Yeast colony survival depends on metabolic adaptation and cell differentiation rather than on stress defense

Abstract

Enzymes scavenging reactive oxygen species (ROS) are important for cell protection during stress and aging. A deficiency in these enzymes leads to ROS imbalance, causing various disorders in many organisms, including yeast. In contrast to liquid cultures, where fitness of the yeast population depends on its ROS scavenging capability, the present study suggests that Saccharomyces cerevisiae cells growing in colonies capable of ammonia signaling use a broader protective strategy. Instead of maintaining high levels of antioxidant enzymes for ROS detoxification, colonies activate an alternative metabolism that prevents ROS production. Colonies of the strain deficient in cytosolic superoxide dismutase Sod1p thus developed the same way as wild type colonies. They produced comparable levels of ammonia and underwent similar developmental changes (expression of genes of alternative metabolism and center margin differentiation in ROS production, cell death occurrence, and activities of stress defense enzymes) and did not accumulate stress-resistant suppressants. An absence of cytosolic catalase Ctt1p, however, brought colonies developmental problems, which were even more prominent in the absence of mitochondrial Sod2p. sod2Delta and ctt1Delta colonies failed in ammonia production and sufficient activation of the alternative metabolism and were incapable of center margin differentiation, but they did not increase ROS levels. These new data indicate that colony disorders are not accompanied by ROS burst but could be a consequence of metabolic defects, which, however, could be elicited by imbalance in ROS produced in early developmental phases. Sod2p and homeostasis of ROS may participate in regulatory events leading to ammonia signaling.

FIGURE 1.

Colonies formed by strains deficient in individual stress defense enzymes differ in ammonia signaling and in related metabolic changes. A, ability of wt, sod1Δ, sod2Δ, and ctt1Δ colonies to produce ammonia over 21 days (x axis). *, p < 0.05; **, p < 0.01 when mutant and wt values were compared. B, morphology of colonies growing on GMA-bromcresol purple. Intensity of violet coloring of pH indicator bromcresol purple correlates with the extent of alkalization. C, colony ability to activate adaptive changes in metabolic and transporter genes demonstrated as average change in expression of CIT3, POX1, ATO1, ATO3, and JEN1 genes. D, expression profile of stress-related genes demonstrated as average change in expression of MSN4, HSP30, and CTT1 genes. For each gene in C and D, the time point exhibiting the highest expression value was set as 100%. *, p < 0.05; **, p < 0.01; changes of particular genes are shown in supplemental Fig. S2.

FIGURE 2.

Center margin cell differentiation does not occur in sod2Δ and ctt1Δ colonies but does in sod1Δ colonies. A, presence of cells with diffuse fragmented or dispersed chromatin (FC) and partially digested shrunken cells (SH) (representatives of cell types are shown in supplemental Fig. S3A). B, level of cellular superoxide (measured with DHE) (comparison of DHE determination by spectrofluorometer with fluorescence microscopy is shown in Fig. S3B). C, level of mitochondrial superoxide (measured with MitoSOX Red). The values in the central and margin regions of wt and sod1Δ colonies significantly differ (p < 0.05 or lower) after day 10 in A and after day 15 in B and C. D, activity of Ctt1p. *, p < 0.05; **, p < 0.01. E, activity of Sod1p. *, p < 0.05; **, p < 0.01. F, heat shock tolerance (survival of untreated cells is set as 100%) in central and outer cells in developing wt, sod1Δ, sod2Δ, and ctt1Δ colonies. x axis, days of colony development ([d]). NA, no activity detected as the gene for the particular enzyme is absent; CPS, counts/s; cen, central cells; out, outer margin cells.

FIGURE 3.

GMA-grown sod1Δ colonies do not accumulate stress-resistant suppressor mutants. A, wt and sod1Δ cells grown on solid media, as indicated, were directly assayed by drop test for the presence of Met⁺ and PQ^r suppressants and for Mn²⁺ sensitivity. B, ability of GMA-sod1Δ (red) and YPDA-sod1Δ (blue) cells, respectively, of various ages (as indicated on x axis) to form Met⁺ and PQ^r suppressants when subsequently growing in either SD (SD-Lq) or YPD (YPD-Lq) liquid media for 4 days (assayed by drop test). The number of cells detected by drop test on control YPDA plates without Paraquat was set as 100% at each time point. A representative experiment of four is shown. S.D. values were calculated from measurements of the three independent samples. C, capacity of sod1Δ cells pregrown for 5 or 20 days (d) either in liquid or on solid glucose (YPD or YPDA) or glycerol (GM or GMA) media to form Met⁺ cells in SD over 4 days (assayed by drop test). Pictograms under the x axis symbolize pregrowth conditions. The total number of cells determined by drop test on control YPDA was set as 100% (usually 10⁵ cells, approximately). D, the scheme of the experimental set-up.

FIGURE 4.

GMA-sod1Δ-derived liquid cultures differ in viability and pH from YPDA-sod1Δ cultures. A, survival of GMA-sod1Δ cells from colonies of various ages (days, x axis) when subsequently growing in liquid complex glucose (YPD-Lq), minimal glucose (SD-Lq), and complex glycerol (GM-Lq) media for 4 days (assayed by drop test). Survival of YPDA-sod1Δ cells of the same age is given in parallel. *, p < 0.05; **, p < 0.01. GMA-sod1Δ versus YPDA-sod1Δ. The number of YPDA-wt (wt cells from YPDA) living cells is set as 100% for each time point. The ratio of GMA-sod1Δ survival in comparison with that of the YPDA-sod1Δ cells is given in supplemental Fig. S6. B, pH of the YPD after 1–7 days of cultivation of GMA-sod1Δ cells of various ages (x axis) and 3-day-old wt and YPDA-sod1Δ cells. C, survival of 20-day-old GMA-sod1Δ cells in SD (SD-Lq) with 2% or 4% glucose as carbon source, buffered to higher pH (+KPi) or unbuffered. Survival was estimated after 7 days of cultivation. The pH of each 7-day-old culture is given.

FIGURE 5.

Properties of individual clones obtained from GMA-grown sod1Δ colonies of various ages. A, average survival of clones obtained from central (c) or outer (o) margin regions of GMA-sod1Δ colonies of various ages (x axis) after 7 days of cultivation in liquid SD (SD-Lq), as determined by drop assay. Survival of clones derived from 3-day-old YPDA-sod1Δ strain is given for comparison. The data are the means ± S.D. of values obtained for eight independent clones at each time point. Survival of the particular clone after 24 h of cultivation in SD is set as 100%. B, survival of the eight individual clones obtained from central GMA-sod1Δ colony regions under the same conditions as in A. Survival of eight individual clones of 3-day-old YPDA-sod1Δ colony is given for comparison.

FIGURE 6.

Model of involvement of stress defense and adaptive metabolism in development and survival of different yeast populations. The cultivation of all population types starts with “chronologically young” cells. Stress defense is essential in liquid cultures to scavenge ROS. Its absence can be overcome by accumulating stress-resistant suppressants. In colonies, independently of stress defense enzymes, cells capable of ammonia signaling undergo a development leading to their differentiation, activation of adaptive metabolism (AM) and long term survival of healthy cells located at margin colony regions. This is the case of wt and sod1Δ colonies. Lack of ammonia signaling causes developmental problems, i.e. no differentiation and reduced fitness of the whole colony population both in the presence and absence of stress defense mechanisms. This occurs in colonies of stress defense mutants sod2Δ and ctt1Δ as well as in colonies of the sok2Δ mutant defective in pleiotropic regulator Sok2p (17).