Transcript expression in Saccharomyces cerevisiae at high salinity* S. Jaqueline Yale and Hans J. Bohnert** 0088, U.S.A.

Transcript

1 JBC Papers in Press. Published on February 14, 2001 as Manuscript M Transcript expression in Saccharomyces cerevisiae at high salinity* S Jaqueline Yale and Hans J. Bohnert** Department of Biochemistry, University of Arizona, Biosciences West, Tucson, AZ , U.S.A. Page title: Time course of yeast salinity stress responses Footnotes *This work was supported by a grant from the US National Science Foundation, Plant Genome Initiative (DBI ). The costs of publication were defrayed in part by the payment of page charges. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. **To whom correspondence should be addressed: Dept. of Biochemistry, University of Arizona, 1041 E. Lowell St. Tucson, AZ , USA. Tel.: ; Fax: ; bohnerth@u.arizona.edu 1 Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

2 S - Supplemental material also available at Summary Transcript expression of Saccharomyces cerevisiae at high salinity was determined by microarray analysis of 6,144 open reading frames (ORFs). From cells grown in 1M NaCl for 10, 30, and 90 minutes changes in transcript abundance >2-fold were classified. Salinity-induced ORFs increased over time: 107 (10 ), 243 (30 ), and 354 (90 ). Upregulated, functionally unknown ORFs increased from 17 to 149 over this period. Expression patterns were similar early, with 67% of upregulated transcripts after 10 identical to those at 30. The expression profile after 90 revealed different upregulated transcripts (identities of 13% and 22%, respectively). Nucleotide and amino acid metabolism exemplified the earliest responses to salinity, followed by ORFs related to intracellular transport, protein synthesis and destination. Transcripts related to energy production were upregulated throughout the time course with respiration-associated transcripts strongly induced at 30. Highly expressed at 90 were known salinity stress-induced genes, detoxification-related responses, transporters of the major facilitator superfamily, metabolism of energy reserves, nitrogen and sulfur compounds, and lipid, fatty acid/ isoprenoid biosynthesis. We chose severe stress conditions to monitor responses in essential biochemical mechanisms. In the mutant, gpd1/gpd2, lacking glycerol biosynthesis, the stress response was magnified with a partially different set of upregulated ORFs. Abbreviations: MAPK - Mitogen-activated protein kinase; STRE - Stress Response Element; EST - Expressed Sequence Tag; HOG - High Osmolarity Glycerol. 2

3 Introduction High salinity represents a stress for organisms because the excess of sodium or other monovalent cations imbalances the osmotic potential, generates water deficit, and the influx of sodium may lead to metabolic toxicity [1,2]. Protective biochemical reactions range from the synthesis of osmolytes, to increased chaperone activity, enhanced radical oxygen scavenging, changes in redox control, increased proton pumping activity, adjustments in carbon/nitrogen balance, and altered ion and water uptake [2-7]. These biochemical activities have been documented in a wide range of organisms, from bacteria to specialized vertebrate tissues, and suggest that the responses, with species-specific adjustments, utilize common cellular defense programs that balance water deficit and ion excess. In yeast, many components underlying the signaling pathways that control these biochemical entities are known [4, 8-12]. Most information is available about signal transduction altering carbohydrate metabolism, where MAPK phosphorelays, exemplified by protein kinase Hog1p (High Osmolarity Glycerol), transmit osmotic changes [13, 14] leading to the induction of transcription that activates downstream biochemical functions [10, 15-18]. Activation of Hog1p constitutes an early phase of the salinity stress response, which then seems to diverge into different pathways [18-20]. One pathway is mediated by the transcription factors Msn2p/Msn4p binding to stress response elements (STREs) and acting in signal amplification [21-23]. Hot1p, another transcriptional activator, has recently been identified as a Hog1p partner in this signaling [12, 20]. In addition, the derepression of genes, for example through the regulated action of Sko1p, seems to add another level of complexity controlling the salinity stress response machinery [24, 25]. Partially interacting with HOG-based signal transduction is a pathway associated with the action of the protein phosphatase calcineurin, a mediator for many cellular 3

