Previous studies exposing yeast to different stressors have shown that although the actual stress responses elicited are unique, substantial areas of overlap, especially in patterns of gene expression, also exist. These observations are in accord with the well-studied phenomenon called “cross-protection,” which occurs when cells are exposed to a mild stress develop tolerance not only to the higher level of the same stress but also to those caused by other stress agents (Mitchel and Morrison, 1982
; Barnes et al., 1990
; Jamieson, 1992
; Flattery-O'Brien et al., 1993
; Davies et al., 1995
; Swan and Watson, 1999
; Estruch, 2000
; Pereira et al., 2001
; Palhano et al., 2004
). Overlap in the stress response at the transcript level involves about 900 genes whose expression changes across a wide range of harmful conditions (Gasch et al., 2000
). More recent studies have also revealed that the yeast growth rate profoundly impacts gene expression (Regenberg et al., 2006
; Castrillo et al., 2007
; Brauer et al., 2008
). Perhaps the most striking overlap is seen between genes that are affected by the differences in growth rate and the genes observed by Gasch et al. (2000)
to change in most or all stresses. These observations raised the possibility that much of gene expression response might be secondary to diminished growth rate caused by stress (Castrillo et al., 2007
; Brauer et al., 2008
Four Subsets of Heat-responsive Genes
We set out to classify genes on the basis of independent experimental assessment of their response to changes in temperature and growth rate. We took advantage of the chemostat to control the growth rate of the culture. We applied a short heat pulse to steady-state yeast cultures growing with doubling times ranging from 14 to 2.8 h. On the basis of their transcriptional responses, we could subdivide heat shock-responsive genes into subsets based on their response to these two variables. Not surprisingly, many genes (~200) known to be induced in response to heat shock were also highly expressed at low
growth rate. Similarly, ~200 genes that are repressed by heat shock are highly expressed at high
growth rate. These results corroborated findings of Castrillo et al. (2007)
and Brauer et al. (2008)
. However, in both cases, we also identified comparable numbers of genes that are heat-shock specific but were not affected by differences in growth rate. It is tempting, but not necessary, to interpret these results as dividing heat-shock–specific responses from more generic responses to environmental perturbations.
For the heat-repressed genes, we found significant Gene Ontology enrichment in both subsets S4 and S6 in : heat-repressed genes that are not affected by growth rate (S6) and those that are positively correlated with changes in growth rate (S4). Although both subsets are enriched for genes involved in the machinery of protein synthesis, there are differences between the two subsets. Subset (S6) is enriched for nucleotide biosynthesis and genes involved in translation functions like tRNA processing, whereas subset (S4) is dominated by ribosomal protein-encoding genes. Because cellular growth rate directly depends upon the rate of production of proteins, it is to be expected that the ribosomal protein gene expression is positively correlated with changes in growth rate, particularly if one supposes that the number of ribosomes is rate-limiting. In this view, the growth rate-insensitive subset contains genes whose expression (or the gene products themselves) are not growth rate limiting. These results are consistent with the conclusion that the decrease in transcription of ribosomal genes that accompanies virtually every kind of stress (cf. Gasch et al., 2000
) simply reflects decreased demand for ribosomes as cells divide more slowly.
Effect of Growth Rate on Heat-Shock Response
The pervasive effect of growth rate on gene expression strongly suggests that different internal conditions prevail at different steady states. We therefore compared the magnitude of the heat shock transcriptional response as a function of the growth rate to see whether growth rate has any impact. We found that the growth rate has little if any impact on the gene expression changes in response to the heat pulse. However, we did observe significant deviations for those genes that are most highly induced by the heat shock (e.g., HXT5, the representative gene for subset S2 in ). For these genes, the magnitude of the change in gene expression to the heat pulse is more dramatic at higher growth rate. Furthermore, we observed that these genes, for the most part, are already highly expressed at low steady-state growth rate, before the heat pulse. We therefore conclude that the magnitude of expression changes is influenced by expression levels before the heat shock.
It is worth noting that a related observation was made by Berry and Gasch (2008)
, who found that the gene expression response to stress is smaller if cells have been exposed previously to stress. Because a substantial fraction (~50%) of the genes are the same in the two studies, this suggests that slow growth might be seen as a mild stress. In contrast, the mild stress might have impacted growth rate, and thus this observation, consistent between the two studies, is unhelpful in distinguishing whether the ultimate reason for the gene expression changes is “stress” or “growth rate.”
