genetic analysis of desiccation tolerance in saccharomyces...

INVESTIGATION

Genetic Analysis of Desiccation Tolerancein Saccharomyces cerevisiae

Dean Calahan,*,† Maitreya Dunham,‡ Chris DeSevo,‡ and Douglas E. Koshland§,1

*Department of Biology, The Johns Hopkins University, Baltimore, Maryland 21218, †Department of Embryology, CarnegieInsitution for Science, Baltimore, Maryland 21218, ‡Department of Genome Sciences, University of Washington, Seattle,Washington 98195, and §Howard Hughes Medical Institute and Department of Molecular and Cell Biology, University of

California, Berkeley, California 94720

ABSTRACT Desiccation tolerance, the ability to survive nearly total dehydration, is a rare strategy for survival and reproductionobserved in all taxa. However, the mechanism and regulation of this phenomenon are poorly understood. Correlations betweendesiccation tolerance and potential effectors have been reported in many species, but their physiological significance has not beenestablished in vivo. Although the budding yeast Saccharomyces cerevisiae exhibits extreme desiccation tolerance, its usefulness hasbeen hampered by an inability to reduce tolerance more than a few fold by physiological or genetic perturbations. Here we report thatfewer than one in a million yeast cells from low-density logarithmic cultures survive desiccation, while 20–40% of cells from saturatedcultures survive. Using this greatly expanded metric, we show that mutants defective in trehalose biosynthesis, hydrophilins, responsesto hyperosmolarity, and hypersalinity, reactive oxygen species (ROS) scavenging and DNA damage repair nevertheless retain wild-typelevels of desiccation tolerance, suggesting that this trait involves a unique constellation of stress factors. A genome-wide screen formutants that render stationary cells as sensitive as log phase cells identifies only mutations that block respiration. Respiration asa prerequisite for acquiring desiccation tolerance is corroborated by respiration inhibition and by growth on nonfermentable carbonsources. 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 ofthese regulators and their targets should provide important clues to the sensors and effectors of desiccation tolerance.

DESICCATION tolerance is the ability of an organism towithstand removal of its intracellular liquid water and

then resume normal metabolism after rehydration (Croweet al. 1992). This trait is common in the seeds of mostgametophytes, which contain desiccated plant embryos(Finkelstein et al. 2008). However, rare desiccation tolerant“adult” species exist such as baker’s yeast (Lesaffre YeastCorporation 2010), resurrection plants (Bartels 2005), andtardigrades (Sømme 1996), which are broadly distributedamong the taxa of unicellular microbes and multicellularplants and animals, respectively. The ability of seeds andthese extremophiles to survive desiccation is thought to de-pend upon their unique ability to mitigate the deleterious

consequences of possibly multiple stresses occurring at allscales, from biochemical reactions to interactions amongtissues (Tunnacliffe and Ricci 2006). However, the molecu-lar basis of desiccation tolerance remains unknown. Indeed,fundamental questions remain unanswered. What are thestresses imposed by desiccation? Which of these stressesare lethal to sensitive organisms? What differences in thephysiology or stress responses of tolerant organisms allowsthem to prevent or overcome the damage imposed by des-iccation? How is desiccation tolerance regulated? Address-ing these questions will contribute to the understanding ofthis remarkable trait and potentially reveal basic insightsinto water homeostasis in all cells.

The impact of desiccation on cells can potentially beunderstood as a composite of several component stresses.Evaporation of water from the medium surrounding a cellincreases external solute concentrations, leading to hyper-osmotic stress and hypersalinity. Unmitigated, these stresseswould lead to increased concentrations of intracellularmacromolecules and ions potentially generating cellular

Copyright © 2011 by the Genetics Society of Americadoi: 10.1534/genetics.111.130369Manuscript received May 4, 2011; accepted for publication July 16, 2011Supporting information is available online at http://www.genetics.org/content/suppl/2011/08/12/genetics.111.130369.DC1.1Corresponding author: Department of Molecular and Cell Biology, University ofCalifornia, 16 Barker Hall, no. 3202, Berkeley, CA 94720-3202.E-mail: [email protected]

Genetics, Vol. 189, 507–519 October 2011 507

http://www.genetics.org/content/suppl/2011/08/12/genetics.111.130369.DC1


mailto:[email protected]

toxicity through aggregation, altered reaction kinetics, orproduction of toxins such as reactive oxygen species (ROS)(Jiang and Zhang 2002; Kranner and Birtić 2005). Thesetoxic molecules may in turn damage DNA, proteins, or mem-branes. As a result, a number of stress-response pathwaysand molecules have been hypothesized to be important fordesiccation tolerance, including osmoregulation, ion homeo-stasis, DNA damage repair, and protein folding.

Indeed, several potential stress-response molecules andpathways appear to be induced in the desiccation-tolerantstate of desiccation-tolerant seeds and extremophiles (Goyalet al. 2005). One class of induced molecules consists of non-reducing disaccharides such as trehalose and sucrose. Thesecompounds are thought to act as compatible solutes, main-taining osmotic balance during moderate water deficit, andperhaps exhibiting protective effects as water removalbecomes extreme. Another class of induced molecules isa family of inherently unstructured hydrophilic proteinsknown as hydrophilins or late embryogenesis abundant(LEA) proteins (Garay-Arroyo et al. 2000; Battaglia et al.2008). Some hydrophilins are proposed to stabilize mem-branes and native protein structures, either alone or in con-cert with trehalose (Sales et al. 2000). In addition to thesesmall molecules, proteins dedicated to stress responses arealso induced. For example, heat shock proteins that correctprotein misfolding are induced during seed desiccation(Almoguera and Jordano 1992; Wehmeyer et al. 1996). Des-iccation also triggers the conserved high osmolarity glycerol(HOG) pathway in yeast (O’Rourke et al. 2002) and otherspecies (Al-Rubeai et al. 2007).

However, the functional links between desiccation toler-ance and these correlated stress-response pathways andmolecules are either absent or controversial. For example,desiccation tolerance in the budding yeast Saccharomycescerevisiae was interpreted to be dependent on trehalose ac-cumulation in one study (Gadd et al. 1987), but indepen-dent of it in another (Ratnakumar and Tunnacliffe 2006).Specific genetic tests for the role of other candidate effectorssuch as hydrophilins or stress-response regulators of osmo-larity, DNA damage repair, ROS, or salinity have not beenperformed. Therefore, it is unclear what stresses cause des-iccation sensitivity and what stress responses are necessaryfor desiccation tolerance. Many of the stress factors corre-lated with desiccation are induced in response to most otherstresses as well (Gasch et al. 2000), suggesting that desic-cation stress factors may be neither unique nor novel. Fur-thermore, while desiccation tolerant organisms appear toexist in both desiccation sensitive and tolerant states (Crowe1972; Gadd et al. 1987; Wright 1988), how they regulatethese states is unknown. One clue may come from adultplants for which water reduction leads to a transcriptionalresponse initiated by the phytohormone abscisic acid (Zhu2002). However, how this response to mild water reductionrelates to true desiccation tolerance is unknown.