4 responses to calcium signals [8, 11, 26]. The calcineurin pathway responds predominantly to challenges in the ionic environment. The rationale for focussing on transcriptional reactions of yeast to salt stress through a genome-wide expression analysis is based on our interest in plant salinity stress responses [2, 6, 27]. Similar abiotic stress-induced gene expression programs seem to exist in yeasts and plants, including conserved signaling pathways and biochemical defense determinants [28, 29]. Comparative studies promise to reveal the similarities and distinctions between cell-specific and organismal components involved in tolerance acquisition. An experimental outline that describes the portion of the yeast genome required for osmotic stress tolerance will aid in delineating the conserved cellular functions of homologous elements in multicellular organisms. Yeast microarrays provide information about the transcription of all genes. Genome-wide monitoring of transcript changes in yeast could show previously unrecognized cellular aspects of stress protection, and reveal genes that represent a yeast-specific solution to survival in high sodium. Such studies with yeast have recently become available [25, 30]. The three times replicated time-course experiments reported here add to these analyses. One novel aspect is the description of a succession of biochemical categories that are progressively upregulated. Early stress responses, affecting mainly nucleotide and protein biosynthetic pathways, are different from later responses, which included intracellular protein and metabolite transport activities and increased energy consumption for metabolic and ion homeostasis. Transcription after prolonged stress also exemplifies ascending functions in cell rescue, in aging (cell death) and defense-related roles, and reveals a large number of functionally unknown ORFs. A yeast genome array, 6,144 coding regions deposited on nylon filters, was used for a complete analysis of changes in transcript expression following hyper-osmotic stress. The results confirm many of the ORFs and proteins previously reported as stress-regulated [3, 10, 15, 23] and adds a number of stress-regulated transcripts that had not been recognized before. We describe early response transcripts 4

5 distinguished from those that act at later times in different functional categories that seem to maintain cell integrity. We identify a set of approximately 200 salt stress-regulated functionally unknown ORFs. Some of the unknown ORFs have orthologs in cdna libraries from salt-stressed plants. Our results complement and extend through the use of a salt stress-sensitive mutant, which is unable to synthesize glycerol, recent reports that have targeted yeast salinity stress responses though the analysis of arrayed ORFs [25, 30]. Materials and Methods Yeast Strains, Growth and Stress Conditions. Saccharomyces cerevisiae strain S150-2B (MATα ura3-52 his3 leu2-3,112 trp1-289) was grown at 30 0 C to mid-log phase (OD 600 = 1.0) in standard rich media, YPD (1% yeast extract/ 2% peptone/ 2% glucose, ph 5.5). Control (no salt) cultures were harvested immediately, and salt-stressed yeast cultures were harvested after 10, 30, and 90 minutes. To salt stress the cells, an equal volume of YPD containing 2M NaCl was added directly to the yeast cultures. Cell count, OD measurements, and streaking of cell on non-selective media during the stress experiments indicated a decline in cell numbers by approximately 25% during the 10 time point but cell number remained constant thereafter and increased after approximately 4h of stress. Cells were collected by centrifugation at 3,000 rpm for 5, rapidly washed once in sterile water, and the cell pellet was frozen and stored at C until RNA extraction. The experiment was repeated three times with independently grown cultures. As an additional control, yeast strain W3031A (MATα leu2-3,112 ura 3-1 trp1-1 his3-11,15 ade2,1 can1-100 SUC2 GAL mal0 GPD1::URA3 GPD2::TRP1), which is defective in glycerol biosynthesis (kindly provided by Dr. S. Hohmann, Gøeteborg, Sweden) was grown to mid-log phase in rich medium and stressed with 0.5M NaCl for 60 min. [31]. 5

6 Yeast Gene Filters. Yeast Gene Filters (Research Genetics Inc.) contain 6,144 PCR products bound to nylon filters ( The size of the PCR products ranged from 300 bp to 4 kb. The filters are missing approximately 300 PCR-products from chromosome 16. Average changes in transcript abundance for each time point were calculated relative to the no salt control using "Pathways" software ( Approximately 30% of the ORFs whose hybridization signal varied at background levels under all experimental conditions were eliminated from the final analysis. The filters were used only once to avoid variations caused by unequal stripping of probe or DNA from the membranes. ORFs with (partially) overlapping reading frames are identified in the supplemental material. Averaging reduced the number for absolute fold-induction but the spread over all hybridizations indicated that a two-fold induction was significant. Preparation of mrna and Hybridizations. Total RNA was isolated from frozen cell pellets by extraction with hot acidic phenol [32]. Complementary DNA was prepared using oligo(dt) primers and 5µg of total RNA labeled with α 33 P-dCTP and purified through a Qiaquick column (Qiagen Inc.). The cdnas (three for each time point) were hybridized to 12 individual sets of gene filters. Detailed experimental procedures for treatment of the filters can be found at Microarray Analysis. Phosphorimages of the yeast microarray filters were captured with a resolution of 50 microns on a Storm phosphorimager (Molecular Dynamics Inc.) and analyzed using Pathways software, version 2.01 which provided 16-bit imaging capability (Research Genetics Inc.). Normalization between sets of filters was based on the average of the signal intensities of all the datapoints on the individual filters. Comparisons for each of the experimental conditions (10, 30 and 90 minutes of NaCl stress) were calculated relative to the no-stress control. The relative fold-changes in transcript abundance for 10, 30, and 90 minutes of salt stress represent the average changes in gene expression for 3 experiments each. We considered expression levels >2.0 fold as induced, <2.0 fold as repressed, and between -1.9 and +1.9 fold as unchanged. Induced ORFs were categorized based on the MIPS 6