The heat-resistance experiments we carried out demonstrated conclusively that slow growth confer cross protection to heat shock, and presumably other types of stress. Typically, cross-protection has been studied between oxidative, salt, osmolarity, and heat stresses. Starvation, a condition that approximates low growth rate, has also been studied. Our observation of cross-protection from heat shock suggests, at the surface, that the relevant protecting genes and functions might be found among subset S2, i.e., those genes that are induced both by heat and slow growth. Thus, one could make the argument that the similar phenotype (i.e., cross-protection) of slow growing cells with those that have been stressed by traditional means seems to suggest that the slow-growing cells behave as if they are under stress.
Role of Oxidative Metabolism in Gene Expression
This line of reasoning then leads to the question of what might be the reason that slow growth induces protective functions. One appealing possibility is that slow growth, even on a fermentable carbon source, differs from rapid growth in that it involves higher levels of respiration. With respect to the bulk of the gene expression changes associated with slow growth, on the one hand, and stress, on the other, our data strongly support this view. A respiratory-deficient ([rhoo]
) culture shows a markedly reduced change in expression of most of the genes associated with the generic stress response (Gasch et al., 2000
) compared with an isogenic wild-type culture growing under the identical conditions.
Additional features of our results are relevant to the role of oxidative metabolism in the influence of growth rate on gene expression. At the physiological level, we observed that at steady-state, slow growing cultures are more dense, produce less ethanol and consume more glucose. This decrease in the production of ethanol, coupled with an increase in the consumption of glucose, is consistent with the assumption that slow growing cells are shifting their carbon flow more toward respiration. At the gene expression level, we also observed an enrichment in genes that participate in the respiratory process and peroxisomal localization at the low growth rate, a result also seen by previous study (Brauer et al., 2008
). Also, we observed in these data, as we had seen before during experiments involving the diauxic shift (Brauer et al., 2005
), that the enzymes of the tricarboxylic acid cycle are induced in slow growth as well (Supplemental Table 2.
These observations provide indirect evidence of a metabolic shift that accompanies a reduction in growth rate. We are left with the general picture that at high growth rates and in the presence of excess glucose fermentation is the predominant mode of energy production, whereas at low growth rates the cell population shifts to increasing dependence on mitochondrial function. It is known that actively respiring cells are susceptible to oxidative damage caused by ROS produced by the electron transport chain, the principal site being close to the cytochrome c
oxidase complex (Guidot et al., 1993
). The increased oxidative damage experienced by cells would elicit a stress response. Not surprisingly, respiring cells grown on nonfermentable carbon source have been observed to be more resistant to oxidative stress than those grown on glucose (Jamieson, 1992
; Flattery-O'Brien et al., 1993
). Correspondingly petite ([rhoo]
) mutants, whose mitochondrial functions have been impaired, are unable to respire and thus have been observed to be more sensitive to various type of oxidative stress (Grant et al., 1997
; Maris et al., 2001
). Furthermore, because the stress response elicited by ROS in the respiring cells is very similar to that induced by heat shock (Godon et al., 1998
; Lee and Park, 1998
; Sugiyama et al., 2000
), Moraitis and Curran (2004
have demonstrated that heat shock response and thermotolerance are strongly influenced by the level of ROS.
No Difference in Cross-Protection in Respiratory-deficient Cultures
With this result in hand, it is logical to expect differences in cross-protection against heat killing in respiratory-deficient and respiratory-competent strains, because most (but not all) of the genes in subsets S2 and S4 are expressed differently in the petite strain. Surprisingly, however, when we tested the cross-protection in both respiratory-deficient and respiratory-competent strains at 50°C and at two different growth rates, we found that the survival curves between the two strains with equal growth rate were virtually identical. Thus, we are forced to conclude that the genes and functions responsible for cross-protection are not being induced by some by-product of respiratory metabolism.
We can readily envision a relatively simple explanation for these apparently conflicting results. Among the many genes in subset S2, there may well be quite a few that are induced equally in wild-type and respiratory-deficient cells. The basis for their induction would have to be some direct readout of growth rate unrelated to oxidative metabolism. Alternatively, there might be a threshold of activity that is passed by even the moderate induction found in the respiratory-deficient strain.
Is Slow Growth Stressful?