To address many of these fundamental questions aboutdesiccation tolerance, the budding yeast S. cerevisiae

appears to be an ideal model organism. In its desiccation-tolerant state it shares many of the correlates of other des-iccation-tolerant species or tissues such as the increasedpresence of trehalose and hydrophilins (Gadd et al. 1987;Sales et al. 2000; Singh et al. 2005). Furthermore, this or-ganism is used intensively to study the deleterious effects ofa number of stresses like heat shock, high osmolarity, andDNA damage as well as the highly conserved pathways thatrespond to those stresses. However, to date the use of thesepotential attributes of yeast have been limited. No system-atic study of the role of known stress responses in desicca-tion tolerance has been reported. A few studies haveperformed unbiased screens for reduced desiccation toler-ance of deletion mutants of nonessential genes of yeast(D’Elia et al. 2005; Shima et al. 2008). These studieshave identified many different mutants that reduce the sur-vival of desiccated cells by up to 20-fold. While potentiallyinformative, the relatively small effect on viability makesfurther phenotypic and genetic studies of these mutantschallenging.

Here our studies of desiccation tolerance of wild-typeyeast reveal that exponential and saturated yeast culturescan exhibit as much as a one-million-fold difference indesiccation tolerance. Using this more robust metric, wesystematically assess the causal role of stress-responsepathways in desiccation tolerance and perform unbiasedscreens for mutants that promote desiccation sensitivity ortolerance. Our results establish budding yeast as a powerfulsystem to elucidate the molecular basis of desiccationtolerance.

Materials and Methods

Yeast strains

Standard methods were used for strain construction (Table 1)(Rose et al. 1990).

Media and growth conditions

Standard rich media were prepared as described previously(Rose et al. 1990). Assay buffer for desiccation toleranceassays was either deionized reverse-osmosis water or a ·8dilution of phosphate buffered saline: NaCl, 17.1 mM; KCl,0.338 mM; Na2HPO4, 1.25 mM; KH2PO4, 0.220 mM; pH 7.4.

Anaerobic culture was performed in an Invivo2 400 hyp-oxic workstation (Biotrace) at 23�. A test-tube rack securedto a vortexer was used to agitate 5-ml cultures. Reagentsand equipment were deoxygenated by preconditioning for24 hr in the workstation. To support anaerobic growth of S.cerevisiae, YEPD was supplemented with 0.5% Tween-80(Cole-Parmer) and 20 mg/ml ergosterol (Sigma) (Andrea-sen and Stier 1953, 1954). Log-phase cultures were initiatedfrom overnight cultures grown anaerobically. Saturated an-aerobic cultures were obtained by 48 hr of anaerobic incu-bation. Desiccation was performed by anaerobic air dryingin 96-well plates.

508 D. Calahan, M. Dunham, and D. E. Koshland

Quiescent and nonquiescent fractions of a 7-day culturewere obtained as described previously (Allen et al. 2006),except that fractions were washed in dilute PBS. Generationof r2 strains by treatment with ethidium bromide was per-formed as previously described (Goldring et al. 1971).

Quantitative assay

Cultures were incubated for 48 hr at 30�. Samples wereprepared by washing �107 cells twice in 1 ml assay bufferand bringing the final volume to 1 ml. Undesiccated controlswere plated for colony counting. For vacuum desiccation,two 100-ml aliquots were transferred to 1.5-ml microcentri-fuge tubes and pelleted at 14,000 rpm in a microcentrifuge.A total of 85 ml of supernatant was removed, and the tubeswere subjected to vacuum desiccation (Speedvac Concentra-tor SVC 100H, Savant Systems), without added heat, forbetween 5 and 24 hr. For air drying, two 100-ml aliquotswere transferred to wells of a 96-well plate (Becton Dick-inson, 353075), and allowed to desiccate in a 23� incubator,with the lid raised (Supporting Information, Figure S2), for48 hr. Samples were resuspended in assay buffer and platedfor colony counting. Data were entered into a spreadsheet(Microsoft Excel 2004 for Mac version 11.2), and the num-ber of colony forming units per milliliter (CFU/ml) for eachplate was computed. For each experiment, CFU/ml for thetwo controls was averaged. The relative viability of each ofthe two experimental samples was determined by dividingthe CFU/ml for that sample by the average CFU/ml of thecontrol plates. These two relative viability values were thenaveraged and their standard deviation was computed usingthe STDEVP worksheet function.

Semiquantitative assay

Samples were prepared and desiccated as described (seeMaterials and Methods, Quantitative assay). Five serial ·10dilutions of each control or sample were prepared in 96-wellplates, and 5 ml of each of the six dilutions were sequentiallyspotted to YEPD/agar.

Data normalization

To combine values obtained from more than one experi-ment, relative viability numbers for experimental samplesare divided by the relative viability obtained from the wild-type internal control assayed in the same experiment, andare reported as “normalized percent relative viability.”

Comparative genomic hybridization

Genome-wide detection of single nucleotide polymorphismswas performed as described previously (Gresham et al.2006).

Determination of complementation groups

Each of the 14 revertant MATa r2 strains was crossed witheach of the 12 revertant recessive MATa r2 strains. Thediploid progeny of these crosses were assayed semiquantita-tively (see Materials and Methods, Semiquantitative assay)

with vacuum desiccation. The parent strains were assignedto the same complementation group if the diploid was des-iccation tolerant.

Genome-wide screen

Using a replicate of the MATa deletion collection in 96-wellplate format, each plate was replicated with a pronged man-ifold into YEPD (75 ml/well). After 2 days of growth at 30�,each culture was replicated onto YEPD/agar as an undesic-cated control. The lid of the plate was then elevated to allowthe culture to air dry (Figure S4). The desiccated wells wererehydrated and replicated to YEPD/agar (Figure S3).

Respiration inhibition

Myxothiazol (Thierbach and Reichenbach 1981) was ap-plied at a concentration of 30 mg/ml from a 1 mg/ml stocksolution of the inhibitor in methanol. To phenocopy the r2

mutation, a single colony of wild-type yeast was inoculatedinto 5 ml of treated YEPD and grown to saturation, and theassay buffer used to wash and desiccate the cells was alsotreated with inhibitor. To inhibit respiration during growthonly, 1 ml of the inhibited culture was washed twice in 1 mlof inhibitor-free assay buffer before desiccation. To inhibitrespiration during desiccation only, a sample prepared froman untreated saturated culture was washed and desiccatedin assay buffer containing inhibitor.

Generation of spontaneous revertants

A total of 5 ml YEPD was inoculated from a single colony ofr2 yeast. Cultures were incubated for 48 hr, then desiccatedunder vacuum. A total of 100 ml of a 101· dilution of thedesiccated sample was spread on YEPD agar and incubatedat 30� for 48 hr. The colonies that developed were pooled bytransferring 1 ml YEPD to the surface of the plate and sus-pending the colonies in that liquid. A total of 50 ml of thissuspension was used to inoculate 5 ml of liquid YEPD andthe growth/desiccation cycle was repeated. A total of 5 mg/ml ampicillin was added in further rounds to inhibit contam-inaion. When hundreds or thousands of colonies developedon a plate after desiccation rather than the dozens usuallyobserved, single colonies from that plate were cultured in-dividually and assayed for desiccation tolerance.

Inhibition of translation and transcription

Samples were prepared as described (see Materials andMethods, Quantitative assay). Transcription: 1 ml each ofDMSO and a 5 mg/ml stock solution of thiolutin (Pfizer)in DMSO were added to produce a final concentration of5 mg/ml thiolutin and 0.2% DMSO. Translation: 2 ml ofa 5 mg/ml stock solution of cycloheximide in DMSO wereadded to produce a final concentration of 10 mg/ml cyclo-heximide and 0.2% DMSO. Untreated controls were broughtto 0.2% DMSO by adding 2 ml solvent to the sample. Trea-ted and untreated samples were then assayed using vacuumdesiccation.