7 classification of yeast ORFs ( Approximately 30% of the 6144 signal intensities were removed from the analysis because they were equal to background intensities under all experimental conditions. The complete data sets can be found at Northern Blots and Densitometry. Total RNA was isolated from mid-log phase, (OD 600 =1.0), yeast cultures after 0, 10, 30 and 90 minutes exposure to 1M NaCl. Probes were made by PCR amplification of specific ORFs from genomic DNA and random primer labeled with α 32 P-dCTP. Probes were chosen based on the microarray data. We chose 7 ORFs with no change in gene expression and 32 ORFs with induced gene expression (see for a list). Standard procedures for RNA blot hybridizations were followed [33]. Phosphorimages of the RNA hybridizations were created on the Storm phosphorimager and densitometry was calculated using Imaquant software (Molecular Dynamics Inc.). Statistical analyses used standard commercial software programs. Results and Discussion Microarray Evaluation. Global gene expression during salinity stress was determined using Yeast Gene Filters (Research Genetics Inc.), assembled from PCR-amplified open reading frames (ORFs) for 95% of the yeast putative and confirmed genes. Yeast cultures were grown to mid-log phase in complete media supplied with 1M NaCl for 0, 10, 30, and 90 minutes. Exploratory experiments at different time points, which have been performed only once, are not included. Compared to previous microarray experiments [25, 30], the lag phase for recovery of growth in our experiments was approximately 2 times longer. Each hybridization was repeated 3 times. The α 33 P-dCTP signal intensities for all hybridizations ranged from background levels (3 to 65 dpm) to high intensity (~72,000 dpm). The most highly expressed transcripts for both stressed and unstressed yeast represented approximately 10% of the yeast genome with an average signal intensity value of 4,500 dpm, whereas 60% of the transcripts had an average signal 7

8 intensity value of 169 dpm (Figure 1). Standard deviations which included all hybridization were within +/-1.5 SD for the most highly expressed transcripts (614 ORFs) and slightly lower for transcripts expressed in the medium to low range (~1,000 ORFs). High salinity affected expression levels of approximately 10% of the yeast ORFs. During salt stress the number of ORFs induced increased more than two-fold from 107 (10 ) to 354 (90 ) (Figure 1). After 10 and 30, 27 and 78 ORFs, respectively, were induced more than 3-fold, and an additional 87 (10 ) and 165 (30 ) ORFs had average changes in transcript levels between 2- and 3-fold. After 90 of salt stress, 170 ORFs (>3-fold) and 185 ORFs (2- to 3-fold) showed increased average changes in transcript abundance. The upregulated ORFs in major MIPS categories are shown (Figure 2). The nature of regulated transcripts over time changed, suggesting that different functions needed to be activated at different time points. As a control, 39 ORFs were examined (Figure 3, not all data included) by RNA blot analysis, to independently verify changes for transcripts in different abundance categories. Open reading frames were chosen with varying levels of transcript abundance in microarrays; 11 ORFs >3.0 fold, 13 ORFs > fold, 7 ORFs with no change in message levels, and 8 with decreases of more than 2- fold. Imagequant software was used to determine changes in transcript levels in northern hybridizations, which were compared to the average changes in gene expression from the microarrays. Among the selected ORFs, 36 of 39 agreed with the microarray data (3 ORFs predicted to be upregulated by a factor of less than 2 showed no change in RNA blot hybridizations) (Figure 3). Overall, low and moderate changes in transcript abundance in the comparison between the RNA blots and the averaged microarray data differed by less than 2-fold, but large changes in abundance can differ by >10-fold, mainly attributable to low basal transcript levels (e.g., YGL037C, YGR243W, YHR087W). Correlation with known Yeast Stress Responses. The behavior of many transcripts in the analyses correlated with known biochemical hyper-osmotic stress responses. Glycerol, for example, an osmoprotectant known to accumulate rapidly in response to stress in yeast [31], accumulated as 8