The evidence presented in this paper demonstrates an inverse relationship between the rate at which cells divide and how resistant they are to heat stress. This behavior bears striking resemblance to cross-protection, in which exposure to one form of stress often confers resistance to another. This leads to the idea that cell with long doubling time seem to be stressed. Three features of slowly growing cells support this notion (Castrillo et al., 2007
; Brauer et al., 2008
). At the level of gene expression, most of the known stress response genes are highly induced at slow growth. At the level of cell cycle regulation, slower growing cells spend larger fractions of their cell cycle in the G0/G1 (i.e., unbudded) phase. At the physiological level, as we have shown here, slow-growing cells are more resistant to lethal heat challenge.
We have dealt with the transcriptional regulation above, by clearly demonstrating that most of the commonly known stress genes are induced during slow growth in the chemostat. Furthermore, we also observed that known stress genes are expressed at a lower level in respiratory-defective [rhoo]
mutants than in wild type at equal growth rate. This implies that there may well be a role for respiratory metabolism or its consequences in activating the regulators of these genes. This might actually be oxidative damage itself, or, alternatively, it might be some other feature of oxidative metabolism such as the proton flux across the mitochondrial membrane or a metabolite associated with a peroxisomal function. In this regard, we must also consider the possibility of metabolic cycling, wherein it is thought that there might be an alternation of oxidative and fermentative metabolism in wild-type cells (Klevecz et al., 2004
; Tu et al., 2005
). Obviously, this explanation will not account for respiration-independent induction of cross-protection.
The cell cycle presents an attractive possibility for a respiration-independent readout of growth rate. It has been reported in the literature that thermal stress induces a transient arrest in the G1 phase of the cell cycle which causes the accumulation of unbudded cells (Johnston and Singer, 1980
). Subsequent studies showed that this heat-induced arrest at the cell cycle regulatory step of START was related to the decrease in transcript abundance of G1 cyclins CLN1
(Rowley et al., 1993
). The accumulation of cells in G1 in response to stress and slow growth suggests that cells in G1, in which no replication or division is taking place, are more stress resistant, suggesting a possible relationship between thermotolerance and cell cycle (Plesset et al., 1987
). However, other studies have shown that thermotolerance is largely independent of cell cycle position (Barnes et al., 1990
; Elliott and Futcher, 1993
). Our results with growth rate-dependent cross protection against heat challenge in respiration-deficient cells may well motivate a reexamination of the possibility of a cell cycle-dependent readout of growth rate.
At the physiological level, a promising avenue is the link between nutrient sensing and stress response. In yeast, the cAMP-dependent protein kinase (PKA) pathway has a strong influence on stress resistance. Mutants with an elevated cAMP–PKA pathway display lower stress resistance, whereas mutants with this pathway repressed show higher stress resistance (Toda et al., 1987
; Cameron et al., 1988
; Park et al., 1997
). Such mutants, although highly stress resistant, also exhibits longer lag phase and slow growth. However, a mutant fil
1 (fermentation-induced loss of stress resistance) with a mutation in adenylate cyclase was isolated and found to have a 10-fold drop in adenylate cyclase activity (Van Dijck et al., 2000
). What made this mutant interesting was that although it exhibited more freeze and drought resistance than that of the wild type, its fermentative growth rate with the wild type is comparable, demonstrating that high stress resistance and normal growth are not mutually exclusive. Interestingly it was also observed that the presence of fil
1 mutation markedly increased the stress resistance of strains deleted for MSN2
, and TPS1
(Versele et al., 2004
). This suggests that there are factors beyond trehalose, Hsp104 and the Msn2/4-controlled genes that play a role in conferring general stress resistance.
Finally, we raise the possibility that slow growth may be the default mode of growth, i.e., the environment in which the genome of yeast was forged by selection over evolutionary time. In this view, all genes that provide cross protection and are viewed by the literature as indications of unusual environmental stress are actually normally expressed, because yeast evolved in slow growth conditions. In this view, the normal scientific laboratory conditions (rich medium, excess glucose) are unusual, and yeast, normally expecting slow growth, heat pulses, radiation exposure, changes in osmolarity and salt provide themselves with the means to resist them all. Under this view, we should require an explanation of why at fast growth rates in rich media these genes are turned off. It could well be that evolution favored populations that could temporarily increase their growth rate by abandoning their defenses when conditions such as food supply and temperature are optimal. Under this view, favorable environmental conditions represent rare but evolutionarily important opportunities for rapid population expansion, and what we currently think of as “stressful” is probably the default lifestyle to which the yeast has been accustomed.