Genetic Analysis of Desiccation Tolerance in Saccharomyces cerevisiae

Desiccation tolerance, the ability to survive nearly total dehydration, is a rare strategy for survival and reproduction observed in all taxa. However, the mechanism and regulation of this phenomenon are poorly understood. Correlations between desiccation tolerance and potential effectors have been reported in many species, but their physiological significance has not been established in vivo. Although the budding yeast Saccharomyces cerevisiae exhibits extreme desiccation tolerance, its usefulness has been hampered by an inability to reduce tolerance more than a few fold by physiological or genetic perturbations. Here we report that fewer than one in a million yeast cells from low-density logarithmic cultures survive desiccation, while 20–40% of cells from saturated cultures survive. Using this greatly expanded metric, we show that mutants defective in trehalose biosynthesis, hydrophilins, responses to hyperosmolarity, and hypersalinity, reactive oxygen species (ROS) scavenging and DNA damage repair nevertheless retain wild-type levels of desiccation tolerance, suggesting that this trait involves a unique constellation of stress factors. A genome-wide screen for mutants that render stationary cells as sensitive as log phase cells identifies only mutations that block respiration. Respiration as a prerequisite for acquiring desiccation tolerance is corroborated by respiration inhibition and by growth on nonfermentable carbon sources. Suppressors bypassing the respiration requirement for desiccation tolerance reveal at least two pathways, one of which, involving the Mediator transcription complex, is associated with the shift from fermentative to respiratory metabolism. Further study of these regulators and their targets should provide important clues to the sensors and effectors of desiccation tolerance. DESICCATION tolerance is the ability of an organism to withstand removal of its intracellular liquid water and then resume normal metabolism after rehydration (Crowe et al. 1992). This trait is common in the seeds of most gametophytes, which contain desiccated plant embryos (Finkelstein et al. 2008). However, rare desiccation tolerant “adult” species exist such as baker’s yeast (Lesaffre Yeast Corporation 2010), resurrection plants (Bartels 2005), and tardigrades (Sømme 1996), which are broadly distributed among the taxa of unicellular microbes and multicellular plants and animals, respectively. The ability of seeds and these extremophiles to survive desiccation is thought to depend upon their unique ability to mitigate the deleterious consequences of possibly multiple stresses occurring at all scales, from biochemical reactions to interactions among tissues (Tunnacliffe and Ricci 2006). However, the molecular basis of desiccation tolerance remains unknown. Indeed, fundamental questions remain unanswered. What are the stresses imposed by desiccation? Which of these stresses are lethal to sensitive organisms? What differences in the physiology or stress responses of tolerant organisms allows them to prevent or overcome the damage imposed by desiccation? How is desiccation tolerance regulated? Addressing these questions will contribute to the understanding of this remarkable trait and potentially reveal basic insights into water homeostasis in all cells. The impact of desiccation on cells can potentially be understood as a composite of several component stresses. Evaporation of water from the medium surrounding a cell increases external solute concentrations, leading to hyperosmotic stress and hypersalinity. Unmitigated, these stresses would lead to increased concentrations of intracellular macromolecules and ions potentially generating cellular Copyright © 2011 by the Genetics Society of America doi: 10.1534/genetics.111.130369 Manuscript received May 4, 2011; accepted for publication July 16, 2011 Supporting information is available online at http://www.genetics.org/content/ suppl/2011/08/12/genetics.111.130369.DC1. Corresponding author: Department of Molecular and Cell Biology, University of California, 16 Barker Hall, no. 3202, Berkeley, CA 94720-3202. E-mail: [email protected] Genetics, Vol. 189, 507–519 October 2011 507 toxicity through aggregation, altered reaction kinetics, or production of toxins such as reactive oxygen species (ROS) (Jiang and Zhang 2002; Kranner and Birtić 2005). These toxic molecules may in turn damage DNA, proteins, or membranes. As a result, a number of stress-response pathways and