9 documented by HPLC analyses (data not shown), and transcripts in the glycerol biosynthetic pathway increased. Indeed, dehydrogenases and phosphatases leading to glycerol production, GPD1/2, GPP1/2, were upregulated at all time points during salinity stress, most strongly as time progressed (Figure 4). Similarly, ORFs for enzymes involved in trehalose metabolism, GLK1, PGM2, HXK1, YKL035W, TPS1, TPS2, and NTH1, were upregulated at 90, but not at 10 and 30 of salt stress. Transcripts for all enzymes of the pathway were among those most highly induced (Figure 4). Trehalose, like glycerol, is implicated in yeast stress responses as an osmoprotectant, although trehalose does not accumulate to osmotically significant concentrations in salt-stressed baker s yeast [34]. PGM2, UGP1, TPS1, TPS2, and the regulatory factor encoded by TSL1, catalyze trehalose biosynthesis, while Nth1p and Ath1p (trehalases) lead to trehalose degradation and the formation of glucose [35]. Completion OF this cycle seems to be indicated by the upregulated transcripts for the kinases HXK1 and GLK1 (Figure 4). The presence of high transcript amounts for Nth1p, Hxk1p and Glk1p may explain why the osmoprotectant trehalose does not accumulate during salt stress. A circular flux of carbon, based on the induction of all ORFs in this pathway, seems to indicate a function for trehalose in a regulatory role, for example in redox control, similar to what has been documented for the functions of the two GPD enzymes [36; 58]. Trehalose synthesis and degradation, in combination with glycerol production, plays a key metabolic role in the protection against high salinity. Such a conclusion, also based on gene expression changes, has recently been put forward [58]. The 1,4-glucan branching enzyme (GLC3) involved in glycogen biosynthesis was only moderately upregulated under our conditions. These results are similar to recently published data with the exception that the high NaCl concentration, 1M, tended to delay up-regulation compared to what has been reported for the yeast transcriptome response in 0.4 M (for 10 and 20 ) or 0.7 M NaCl (45 ) and 0.95 M sorbitol (30 ) [25,30]. At the lower sodium concentration (0.4 M) nearly 1,400 ORFs increased, most of them transiently [30]. The upregulated ORFs shown in the study by Posas et al. [30] (0.4 M NaCl, 10 and 20 ) tended to be early-induced ORFs in our studies (see the supplemental 9

10 material). Induced ORFs reported by Rep et al. [25] at a concentration of 0.7 M NaCl (45 ) are mostly found among those ORFs upregulated after 90 in our experiments. The extent to which transcript increases correlate with protein amount has been verified in some studies. Apart from increases in the activity of enzymes and the phenotype of knockout mutants, twodimensional electrophoresis of proteins and partial sequencing of upregulated peptides indicated general proportionality between RNA and protein amounts for metabolic enzymes and this also extended to the downregulation of, for example, enolases (ENO1/2) [60-62; see Supplemental table 3]. Induced Gene Expression during hyper-osmotic Stress. Global gene expression patterns were determined for ORFs induced more than 2-fold after 10, 30, and 90 minutes of exposure to 1M NaCl (Figure 2). Expression patterns for early-induced transcripts, at 10 and 30 following stress, were similar, with 67% of the ORFs induced after 10 being identical to those induced after 30. The profiles are characterized by rapid transcript increases for ORFs in protein metabolism, mainly attributable to transcripts for components of protein synthesis, protein destination and the regulation of protein fate. Nearly half of all upregulated transcripts (42%; 44 ORFs) originated from the categories "protein destination" (ORFs related to protein modification, transport and targeting), "intracellular transport" (cellular import, protein trafficking, and vesicular transport), and "protein synthesis" (ribosomal proteins). These three categories represented 13% (53 ORFs) of the ORFs upregulated after 90. Based on a relative scale, the difference seems to indicate the significance of these ORFs for the adjustment of metabolism early during the salt stress. ORFs for ribosomal Proteins. Ribosomal proteins (RPO) account for the majority of induced ORFs after 10 (17 ORFs) and 30 (45 ORFs), and the 17 ORFs found after 10 are included in the group of 45 ORFs found at 30 (Table 1). This is in contrast to the expression patterns of all ORFs for ribosomal proteins at 90 of salt stress when 93 of the 176 (55%) strongly downregulated ORFs encoded ribosomal proteins. At the late time point, only three RPO were upregulated, all of them encoding mitochondrial 10