Yeast Desiccation Tolerance Genetics 509

http://www.genetics.org/cgi/data/genetics.111.130369/DC1/1


http://www.genetics.org/content/suppl/2011/08/12/genetics.111.130369.DC1/FigureS4.pdf


Optical density

Measurements were taken on a Gilford Stasar III spectro-photometer at a wavelength of 600 nm. Dense cultures werediluted to achieve an optical density of between 0.100 and0.500.

Results

Characterization of desiccation tolerance in wild-typebudding yeast

To measure desiccation tolerance in S. cerevisiae, we devel-oped a quantitative colony counting assay to determine theproportion of cells in a growing culture that survive desic-cation. Briefly, cultures are harvested and brought to a celldensity of between 0.5 · 108 and 1.0 · 108 cells/ml byeither dilution or concentration, depending on the originalcell density. An aliquot is removed to determine cell viabilityprior to desiccation. The remaining culture is desiccated,samples are rehydrated and then assayed for viability. Theratio of the viability of the desiccated sample to undesic-cated control provides a measure of the desiccation toler-ance and is expressed as percent relative viability.

The percent relative viability after desiccation is �5–20%for saturated cultures of typical laboratory wild-type yeaststrains grown for 48 hr in YEPD. During development of thisassay we noticed that there is no difference in relative viabil-ity obtained by desiccating samples rapidly under vacuumcentrifugation or slow air drying (Figure S1). However, thepercent relative viability can differ two- to five-fold for wild-type cultures in different desiccation experiments, potentiallyreflecting subtle differences in growth or desiccation condi-tions. Therefore, all desiccation experiments include a wild-type saturated culture as an internal standard. Normalizationto the wild-type culture in each experiment allows compari-son between cultures in different desiccation experiments. Itshould be noted that these normalizations are usually irrele-vant as our conclusions are based upon differences in rela-tive viability of many orders of magnitude betweencultures. With this assay, we began our studies by charac-terizing how desiccation tolerance varies with cell growth,the time spent in the desiccated state, and speciation.

To address how desiccation tolerance varies as a functionof cell growth, we established a wild-type culture in logphase (6.0 · 106 cells/ml), and quantitatively assayed it fordesiccation tolerance at intervals until it reached late log

Table 1 Saccharomyces cerevisiae and other strains used in this study

Strain Genotype Source

BY4742 MATa his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 ATCCcat1Δ MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 cat1::KanR ATCCcta1Δ MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 cta1::KanR ATCCgre1Δ sip18Δ MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 gre1::KanR sip18::KanR This studygre1Δ hsp12Δ sip18Δ MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 gre1::KanR sip18::KanR hsp12::HygR This studygre1Δ MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 gre1::KanR ATCChal1Δ MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 hal12::KanR ATCChal5Δ MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 hal5::KanR ATCChsp12Δ MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 hsp12::KanR ATCChsp104Δ MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 hsp104::KanR ATCChog1Δ MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 hog1::KanR ATCCmet22Δ MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 met22::KanR ATCCmrpl16Δ MATa his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 mrpl16::KanR

msb2Δ MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 msb2::KanR ATCCmsn2Δ MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 msn2::KanR ATCCmsn4Δ MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 msn4::KanR ATCCmsn2Δ msn4Δ MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 msn2::KanR msn4::HygR This studynst1Δ MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 nst1::KanR ATCCopy2Δ MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 opy2::KanR ATCCpbs2Δ MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 pbs2::KanR ATCCrad52Δ MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 rad52::KanR ATCCrck2Δ MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 rck2::KanR ATCCsat4Δ MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 sat4::KanR ATCCsip18Δ MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 sip18::KanR ATCCsod1Δ MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 sod1::KanR ATCCsod2Δ MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 sod2::KanR ATCCssk2Δ MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 ssk2::KanR ATCCssk22Δ MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 ssk22::KanR ATCCtps1Δ MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 tps1::KanR ATCCtps2Δ MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 tps2::KanR ATCCtps3Δ MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 tps3::KanR ATCCS. pastorianusC. albicans (BC15) Clinical isolate Brendan CormackC. glabrata (BC615) Clinical isolate Brendan Cormack



phase (1.7 · 108 cells/ml) (Figure 1A). The percent relativeviability is ,1025 at cell densities ,4.6 · 107 cells/ml.Above this density, the percent relative viability rises withincreased cell density, eventually reaching a value of 0.5%.These data establish a dramatic increase in desiccation tol-erance at late log phase.

Since desiccation tolerance increases from late log phaseto saturation, reaching 20% after 48 hr, we wonderedwhether the proportion of tolerant cells would continue toincrease in cultures grown for longer than 48 hr. Weinoculated a culture of wild-type cells in YEPD and assayedit every 24 hr for 7 days (Figure 1B). Percent relative via-bility rises to a maximum of 69% at 3 days, then slowlydeclines to 20%. Since cells in a saturated culture havestopped dividing, this finding indicates that changes in me-tabolism within the cell can have a measurable effect ondesiccation tolerance. Furthermore even after prolonged in-cubation, the saturated culture persists as a mixture of des-iccation tolerant and sensitive cells.

Precedence exists for two populations of cells withinsaturated yeast cultures. Density sedimentation of saturatedcultures reveals a light and heavy cell population; the latterhas severely reduced metabolism and has been character-ized as quiescent (Allen et al. 2006). We wondered whetherthe quiescent population might correspond to the desicca-tion tolerant cells. We separated a 7-day-old culture of wild-type yeast into light and heavy bands and assayed each bandseparately (Figure 1C). Cells in the heavy and light bandsshow equivalent desiccation tolerance. Thus desiccation tol-erance appears to be independent of quiescence.

We next addressed how stable the desiccation tolerant stateis in saturated cultures. First we asked whether desiccated cells

lose viability with time after they are desiccated. We simulta-neously desiccated multiple samples of a saturated culture ofwild-type yeast, then rehydrated them at different intervalsover 1 month and assayed them for viability (Figure 1D). Thepercent relative viability remains �20% independent of thetime spent in the desiccated state. We conclude that the des-iccated state is indeed stable and that desiccated cells are ableto prevent or mitigate any further damage induced duringstorage under ambient conditions.

Second, we asked how stable are the desiccation tolerantcells in saturated cultures upon return to exponential growth.Saturated cultures are used as inocula for exponentialcultures. Since the exponential cultures are very desiccationsensitive, the desiccation tolerant cells in the saturatedinocula must either lose their tolerance or retain theirtolerance and be diluted out by newly generated daughtercells and their progeny. To distinguish between thesepossibilities, we diluted a saturated culture to a density of4.1 · 106 cells/ml in fresh media and then maintained theculture at this density for multiple divisions by rediluting theculture each time it doubled. We assayed desiccation toler-ance of the culture immediately upon dilution from saturationand at regular intervals thereafter. Cells rapidly begin to losetheir desiccation tolerance, dropping by two orders of mag-nitude within 2 hr, then slowly continuing to lose tolerancefor several more hours, a biphasic or perhaps logarithmic re-sponse possibly related to the concentration of dextrose orethanol in the medium. The proportion of unbudded cellsdeclines from �70% to just ,50%, indicating that cell divi-sion had begun (Figure 1E). To obtain a higher resolutionmeasure of the kinetics of tolerance loss, we repeated theexperiment, taking samples at more frequent intervals

Figure 1 Culture of yeast con-sists of sensitive and tolerantpopulations, whose relative pro-portions are governed by growthphase. (A) Log-phase culture ofBY4742 was established in YEPDat �106 cells/ml, then assayed fordesiccation tolerance at varioustimes as it grew to 1.8 · 108

cells/ml. Dashed line indicateslower limit of relative viability de-tectable by the assay as per-formed. Desiccation tolerance isexpressed as the percent relativeviability, the colony forming unitsof the desiccated and rehydratedculture divided by the undesic-cated control (see Results). (B)Yeast culture was inoculated inYEPD then assayed for desicca-tion tolerance at 1-day intervalsfor 7 days. (C) Seven-day-old cul-ture of BY4742 was separated in-

to heavy (H) (quiescent) and light (L) populations (seeMaterials and Methods), which were then assayed for desiccation tolerance. (D) Several desiccatedsamples of yeast were prepared from a saturated culture and assayed at intervals of several days for �1 month. (E) Saturated yeast culture wasinoculated into YEPD at 2 · 106 cells/ml and assayed for desiccation tolerance at various times for several hours. (F) Saturated yeast culture wasinoculated into YEPD at 2 · 106 cells/ml and then assayed for desiccation tolerance at �15-min intervals for 2 hr.