molecules have been hypothesized to be important for desiccation tolerance, including osmoregulation, ion homeostasis, DNA damage repair, and protein folding. Indeed, several potential stress-response molecules and pathways appear to be induced in the desiccation-tolerant state of desiccation-tolerant seeds and extremophiles (Goyal et al. 2005). One class of induced molecules consists of nonreducing disaccharides such as trehalose and sucrose. These compounds are thought to act as compatible solutes, maintaining osmotic balance during moderate water deficit, and perhaps exhibiting protective effects as water removal becomes extreme. Another class of induced molecules is a family of inherently unstructured hydrophilic proteins known as hydrophilins or late embryogenesis abundant (LEA) proteins (Garay-Arroyo et al. 2000; Battaglia et al. 2008). Some hydrophilins are proposed to stabilize membranes and native protein structures, either alone or in concert with trehalose (Sales et al. 2000). In addition to these small molecules, proteins dedicated to stress responses are also induced. For example, heat shock proteins that correct protein misfolding are induced during seed desiccation (Almoguera and Jordano 1992; Wehmeyer et al. 1996). Desiccation also triggers the conserved high osmolarity glycerol (HOG) pathway in yeast (O’Rourke et al. 2002) and other species (Al-Rubeai et al. 2007). However, the functional links between desiccation tolerance and these correlated stress-response pathways and molecules are either absent or controversial. For example, desiccation tolerance in the budding yeast Saccharomyces cerevisiae was interpreted to be dependent on trehalose accumulation in one study (Gadd et al. 1987), but independent of it in another (Ratnakumar and Tunnacliffe 2006). Specific genetic tests for the role of other candidate effectors such as hydrophilins or stress-response regulators of osmolarity, DNA damage repair, ROS, or salinity have not been performed. Therefore, it is unclear what stresses cause desiccation sensitivity and what stress responses are necessary for desiccation tolerance. Many of the stress factors correlated with desiccation are induced in response to most other stresses as well (Gasch et al. 2000), suggesting that desiccation stress factors may be neither unique nor novel. Furthermore, while desiccation tolerant organisms appear to exist in both desiccation sensitive and tolerant states (Crowe 1972; Gadd et al. 1987; Wright 1988), how they regulate these states is unknown. One clue may come from adult plants for which water reduction leads to a transcriptional response initiated by the phytohormone abscisic acid (Zhu 2002). However, how this response to mild water reduction relates to true desiccation tolerance is unknown. To address many of these fundamental questions about desiccation tolerance, the budding yeast S. cerevisiae appears to be an ideal model organism. In its desiccationtolerant state it shares many of the correlates of other desiccation-tolerant species or tissues such as the increased presence of trehalose and hydrophilins (Gadd et al. 1987; Sales et al. 2000; Singh et al. 2005). Furthermore, this organism is used intensively to study the deleterious effects of a number of stresses like heat shock, high osmolarity, and DNA damage as well as the highly conserved pathways that respond to those stresses. However, to date the use of these potential attributes of yeast have been limited. No systematic study of the role of known stress responses in desiccation tolerance has been reported. A few studies have performed unbiased screens for reduced desiccation tolerance of deletion mutants of nonessential genes of yeast (D’Elia et al. 2005; Shima et al. 2008). These studies have identified many different mutants that reduce the survival of desiccated cells by up to 20-fold. While potentially informative, the relatively small effect on viability makes further phenotypic and genetic studies of these mutants challenging. Here our studies of desiccation tolerance of wild-type yeast reveal that exponential and saturated yeast cultures can exhibit as much as a one-million-fold difference in desiccation tolerance. Using this more robust metric, we systematically assess the causal role of stress-response pathways in desiccation tolerance and perform unbiased screens for mutants that promote desiccation sensitivity or tolerance. Our results establish budding yeast as a powerful system to elucidate the molecular basis of desiccation tolerance. Materials and Methods