11 RPO (MRP8, MRPS18, MET13). The number of transcripts strongly repressed after 90 included 31 of the early-induced RPO (Table 1). The upregulated transcripts (30 ) represented 22 subunit proteins of small and 22 subunit proteins of large subunits of cytoplasmic ribosomes and, in addition, one subunit protein for mitochondrial ribosomes (MRP8). In only six cases (RPL1A/B, RPL19A/B, RPL34A/B, RPS25A/B, RPS26/A/B and RPS27A/B) were both isoforms of a ribosomal protein upregulated (possibly because of cross-hybridization) while only one of two isoforms showed increased transcript levels for the other RPO. Of the 137 yeast cytoplasmic ribosomal proteins of which 59 are duplicated [37], the 44 upregulated cytosolic RPO suggest specific promoter and possibly also signaling events. Their upregulation may also indicate a requirement for the presence of specific RPO under stress conditions. We have found a similar theme in the salt stress response of rice (Oryza sativa), recorded by microarrays of 1,728 transcripts. The early time points of salinity stress in rice lead within one hour to the upregulation of a large number of rice transcripts encoding plant cytosolic ribosomal proteins [38]. our hybridizations with yeast distinguished isoforms at identity scores of approximately 90% at the 2-fold increase threshold that was chosen. Relaxing the stringency to, for example, 1.6-fold increase would have included an even larger number of Rpo-transcripts. RPO upregulation has also been observed by Posas et al. [30]. Other upregulated Functions. Other differences in gene expression between 10 and 30 of salt stress were related to amino acid and nucleotide biosynthesis. At 10, induced ORFs in the categories amino acid (11.2%) and nucleotide metabolism, (9.3%) are higher relative to 30, (6.2% and 3.3%, respectively). Strongly upregulated are 10 of 12 ORFs associated with amino acid biosynthesis (YAL004w, THR4, HOM2, YER081w, PRS3, THR1, YIL074c, MET25, GLN1, and LYS21). Five of 11 are related to nucleotide metabolism: URA1, ADE1, PRS3, SHM2 and ADE13. The earliest metabolic activities, it seems, in need of upregulation include amino acid biosynthesis and ribonucleotide metabolism. Between 10 and 30 the transcriptional and translational machineries generate, modify and transport proteins 11

12 within the cell and this process seems to be completed by 90. At that time, ORFs involved in protein synthesis have declined precipitously and the majority of gene expression changes now target different functions, such as chaperone and detoxification increases and intracellular transport. The category energy supply showed a peak of induction in the number of regulated transcripts, specifically in the subcategories respiration and the metabolism of energy reserves (Figure 2 and supplemental material). After 30 of salt stress, 9.1% of the induced ORFs are components of the electron transport chain, such as cytochromes, or subunits of the ATP synthase complex, with the comparable percentages of induced ORFs, 5.6% and 4.0% after 10 and 90, respectively. Signal transduction components. Transcriptional regulation under hyper-osmotic conditions by the HOG1 protein kinase and the CNB1 protein phosphatase (calcineurin) has been well documented [10, 15, 17, 39, 40]. Especially well studied is the signaling pathway terminating at HOG1, with the sensors Sln1p and Sho1p converging in the MAPK cascade from Ssk2/22p through Pbs2p to Hog1p [10, 18, 24, 41-43]. Additional evidence for genes regulated downstream of Hog1p and the transcriptional activators MSN2/MSN4 has recently been provided in microarray experiments comparing wild type and deletion mutants of Hog1p and Msn2p/Msn4p [25, 30]. Our results indicated that calcineurin was induced during the 10 and 30 time points, but HOG1 was not found among the significantly upregulated transcripts. Several other protein kinases, phosphatases, and several transcription factors were consistently upregulated. These included ORFs with established functions in cell signaling (MFA1, SRA3, and HAC1), cell cycle control and mitosis (PPH22, PHO85, PPH21, and CIN5), and mrna transcription (PHO4, PHO85, GCN4, CUP2, TIS11, YHR056C, and YER130C). Functions in Cell Defense. Not surprisingly, the number of ORFs in the categories cell rescue, defense, cell death and aging increased during the stress (Table 2). These included the ORFs for glycerol and trehalose production, and five genes involved in cellular detoxification [GRX1 and TTR1 (glutaredoxin), the duplicated CUP1A/CUP1B (metallothionein), and CCP1 (cytochrome c peroxidase 12

13 precursor)]. In the case of CUP1A/B, their high sequence homology led to cross-hybridization and only one of the genes may be upregulated. YGP1 (gp37, glycoprotein secreted in response to nutrient limitation) is also induced, although its function during salt stress is not clear. It may be associated with cell wall biogenesis since it is 47% homologous to SPS100, which is involved in cell wall formation [44]. The percentages of ORFs related to the defense response (Figure 2) are 10% (10 ), 5 % (30 ), and 7% (90 ). The lower percentage at 30 relative to other times can be attributed to heat shock proteins (HSPs) (Table 2), which have been reported to be upregulated in a variety of stresses such as oxidative stress (H 2 O 2 ), MMS (methyl methanesulfonate), and heat shock [45-47]. Four HSPs (SSA1, HSC82, YGL128C, and YDR033W), are induced within 10 ; one HSP (HSP12) after 30, and nine HSPs after 90 of exposure to salt (HSP12, YRO2, HSP26, SSE2, HSP78, SSA4, MDJ1, HSP104, and DDR48). Only HSP12 was induced during two time points (30 and 90 ). The groups of HSPs induced during 10 and 90 are distinct, indicating that different sets of HSPs could have different functional targets in the early and later responses to salinity stress. Also, the majority (5 of 6) of induced genes involved in DNA repair (Table 2) showed increased transcript amounts only during the later stress response (90 ). These included HSP12, FUN30, HEL1, THI4, and RNR4. Transport Functions. During the early response to salinity stress, few induced ORFs were observed in the categories of the major facilitator superfamily (MFS) (3 ORFs), nitrogen and sulfur metabolism (1 ORF), and in lipid, fatty acid and isoprenoid metabolism (6 ORFs) (Figure 2). This changed during the 90 time point: 17 upregulated ORFs encode MFS transport proteins, and 9 and 16 ORFs are induced involved in nitrogen and sulfur metabolism and in lipid, fatty acid and isoprenoid metabolism, respectively (Table 3). Members of the MFS are characterized as permeases with 12 transmembrane domains. Many proteins in this class act as sugar, amino acid, or multidrug transporters [49]. Five of the 17 induced MFS genes have been characterized: the iron transporter, FET3; the amino acid transporters FUR4, PUT4, and BAP2; and the low-affinity hexose transporter, HXT1. It remains to be seen whether 13