(Figure 1F). Tolerant cells begin to become sensitive within30 min of being introduced at low density into YEPD. Thisrapid loss of desiccation tolerance when the culture has notyet even doubled demonstrates that the tolerant cells in thesaturated inoculum must lose their tolerance upon exposureto their new growth conditions. Thus yeast cells can transitfrom the tolerant to the sensitive state as well as from thesensitive to the tolerant state (Figure 1A).

Saccharomycotina, the taxon of ascomycetes to whichS. cerevisiae belongs, may be a desiccation-tolerant taxon, orS. cerevisiae may be unique in possessing this trait. We there-fore grew saturated 48-hr cultures of several members ofSaccharomycotina (Figure 2A) and assayed them for desicca-tion tolerance (Figure 2B). The tested members of the cladeare all desiccation tolerant (with between 20 and 80%relative viability). The level of desiccation tolerance doesnot appear to correlate with phylogenetic distance fromS. cerevisiae or relationship to the whole genome duplication.To further investigate the evolutionary relationship betweendesiccation tolerance and phylogenetic distance from S.cerevisiae, log-phase cultures of Caenorhabditis albicans andC. glabrata were established and also found to exhibit sensi-tivity, being four orders of magnitude more sensitive thanwhen saturated (Figure 2C). We conclude that Saccharomy-cotina is likely a highly desiccation tolerant taxon, with tol-erance and sensitivity governed by a similar set of effectors.

Testing the predicted effectors and stress pathways fortheir importance in desiccation tolerance

Loss of water increases the solute to solvent ratio in a cell,thereby changing osmolarity and salt concentration. Yeast

cells maintain homeostasis in salinity and osmolarity throughregulators that induce or change the activity of membranepumps. They also produce small molecules such as trehaloseand hydrophilins that may act as osmolytes and/or watersubstitutes (Yancey et al. 1982; Oliver et al. 2001; Crowe2002). To test whether any of these known functions areimportant for desiccation tolerance we measured desiccationtolerance in deletion mutants that eliminate their function(Figure 3). Desiccation tolerance is either unaffected or re-duced only a few fold in deletions of genes encoding masterregulators or effectors of osmolarity (Figure 3A), salinity (Fig-ure 3B), trehalose biosynthesis (Figure 3C), and three hydro-philins (Figure 3D). These three hydrophilins were selectedbecause a previous study suggested they were highly inducedduring desiccation. Even a triple mutant lacking all threehydrophilins exhibited normal desiccation tolerance. Noneof these deletion strains reduce desiccation tolerance any-where near the five orders of magnitude reduction seen forexponential growth (Figure 1A). Therefore, either changes inosmolarity and/or salinity are not major causes of desiccationsensitivity, or yeast cells have alternative yet-to-be-discoveredpathways redundant with these canonical pathways that mit-igate these stresses during desiccation.

Desiccation has also been proposed to stress cells bycausing DNA double strand breaks (DSB), protein denatur-ation, or ROS. Double strand breaks are repaired bya number of pathways all of which require Rad52p. Proteinaggregates are resolubilized primarily by Hsp104p (Lee et al.2010). However, deletion of neither RAD52 nor HSP104reduces desiccation tolerance (Figure 4A). Oxidative stressis mitigated in yeast primarily by two superoxide dismutasesand two catalases. Individual knockouts of each of thesegenes generated a fivefold reduction in viability (Figure4B). Since multiple knockouts of these ROS scavengers tendto produce very sick strains, this line of exploration was notpursued via a multiple deletion strategy. However, if ROSare the major cause of desiccation sensitivity, we should beable to generate tolerance by propagating yeast anaerobi-cally where the formation of ROS is abrogated (Figure 4C).The absence of oxygen does not rescue the extreme sensi-tivity of log-phase cells and in fact induced three orders ofmagnitude increase in sensitivity in saturated cultures. Fromthese findings we conclude that although reduction of ROSscavenging ability does introduce a mild phenotype, molec-ular oxygen is in fact required to generate significant desic-cation tolerance in saturated cultures of yeast.

Since inactivation of specific stress response pathwayshas at most only a minor impact on tolerance, we askedwhether tolerance would be compromised by the simulta-neous downregulation of multiple stress response pathways.Many of these stress pathways are regulated by the Msn2pand Msn4p paralogs, the primary transcriptional regulatorsof the general stress response in yeast. While the msn2Δ andmsn4Δ strains exhibit no defect in desiccation tolerance,tolerance is reduced about fivefold in the msn2Δ msn4Δdouble mutant (Figure 4D). Thus at least one factor

Figure 2 Desiccation tolerance is conserved among the phylogeneticneighbors of S. cerevisiae. (A) Partial phylogenetic tree of Saccharomyco-tina. “WGD” indicates the branch evolving after the whole genome du-plication. (B) Saturated cultures of Candida albicans, C. glabrata,Saccharomyces cerevisiae, and S. pastorianus were assayed for desicca-tion tolerance. (C) Log-phase cultures of C. albicans and C. glabrata wereassayed for desiccation tolerance.


http://www.yeastgenome.org/cgi-bin/locus.fpl?dbid=S000004494










regulated by the established stress responses contributes todesiccation tolerance. In summary, most of desiccation tol-erance must be mediated by factors either redundant with orindependent of canonical stress responses previously corre-lated with desiccation.

Genome-wide screen for sensitive mutants identifiesrespiration as a prerequisite for desiccation tolerance

Since testing candidates for effectors of desiccation toler-ance failed to identify genes whose deletion producessensitivity approaching that of log-phase cultures, an un-biased genetic screen of mutants for desiccation sensitivitymight reveal pathways that had heretofore been unappre-ciated as contributors to desiccation tolerance. We screenedthe haploid deletion collection of budding yeast for desic-cation sensitivity (Materials and Methods, Genome-widescreen). We originally found �100 deletion strains thatappeared promising, but on further characterization we de-termined that most of these were r2 respiration-incompe-tent strains. Indeed, all r2 strains tested (except thespontaneous revertants described below) were found to bedesiccation sensitive. The mutant mrpl16Δ is r2 and is usedas a representative r2 strain. In addition, we generated r2

wild type by treatment with ethidium bromide. The r2 wild-type cells exhibit the same dramatic decrease in desiccationtolerance (Figure 5A). Thus mitochondrial function is re-quired for desiccation tolerance.

