression under anaerobic conditions. The middle and right panels show the expression profiles under aerobic and anaerobic conditions, respectively. Red indicates stimulated expression and green diminished expression, compared to the average for the prestress time points. Note the presence of a clear trend of induction under anaerobic but not under aerobic conditions.

Anaerobic cultures display more transient Hog1p phosphorylation and target gene activation as well as faster glycerol accumulation. Osmotic stress activates the HOG pathway rapidly during both aerobiosis and anaerobiosis, but the signal is more transient in the anaerobically growing cells, indicative of a faster adaptation (A). Accordingly, induction of the HOG pathway-dependent STL1 gene is strongly reduced during anaerobic conditions, in both amplitude and duration (B). Furthermore, the accumulation of intracellular glycerol occurs faster during anaerobiosis (C). Closed squares represent the aerobic culture, and open squares represent the anaerobic culture. OD₆₁₀, optical density at 610 nm.

GPD1 overexpression mimics anaerobic conditions. Cells overexpressing (O.E.) GPD1, i.e., with an increased intrinsic glycerol production capacity, show a more transient HOG pathway activation (A). Furthermore, these cells show a reduced STL1 induction (B) and a faster accumulation of intracellular glycerol (C). Closed circles represent the wild type, and open circles represent the GPD1-overexpressing strain. OD₆₁₀, optical density at 610 nm.