14 the 12 unknown MFS transcripts are translated into membrane associated proteins, and if their induction is salt-specific. Among the ion transporters known to play a role in sodium detoxification [3, 48, 50, 51], several transcripts were upregulated. The Na-ATPases, ENA1-4, were induced after 30 of salt stress. Vacuolar H + -ATPase subunits were induced during all time points and the H + -ATPase PMA1 was not induced (Figure 2, and supplemental information). It is difficult to determine metabolic functions in stressed cells for the induced ORFs relative to nitrogen, sulfur, lipid, fatty acid and isoprenoid metabolism. However, 12 ORFs related to nitrogen and sulfur metabolism were found to be upregulated following a different stress, MMS [47], and 4 of these are also found during 90 of salt stress (Table 4): NPR1 (serine/threonine kinase), and three unknown ORFs, YFL030W (similar to transaminases), YPL135W and YOR226C (both similar to proteins involved in nitrogen fixation). We also analyzed the regulation of stress response genes in a group of 84 ORFs, which include at least two STRE s in their 5 -upstream regions [23]. A subset of these genes is affected by the Hog1pdependent signal transduction pathway [10, 25, 30]. Transcripts for 34 ORFs containing multiple STREs were upregulated with the majority induced after 90 of salt stress: 3 (10 ), 8 (30 ), and 30 ORFs (90 ) (see the supplemental material). Magnification of the Stress Response in a gdp1, gdp2 Strain. As a control to test filter reliability, hybridizations used RNA from a mutant strain, gpd1 gpd2 [31], which is deficient in glycerol production. The GPD1 and GPD2 transcripts were not detectable (not shown). The inability of these cells to osmotically adjust to high salinity also affected a number of other transcripts. The gpd1/2 strain, when grown for 60 in 0.5 M NaCl, showed a significantly different transcript induction profile compared to wild type grown in 1 M NaCl (Table 4), a concentration that is lethal for this mutant. Upregulated transcripts were mostly comparable to the 90 time point in wild type in 108 of highly induced transcripts (see the supplemental material), including most of the metabolic functions. Upregulated (>2-fold) in the 14

15 deletion mutant though not in wild type, were another 87 transcripts (Table 4). Most conspicuous were transcripts for components of retrotransposons. While one gene related to retrotransposons (YOR344c) was among the upregulated ORFs in wild type, the number increased to 17 (including YOR344c) in gpd1/2. Cellular stress has been implicated as an agent that increases transposition [e.g., 63]. In yeast, FUS3, a mitogen-activated protein kinase inhibits Ty1 retrotransposition [64]. This transcript is strongly downregulated in gpd1/2 (-3.0) but unaffected by 1 M NaCl in wild type (see Supplemental table 4). In addition, another 34 ORFs were induced for which no function is known (or where the putative function has not experimentally been verified). At least 11 of the ORFs strongly upregulated in the mutant are functionally associated with mitochondria, possibly indicating a function in aging or cell death. Also, α-factor receptor, mating pheromone α-2 factor, mating factor α, agglutinin, and transcripts for surface proteins increased. Finally, an ORF with strong similarity to a protein kinase (Stl2p), and ORFs for two transcription factor-like proteins (YLR256w, YKL109w [HAP4]) were also newly upregulated in the glycerol-deficient mutant. The transcriptional behavior in this mutant supports a connection between osmoregulatory and pheromone response pathways: the cross talk in signaling to regulate growth and osmotic stress defense pathways reported before [e.g., 52]. We can only speculate that in the absence of glycerol synthesis and accumulation in this strain the hyper-osmotic condition leads to a severe stress that affects cell integrity to a much larger extent than in the wild type at even higher osmolarity. Functionally unknown salinity-regulated ORFs. Unclassified, putative and unconfirmed ORFs represent approximately 40% of the total yeast genome. Many of these were induced by the stress: 16% (17 ORFs) of all induced transcripts at 10, 24% (57) at 30, and 42% (149) at 90 of salt stress (Figure 2). Reasons for a gradual increase of functionally not studied ORFs are that long-term stress experiments have been few and that these ORFs may only be expressed under severe stress conditions. The program for the systematic elimination and analysis of ORFs [53] has already produced data (for a database of protein-protein interactions, see: None of these 15