To eliminate r2 mutants as a cause of desiccation sensi-tivity, we isolated respiratory competent cells for each can-didate deletion by demanding their growth on glycerol. Ofthese, only three strains were found to generate sensitivityapproaching that of log-phase cells: lip5Δ, mdh1Δ, andoar1Δ (Figure 5B). Introduction of lip5Δ, mdh1Δ, andoar1Δ deletions into the A364A background also results in

a desiccation-sensitive phenotype (Figure S5), indicatingthat desiccation tolerance requires these genes. All threeof these genes have a connection with respiration or mito-chondria: LIP5 codes for lipoic acid synthase; lipoic acid is

Figure 3 Deletion of genes predicted tobe important for desiccation tolerancehave only a minimal impact on the trait.Saturated cultures of strains with dele-tions of genes involved in response to(A) osmolarity and (B) salinity wereassayed for desiccation tolerance. (C)Saturated cultures of strains with dele-tions of genes required for trehalose syn-thesis were assayed for desiccationtolerance. (D) Saturated cultures ofstrains with single and multiple deletionsof hydrophilins reported to be highly in-duced by desiccation were assayed fordesiccation tolerance. All mutants listedin this figure and the remaining figuresharbor deletion alleles constructed bythe whole genome yeast consortium(Winzeler et al. 1999).

Figure 4 Ablation of stress responses predicted to contribute to desicca-tion tolerance minimally impact the trait, but anaerobic growth inducessensitivity. Saturated cultures of strains with deletions of genes responsi-ble for DSB repair (A), heat shock tolerance (A), mitigation of ROS stress(B), and induction of the general stress response (D) were assayed fordesiccation tolerance (C). Anaerobically cultured log-phase and saturatedcultures of wild-type yeast were assayed for desiccation tolerance (seeMaterials and Methods).











an essential coenzyme of pyruvate dehydrogenase, whichcontributes to transforming pyruvate into acetyl CoA.MDH1 codes for the mitochondrial malate dehydrogenase,which performs the penultimate step of the tricarboxylicacid cycle. OAR1 codes for a enzyme involved in mitochon-drial fatty acid metabolism. All three deletion strains, whilecapable of growth on glycerol, do so very slowly. Takentogether, our results suggest that respiration is required forsaturated cultures to acquire desiccation tolerance. This con-clusion explains the requirement for oxygen that we ob-served previously (Figure 4C) and the transition fromsensitivity to tolerance in late exponential cultures (Figure1A), which correlates with the diauxic shift when cellsswitch from fermentation to respiration.

To further explore the connection between respirationand desiccation tolerance, we asked when the ability torespire is required for tolerance: before, during, or bothbefore and during desiccation. For this purpose, we appliedthe respiration inhibitor myxothiazol (Materials and Meth-ods, Respiration inhibition) either during growth only, duringdesiccation only, or for the duration of the entire experi-ment. As expected, inhibition of respiration by myxothiazol

during both growth and desiccation phenocopies the desic-cation sensitivity of r2 cells. A similar level of desiccationsensitivity is observed when respiration is inhibited onlyduring growth. However normal desiccation tolerance is ob-served when respiration is inhibited only during desiccation(Figure 5C). Thus to acquire desiccation tolerance, cellsmust have experienced respiration prior to desiccation butneed not respire during desiccation itself.

To determine how rapidly yeast cells can acquire desic-cation tolerance by respiring, we exploited the fact that ourlog-phase cultures are very desiccation sensitive whengrown on a medium with glucose, a fermentable carbonsource (Figure 1A). We replaced this mediumwith a mediumcontaining glycerol, a carbon source that can be assimilatedonly via respiration. Within 2 hr (within one doubling), thedividing cultures increase in desiccation tolerance by sevenorders of magnitude (Figure 5D). To test that this acquisi-tion of desiccation tolerance is through the respiration ofglycerol rather than some other effect of glycerol, we addedglycerol directly to the medium containing glucose (Figure5E). The presence of glycerol in the medium does not rescuethe desiccation tolerance of the r2 cells, indicating that

Figure 5 Respiration is required for acquisition of desicca-tion tolerance. An unbiased screen of the haploid deletioncollection was performed to identify mutants sensitive todesiccation when grown to saturation. (A) r+ and r–

BY4742 were grown to saturation and assayed for desic-cation tolerance (see Materials and Methods). (B) Satu-rated cultures of mutants recovered from the haploiddeletion collection with at least two orders of magnitudereduction in desiccation tolerance. (C) Saturated culturesof BY4742 were exposed to 0.2 mg/ml myxothiazol duringgrowth, during desiccation, or during both growth anddesiccation (as indicated by the bullet) and then assayedfor desiccation tolerance. (D) Glucose fermenting log-phase culture of BY4742 was forced to respire by switch-ing the growth medium to glycerol, a nonfermentable car-bon source. Samples were taken approximately every20 min and assayed for desiccation tolerance. (E) r+ andr– BY4742 cultures were grown to saturation and assayedfor desiccation tolerance either in YEPD (solid bars) or inYEPD with 3% glycerol added (open bars).




glycerol only induces tolerance when it is used as a carbonsource. In summary, the cellular changes required to acquiredesiccation tolerance can occur within one cell division anddividing cells can exhibit the same level of tolerance as sta-tionary cells (compare Figures 5D and 1A).

Transcriptional regulation of desiccation tolerance

Some molecular component of respiration may be requireddirectly for desiccation tolerance or respiration may providea signal that activates a cascade leading to tolerance. If thelatter is true, then it should be possible to isolate mutationsthat activate desiccation tolerance via bypass of the respira-tion signal. Therefore, starting with 14 independentlyestablished cultures of r2 MATa (BY4741: mrpl16Δ; r2)and r2 MATa (BY4742: mrpl16Δ; r2) strains, we grew eachculture to saturation, desiccated them, and then pooled thecolonies that survived desiccation and repeated the process.After four rounds of this procedure, strains with almost wild-type desiccation tolerance were generated. Of the 28 strainsrecovered, two of the MATa strains were found to be dom-inant mutations while the remaining strains were recessive.The existence of these revertants indicates that a directproduct of respiration is not essential for desiccation toler-ance. None of the revertants were competent for growth onglycerol, indicating that the mutations did not restore re-spiratory competence.

To identify the mutations responsible for rescuing thedesiccation sensitivity of r2 yeast, we assigned 21 of therecessive revertants to five complementation groups (Table2). We then hybridized genomic DNA from each strain toa tiling microarray, a technique capable of identifying locithat are polymorphic relative to the reference genome se-quence. To eliminate irrelevant or false positive polymor-phisms, we focused on polymorphisms that co-occurredwithin a single gene for each complementation group. Wesequenced these polymorphic alleles and in almost all casesthey generated a nonsense codon in the gene. For thesecandidates, we constructed r2 variants of the correspondingdeletion strain from the yeast deletion library and assayedthem for desiccation tolerance. The r2 variants are desicca-tion tolerant, providing further evidence that the inactiva-tion of each of these genes is responsible for the rescue ofthe desiccation sensitivity of r2 strains (Figure 6A).

Two of the deletions, srb8Δ and ssn2Δ, identify subunitsof the Mediator repressive module. Mediator is a large mul-tisubunit complex that regulates transcription of hundredsof genes (Van De Peppel et al. 2005). The repressive modulecontains four subunits that together repress Mediator’s othersubunits required for transcription activation. We assayedr2 strains deleted for the other two components (SSN3and SSN8) of the Mediator repressive module and foundthem to be desiccation tolerant as well (Figure S4). Thusloss of any subunit of the Mediator repressive module leadsto restoration of desiccation tolerance in cells defective forrespiration. This result suggests that factors important for

desiccation tolerance in budding yeast are under transcrip-tional control.

The repressive module inactivates the activator module ofMediator that includes Med2p (Van De Peppel et al. 2005).We hypothesized that inactivation of the repressive moduleallows the activator module to induce genes necessary fordesiccation tolerance. If so, inactivation of the activator mod-ule by deletion of MED2 in the srb8Δ background shouldeliminate the ability of srb8Δ to rescue the desiccation sensi-tivity of a r2 strain. Indeed this is the case (Figure 6B). Todetermine whether the non-Mediator revertants, pex10Δ,ras2Δ, and trr2Δ operate in the same pathway, we assayeddouble knockouts of these genes with med2Δ and found thatonly the med2Δ pex10Δ double mutant loses significantdesiccation tolerance like the srb8Δ med2Δ double mutant(Figure 6C). Thus MED2-independent as well as MED2-dependent pathways control desiccation tolerance. In agree-ment with this, saturated cultures of r+ med2Δ strains are notdesiccation sensitive, indicating that desiccation tolerance canalso be induced by MED2-independent mechanism(s).

Since the revertant deletions rescue sensitivity generatedby damage to the mitochondrial genome in r2 strains, wewere curious whether they also rescued sensitivity in logphase when r+ strains are also extremely sensitive. Wetherefore established log-phase cultures of r+ strains of eachof the deletants and assayed them for desiccation toleranceat cell densities for which wild-type strains are extremelysensitive (Figure 6D). The srb8Δ strain is extremely desicca-tion tolerant in these conditions, whereas none of the otherdeletions rescues sensitivity as they do in saturated r2

strains. We conclude that disruption of the Mediator repres-sive module allows the transcription of genes whose prod-ucts are instrumental in generating desiccation tolerance.

Similar to our earlier studies on respiration, we askedwhether the transcriptional induction by Mediator of desic-cation tolerant factors occurs at the time of desiccation orprior to desiccation. To distinguish between these twopossibilities, we desiccated a saturated yeast culture in thepresence of transcription or translation inhibitors (see Mate-rials and Methods, Translation and transcription inhibition).In both cases, desiccation tolerance was unaffected (Figure6E), supporting the conclusion that all the genes needed fordesiccation tolerance are already transcribed and translatedprior to desiccation. This finding is consistent with the con-cept that Mediator activation module activates expression of

Table 2 Complementation group assignments of recessivespontaneous revertants

Locus Number of mutants Function

SRB8 5 Mediator repressive moduleSSN2 4 Mediator repressive modulePEX10 1 Peroxisome importRAS2 5 Small GTP binding proteinTRR2 6 Mitochondrial thioredoxin reductaseUnknown 5


























desiccation-relevant genes when respiration begins duringthe diauxic shift.

Discussion

Here we characterize the effect of growth conditions ondesiccation tolerance in budding yeast. The fraction of cellssurviving desiccation ranges from 20–70% in saturated cul-tures to fewer than one in a million in early log-phase cul-tures. This dramatic difference is observed in many differentbackgrounds, related species, and under both slow andrapid desiccation regimens. Previous studies have found thatlog phase are only 20-fold more sensitive than saturatedcultures (Beker and Rapoport 1987; Ratnakumar and Tun-nacliffe 2006) (Ratnakumar et al. 2011). Why these studiesdid not observe the six-orders-of-magnitude differencereported here is unclear, but the magnitude of the differenceis critical, shaping our interpretation of results in this studyand previous studies. It also makes the study of desiccationtolerance in yeast amenable to powerful genetic screens andselections as exemplified in this study.

With this new metric, we assessed candidate effectors ofdesiccation tolerance implicated by prior studies in yeastand other organisms. Remarkably, mutants lacking these

proteins exhibit, at most, only small decreases in desiccationtolerance. We corroborate previous studies showing thatneither trehalose biosynthesis (Ratnakumar and Tunnacliffe2006) nor osmoregulation by Hog1p (Ratnakumar et al.2011) are essential for robust desiccation tolerance. Addi-tionally, we show that desiccation tolerance does not requireDNA double strand break repair, the hydrophilins mosthighly expressed during desiccation (Singh et al. 2005),the major protein-folding chaperone Hsp104p, or the com-plete complement of ROS scavengers. The lack of sensitivityin these mutants suggests that cells might possess undiscov-ered pathways that operate in the absence of these canonicalstress responses. Alternatively, these stresses may not be thelethal ones imposed by desiccation. Indeed, contrary to theprevailing consensus, we show that eliminating oxygen andthus ROS stress causes desiccation sensitivity rather thantolerance. If these stresses are not the lethal ones, then theirpotentially relevant stress response factors may serve func-tions other than protection from desiccation. For examplerecent studies suggest that trehalose might accumulate instationary cells as a preferred carbon source to allow rapidexit from G0 when nutrients return (Shi et al. 2010).

Complementing our candidate testing of known stressresponses, we performed an unbiased genome-wide screen

Figure 6 Desiccation tolerancearises from at least two path-ways, at least one of which istranscriptionally regulated. Desic-cation tolerant revertants of r2

BY4742 were isolated, andwhole genome sequencing ofthe revertants revealed putativenull alleles responsible for therestoration of desiccation toler-ance (see Materials and Methodsand Results). (A) Deletion allelesfrom the haploid deletion col-lection that correspond to the re-vertant alleles were identified.Saturated cultures of the r+ andr2 variants of these deletions al-lele were assayed for desiccationtolerance. (B and C) Saturatedcultures of the r+ and r2 variantsof mutants in the Mediator re-pressor (srb8) and activator (med2)submodules were assayed fordesiccation tolerance. (D) Log-phase cultures of r+ BY4742 (di-amond), pex10 (circle), ras2 (X’s),srb8 (square), trr2 (triangle) wereassayed for desiccation tolerance.(E) Saturated cultures of BY4742were desiccated in the presenceof the translation inhibitor cyclo-heximide (cyc) or the transcriptioninhibitor thiolutin (thio) and thenassayed for desiccation tolerance.(F) Summary of the regulation ofdesiccation tolerance in ferment-ing and respiring yeast.




for mutants that are extremely sensitive in saturatedcultures, producing two remarkable findings. First, defectsin most cellular processes do not dramatically sensitize yeastto desiccation. Some minor sensitivity may occur, as otherrecent studies have identified from �300 (Shima et al.2008) to .500 (D’Elia et al. 2005) different deletionmutants in a number of cellular processes that produce smallbut measurable defects in tolerance. However, such smalleffects relative to fermenting cells could also be explainedby indirect effects on fermentation or respiration. Desicca-tion tolerance is refractory to many cellular perturbationsdespite the reliance of nearly every biochemical processupon the presence of water.

Second, in contrast to the general robustness of desicca-tion tolerance, mutants in only one function, respiration,reduce desiccation tolerance by several orders of magnitude.The connection between respiration and tolerance isstrongly supported by additional observations also reportedhere. A saturated culture of wild-type cells fails to exhibitdesiccation tolerance when it is forced to grow to saturationwithout respiring, by either the removal of oxygen or thepresence of respiratory inhibitors. Furthermore, the majorityof wild-type cells acquire desiccation tolerance at a celldensity when cells in the culture transit from fermentationto respiration (this study and Ratnakumar and Tunnacliffe2006). Finally, early log-phase cultures can exhibit the samelevel of tolerance as saturated cultures if the log-phase cul-tures are forced to respire by growth on glycerol. Thus weprovide compelling evidence that respiration triggers bud-ding yeast to undergo a dramatic shift from desiccation sen-sitivity to tolerance.