16 knockout mutants shows a clear phenotype under salt stress conditions (streaking and analysis for survival after long-term growth) [55, 59]. However, several of the KO strains show phenotypes that are related to stresses, for example, the addition of 0.03% SDS or hygromycin, indicating that these ORFs could have a function in cell wall synthesis or integrity (59; see also: A Comparison of Yeast and Plant Salinity Stress Responses. Comparisons of unclassified yeast ORF expression profiles to gene expression changes among unknown genes from other organisms such as bacteria and plants will provide clues to the functions of the unknown yeast ORFs. Table 5 lists ORFs with unknown function in yeast for which homologous plant ESTs have been found. As indicated, several of the plant homologs are significantly upregulated at different time points during stress. A comparison is made with ESTs from a salt-tolerant plant, Mesembryanthemum crystallinum (ice plant), including ESTs that show significant homology to functionally unknown yeast ORFs or questionable ORFs [57]. In addition, more than 50 yeast genes for which functions are known are also upregulated in salt-stressed plants where a similar progression of different upregulated functions has been observed [38, 57, and unpublished]. Several of the plant homologs identified ORFs termed hypothetical in yeast and exhibited similarities to putative serine/threonine-type protein kinases. Yeast knockout strains that eliminated these ORFs did, however, not show a clear phenotype under salt stress conditions [55]. Salinity tolerance - a progression of programs? We have catalogued changes in gene expression in yeast over time following a severe salt shock, which affects growth significantly. Determining number and nature of induced transcripts and the changes in time of transcript categories allowed for several conclusions. The profile of the genes expressed is similar at the early times (10 and 30 ) but changes later (90 ) are considerable. The evidence points towards the initiation of protein synthesis and restructuring of protein composition as a major task for the initial period (ribosomal proteins, transcription machinery, amino acid synthesis and utilization). Another part of the early response is the induction of chaperone-type proteins. Only early on, a few components of signaling pathways are found upregulated; 16

17 we think that more of these components could be induced at even earlier time points or under less severe stress. To place the information into context, we indicate four response types that allow for clear statements. (1) A few transcripts are rapidly and strongly upregulated early. The most dramatically early upregulated ORF (YDR276c) encodes gene PMP3/SNA1. Deletion of PMP3 confers hypersensitivity to sodium in mutants that lack sodium efflux systems (e.g., Pmr2p/Enap and Nha1p). A function for Pmp3p has recently been documented in the control of membrane polarization, such that its deletion leads to membrane hyper-polarization and increased influx of monovalent cations [56]. A plant (Arabidopsis thaliana) PMP3 homolog complements this phenotype. PMP3 homologues, which are salt stressdependently upregulated in microarray analyses [57], have also been found in the halophytic plant Mesembryanthemum crystallinum [27, 57] and in salt-stressed rice [38]. PMP3 transcript induction in our experiments changed from an early induction (17-fold at 10, 14-fold at 30 ) to 2-fold induction at 90. (2) Ribosomal proteins and several other functions exemplify the second response type, gradual upregulation to a peak at 30 followed by the later repression of all RPOs with the exception of a few mitochondrial proteins. Among those upregulated, some if them transiently, in the second phase are also components for mitochondrial functioning, glycolysis, transcription, inter-compartmental protein transport, and protein turnover. (3) The third type comprises ORFs unaffected or downregulated early and gradually induced later. These included many known yeast salinity stress responses.. ORFs in this category have also been reported as upregulated in two previous array analyses [25, 30] with few cases where the induction was close to the cutoff value of two-fold (see supplemental materials). The increases reported in previous studies are generally higher than the fold-increases found in our experiments but all three data sets show the same trend [25, 30]. What might be upregulated 50-fold or 10-fold in previous reports is shown in our analyses an averaged induction of 20- to 5-fold (see Supplementary Table 1). In addition to these 17