While respiration is an important trigger for desiccationtolerance, a significant fraction of respiring cells remainssensitive (this study). Perhaps our laboratory conditions failto mimic natural conditions that are more effective atinducing tolerance. Alternatively, the tolerant state may havea cost, with incomplete conversion of an entire culture totolerance programmed to ensure maximization of survivaloptions with regard to unanticipated changes to its environ-ment. Additionally, desiccation tolerant cells continue toaccumulate in cultures after reaching saturation and switch-ing to respiration. These observations are consistent withprocesses other than respiration contributing to the acquisi-tion of tolerance. Our study argues that quiescence is not oneof these other processes. However, a recent study suggeststhat autophagy is required for desiccation tolerance, asmutants exhibit a 10-fold defect in tolerance (Ratnakumaret al. 2011). The induction of autophagy in saturated culturesand a similar fold increase in desiccation tolerance after sat-uration is consistent with a role for autophagy in tolerance.

Two observations support the notion that respirationtriggers the acquisition of desiccation tolerance rather thandirectly providing a protective reagent or critical energysource. First, respiration inhibitors reduce tolerance whenpresent during the growth of the culture but not whenpresent only during desiccation. Second, we isolate mutants

that achieve robust desiccation tolerance without respiring.Several possibilities potentially explain how respirationtriggers desiccation tolerance. The stresses associated withrespiration may mimic the stress conditions of desiccation.Respiration, a known prerequisite for completion of meiosisand sporulation, may be coupled to desiccation tolerance toensure tolerance of spores. This coupling may be fortu-itously preserved during vegetative growth. Alternatively,desiccation tolerance may be a constitutive state of yeast inthe wild, because of the potential for rapid changes inenvironment (for example, direct sunlight rapidly dehydrat-ing the surface occupied by a yeast colony). The cell onlysuppresses desiccation tolerance and other protectiveshields under favorable fermenting growth conditions, whenthe growth rate is maximized. Clearly, discriminating be-tween these and other possibilities will require better un-derstanding of the regulatory pathway and effectors ofdesiccation tolerance controlled by respiration.

We therefore isolated suppressors that restore desiccationtolerance to a respiration defective mutant. The majority ofour suppressors are loss-of-function mutations, which arehighly biased to inactivate negative regulators of desiccationeffectors. Indeed, our analysis revealed that one suchnegative regulator is the inhibitory submodule of theMediator complex, an important component of the RNApolymerase II holoenzyme (Davis et al. 2002). Furthermore,we show that this submodule likely acts by repressing tran-scription activation by the Med2p activator submodule. Thuswe provide compelling evidence that a transcriptional pro-gram regulates the transition from desiccation sensitivity totolerance in budding yeast (Figure 6F). Intriguingly, whilethe Med2p submodule activates hundreds of genes, it hasbeen implicated in specifically regulating other stressresponses including the response to iron deficiency andthe general stress response pathway controlled by Msn2p/Msn4p (Van De Peppel et al. 2005). Transcriptional regula-tion of water homeostasis has precedent as drought toler-ance in plants is also transcriptionally regulated (Mooreet al. 2009; Wang et al. 2009). Finally, Med2p cannot bethe only positive regulator of desiccation effectors, sincedesiccation tolerance can occur independent of Med2p func-tion in saturated cultures of both wild-type and ras2Δ r2

cells (this study). Thus the regulation of desiccation toler-ance is complex.

Observations from this study provide a foundation toidentify effectors of desiccation tolerance. First we show thatdesiccation sensitivity in respiratory defective cells is res-cued by inactivation of TRR2 and PEX1. TRR2 and PEX10encode proteins important for mitochondrial thioredoxinfunction and peroxisome biogenesis, respectively. Thiore-doxin and perioxisomes both impact cellular redox, suggest-ing that effectors of redox homestasis may be important fordesiccation tolerance. Furthermore, the knowledge that des-iccation tolerance is transcriptionally regulated by Med2p(and likely other transcription programs) is also very excit-ing. Budding yeast has a long track record of successfully














elucidating targets of transcriptional programs, particularlywhen coupled with powerful genetic screens and selectionslike those demonstrated here. Thus exploiting this transcrip-tional control and the connection to redox homeostasis pro-vides important new avenues to identify the elusive effectorsof desiccation tolerance.

Acknowledgments

The authors thank Peter Espenshade for use of his anaerobicworkstation, Kyle Cunningham for the kind gift of myxo-thiazol, and Brendan Cormack for the kind gift of Candidastrains. We thank Hugo Tapia and Aaron Welch for criticalreading of the manuscript. Funding for this work came fromHoward Hughes Medical Institute to D.E.K. and NationalInstitutes of Health grant P50 GM071508 to M.D.

Literature Cited

Al-Rubeai, M., M. Fussenegger, S. T. Sharfstein, D. Shen, T. R. Kiehlet al., 2007 Molecular response to osmotic shock, pp. 213–236in Systems Biology. Springer-Verlag, The Netherlands.

Allen, C., S. Buttner, A. D. Aragon, J. A. Thomas, O. Meirelles et al.,2006 Isolation of quiescent and nonquiescent cells from yeaststationary-phase cultures. J. Cell Biol. 174: 89–100.

Almoguera, C., and J. Jordano, 1992 Developmental and environ-mental concurrent expression of sunflower dry-seed-stored low-molecular-weight heat-shock protein and Lea mRNAs. PlantMol. Biol. 19: 781–792.

Andreasen, A. A., and T. J. B. Stier, 1953 Anaerobic nutrition ofSaccharomyces cerevisiae. I. Ergosterol requirement for growth ina defined medium. J. Cell. Comp. Physiol. 41: 23–36.

Andreasen, A. A., and T. J. B. Stier, 1954 Anaerobic nutrition ofSaccharomyces cerevisiae. II. Unsaturated fatty and requirementfor growth in a defined medium. J. Cell. Comp. Physiol. 43:271–281.

Bartels, D., 2005 Desiccation tolerance studied in the resurrectionplant craterostigma plantagineum1. Integr. Comp. Biol. 45: 696.

Battaglia, M., Y. Olvera-Carrillo, A. Garciarrubio, F. Campos, andA. A. Covarrubias, 2008 The enigmatic LEA proteins and otherhydrophilins. Plant Physiol. 148: 6–24.

Beker, M., and A. Rapoport, 1987 Conservation of yeasts bydehydration, pp. 127–171 in Biotechnology Methods. Springer-Verlag, Berlin.

Crowe, J. H., 1972 Evaporative water loss by tardigrades undercontrolled relative humidities. Biol. Bull. 142: 407–416.

Crowe, J. H., F. A. Hoekstra, and L. M. Crowe, 1992 Anhy-drobiosis. Annu. Rev. Physiol. 54: 579–599.

Crowe, L. M., 2002 Lessons from nature: the role of sugars inanhydrobiosis. Comp. Biochem. Physiol. A Mol. Integr. Physiol.131: 505–513.

D’Elia, R., P. L. Allen, K. Johanson, C. A. Nickerson, and T. G.Hammond, 2005 Homozygous diploid deletion strains of Sac-charomyces cerevisiae that determine lag phase and dehydrationtolerance. Appl. Microbiol. Biotechnol. 67: 816–826.

Davis, J. A., Y. Takagi, R. D. Kornberg, and F. J. Asturias,2002 Structure of the yeast RNA polymerase II holoenzyme:mediator conformation and polymerase interaction. Mol. Cell10: 409–415.

Finkelstein, R., W. Reeves, T. Ariizumi, and C. Steber, 2008 Mo-lecular aspects of seed dormancy. Annu. Rev. Plant Biol. 59:387–415.

Gadd, G. M., K. Chalmers, and R. H. Reed, 1987 The role oftrehalose in dehydration resistance of Saccharomyces cerevisiae.FEMS Microbiol. Lett. 48: 249–254.

Garay-Arroyo, A., J. M. Colmenero-Flores, A. Garciarrubio, andA. A. Covarrubias, 2000 Highly hydrophilic proteins in prokar-yotes and eukaryotes are common during conditions of waterdeficit. J. Biol. Chem. 275: 5668–5674.

Gasch, A. P., P. T. Spellman, C. M. Kao, O. Carmel-Harel, M. B.Eisen et al., 2000 Genomic expression programs in the re-sponse of yeast cells to environmental changes. Mol. Biol. Cell11: 4241–4257.

Goldring, E. S., L. I. Grossman, and J. Marmur, 1971 Petite mu-tation in yeast. J. Bacteriol. 107: 377–381.

Goyal, K., L. J. Walton, J. A. Browne, A. M. Burnell, and A. Tunna-cliffe, 2005 Molecular anhydrobiology: identifying moleculesimplicated in invertebrate anhydrobiosis. Integr. Comp. Biol. 45:702.

Gresham, D., D. M. Ruderfer, S. C. Pratt, J. Schacherer, M. J.Dunham et al., 2006 Genome-wide detection of polymor-phisms at nucleotide resolution with a single DNA microarray.Science 311: 1932–1936.

Jiang, M., and J. Zhang, 2002 Water stress-induced abscisic acidaccumulation triggers the increased generation of reactiveoxygen species and up-regulates the activities of antioxidantenzymes in maize leaves. J. Exp. Bot. 53: 2401–2410.

Kranner, I., and S. Birtić, 2005 A modulating role for antioxidantsin desiccation tolerance. Integr. Comp. Biol. 45: 734–740.

Lee, S., B. Sielaff, J. Lee, and F. T. F. Tsai, 2010 CryoEM structureof Hsp104 and its mechanistic implication for protein disaggre-gation. Proc. Natl. Acad. Sci. USA 107: 8135–8140.

Lesaffre Yeast Corporation, 2010 “Red Star Original” (http://www.redstaryeast.com/ 8/24/2010).

Moore, J. P., N. T. Le, W. F. Brandt, A. Driouich, and J. M. Farrant,2009 Towards a systems-based understanding of plant desic-cation tolerance. Trends Plant Sci. 14: 110–117.

O’Rourke, S. M., I. Herskowitz, and E. K. O’Shea, 2002 Yeast gothe whole HOG for the hyperosmotic response. Trends Genet.18: 405–412.

Oliver, A. E., O. Leprince, W. F. Wolkers, D. K. Hincha, A. G. Heyeret al., 2001 Non-disaccharide-based mechanisms of protectionduring drying. Cryobiology 43: 151–167.

Ratnakumar, S., and A. Tunnacliffe, 2006 Intracellular trehaloseis neither necessary nor sufficient for desiccation tolerance inyeast. FEMS Yeast Res. 6: 902–913.

Ratnakumar, S., A. Hesketh, K. Gkargkas, M. Wilson, B. M. Rashet al., 2011 Phenomic and transcriptomic analyses reveal thatautophagy plays a major role in desiccation tolerance in Saccha-romyces cerevisiae. Mol. Biosyst. 7: 139–149.

Rose, M. D., F. M. Winston, and P. Heiter, 1990 Methods in YeastGenetics: a Laboratory Course Manual. Cold Spring Harbor Lab-oratory Press, Cold Spring Harbor, NY.

Sales, K., W. Brandt, E. Rumbak, and G. Lindsey, 2000 The LEA-like protein HSP 12 in Saccharomyces cerevisiae has a plasmamembrane location and protects membranes against desiccationand ethanol-induced stress. Biochimica et Biophysica Acta(BBA) -. Biomembranes 1463: 267.

Shi, L., B. M. Sutter, X. Ye, and B. P. Tu, 2010 Trehalose is a keydeterminant of the quiescent metabolic state that fuels cell cycleprogression upon return to growth. Mol. Biol. Cell 21: 1982–1990.

Shima, J., A. Ando, and H. Takagi, 2008 Possible roles of vacuolarH+-ATPase and mitochondrial function in tolerance to air-dryingstress revealed by genome-wide screening of Saccharomyces cer-evisiae deletion strains. Yeast 25: 179–190.

Singh, J., D. Kumar, N. Ramakrishnan, V. Singhal, J. Jervis et al.,2005 Transcriptional response of Saccharomyces cerevisiae to


http://www.redstaryeast.com/8/24/2010)

http://www.redstaryeast.com/8/24/2010)

desiccation and rehydration. Appl. Environ. Microbiol. 71:8752–8763.

Sømme, L., 1996 Anhydrobiosis and cold tolerance in tardigrades.Eur. J. Entomol. 93: 349–357.

Thierbach, G., and H. Reichenbach, 1981 Myxothiazol, a new an-tibiotic interfering with respiration. Antimicrob. Agents Chemo-ther. 19: 504–507.

Tunnacliffe, A., and C. Ricci, 2006 Mechanisms of desiccationtolerance, pp. in ESF LESC Exploratory Workshop, edited by A.Tunnacliffe. European Science Foundation, Pembroke College,Cambridge, UK.

van de Peppel, J., N. Kettelarij, H. van Bakel, T. T. J. P. Kockelkorn,D. van Leenen et al., 2005 Mediator expression profiling epista-sis reveals a signal transduction pathway with antagonistic sub-modules and highly specific downstream targets. Mol. Cell 19: 511.

Wang, Z., Y. Zhu, L. Wang, X. Liu, Y. Liu et al., 2009 A WRKYtranscription factor participates in dehydration tolerance in Boeahygrometrica by binding to the W-box elements of the galactinolsynthase (BhGolS1) promoter. Planta 230: 1155–1166.

Wehmeyer, N., L. D. Hernandez, R. R. Finkelstein, and E. Vierling,1996 Synthesis of small heat-shock proteins is part of the de-velopmental program of late seed maturation. Plant Physiol.112: 747–757.

Winzeler, E. A., D. D. Shoemaker, A. Astromoff, H. Liang, K. Andersonet al., 1999 Functional characterization of the S.cerevisiae ge-nome by gene deletion and parallel analysis. Science 285: 901–906.

Wright, J. C., 1988 Structural correletes of permeability and tunformation in tardigrade cuticle: an image analysis study.J. Ultrastruct. Mol. Struct. Res. 101: 23–39.

Yancey, P. H., M. E. Clark, S. C. Hand, R. D. Bowlus, and G. N.Somero, 1982 Living with water stress: evolution of osmolytesystems. Science 217: 1214–1222.

Zhu, J.-K., 2002 Salt and drought stress signal transduction inplants. Annu. Rev. Plant Biol. 53: 247–273.

Communicating editor: F. Winston


GENETICSSupporting Information


Genetic Analysis of Desiccation Tolerancein Saccharomyces cerevisiae

Dean Calahan, Maitreya Dunham, Chris DeSevo, and Douglas E. Koshland

Copyright © 2011 by the Genetics Society of AmericaDOI: 10.1534/genetics.111.130369

genetic analysis of desiccation tolerance in saccharomyces...

Documents