18 ORFs [25, 30], our analyses detected other strongly upregulated transcripts. A secreted glycoprotein (YGP1; YNL160w) for example, reported as 3-fold upregulated [25] was 26-fold upregulated in our experiments. ARE2 (acyl-coa sterol acyltransferase) is strongly upregulated as are a putative resistance protein (YOR273c), PEP4 (aspartyl protease), ERG5 (C-22 sterol desaturase), YLL028c (similarity to multidrug resistance proteins), GYP6 (GTPase-activating protein) or the CUP1A/1B metallothioneins. These have not been reported before [25, 30], indicating strain differences or reflecting the different stress conditions. (4) The fourth response type, consistently upregulated transcripts, falls in many different functional categories. Each of the four response categories includes upregulated functionally unknown, putative and/or questionable ORFs with the absolute numbers increasing at the 90 time point (Figure 2). Among the late-induced ORFs, highly expressed transcripts identify membrane transporters, cell detoxification functions, and a number of unclassified ORFs. Plant ESTs exist with homology not only to functionally identified transcripts but also to ORFs whose functions remain to be determined. Resources that will aid in deciphering the roles of these unknown ORFs include the generation and availability of yeast deletion strains [e.g., 53], programs for protein function determination by protein-protein interactions [54] or in silico, and the microarray expression data from related organisms such as other fungi, bacteria and plants. The advantage provided by the three time points chosen in connection with a strong salt shock seems to lie in a drawn-out stress response that allowed for a separation of successively engaged response pathways. This succession identifies early, strongly upregulated functions, intermediate and late functions. The analysis presented here does not, however, include the equally numerous downregulated functions, which provide additional information about the effects of stress and the responses by which tolerance can be achieved. Further in depth analyses, including additional time points, different salt stress treatments or other abiotic stress factors, and additional mutant strains will be necessary for a complete understanding of the yeast stress response. Our results extend the reported data 18

19 [25, 30] through the inclusion of several time points under severe stress conditions and integrates immediate cellular responses with long-term metabolic and physiological events that seem to assure survival. The kinetics of this response may serve as a paradigm against which plant salinity stress responses could be scaled. Acknowledgements. We thank Ariana Call, Chris Borchert and Robert Hershoff for help, and the people from Research Genetics for advice. We are indebted to Drs. Tracie Matsumoto, Mike Hasegawa (Purdue University), Carol Dieckmann (University of Arizona), Rolf Prade and Patricia Ayoubi (Oklahoma State University) for discussions or help with annotations. Drs. Stefan Hohmann (University of Gøeteborg, Sweden) and Carol Dieckmann generously provided yeast strains and advice. 19

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24 Table 1. Yeast ribosomal protein genes induced at 10 and 30 minutes (>2.0 fold) and repressed at 90 minutes (<2.0 fold) Accession Foldinduction Gene name Description YNL178w 4.1 RPS3 small subunit ribosomal protein, 40S, S3.e YHR203c 2.9 RPS4B small subunit ribosomal protein, 40S, S4.e.c8 YPL090c 3.9 RPS6A small subunit ribosomal protein, 40S, S6.e YNL096c 2.6 RPS7B strong similarity to ribosomal protein S7, 40S YBL072c 2.2 RPS8A small subunit ribosomal protein, 40S, S8.e YPL081w 2.8 RPS9A small subunit ribosomal protein, 40S, S9.e.A YDR025w 3.3 RPS11A small subunit ribosomal protein, 40S, S11.e YDR064w 2.7 RPS13 small subunit ribosomal protein, 40S YOL040c 2.2 RPS15 small subunit ribosomal protein, 40S, S15 YDL083c 2.3 RPS16B small subunit ribosomal protein, 40S, S16.e YDR447c 2.3 RPS17B small subunit ribosomal protein, 40S, S17.e.B YDR450w 2.3 RPS18A small subunit ribosomal protein, 40S, S18.e.c4 YHL015w 2.6 RPS20 small subunit ribosomal protein, 40S YJL190c 4.7 RPS22A small subunit ribosomal protein, 40S, S15a.e.c10 YIL069c 3.3 RPS24B small subunit ribosomal protein, 40S, S24.e YGR027c 3.3 RPS25A small subunit ribosomal protein, 40S, S25.e.c7 YLR333c 2.7 RPS25B small subunit ribosomal protein, 40S, S25.e.c12 YGL189c 2.4 RPS26A small subunit ribosomal protein, 40S, S26e.c7 YER131w 2.9 RPS26B small subunit ribosomal protein, 40S, S26e-c5 YKL156w 3 RPS27A small subunit ribosomal protein, 40S, S27.e YHR021c 2.2 PRS27B small subunit ribosomal protein, 40S, S27.e YLR167w 2.6 UBI3/RPS31 ubiquitin domain signature/ small subunit ribosomal protein, 40S YPL220w 2 RPL1A large subunit ribosomal protein, 60S YGL135w 2.3 SSM2/RPL1B large subunit ribosomal protein, 60S YGL147c 3.2 RPL9A large subunit ribosomal protein, 60S, L9.e YGR085c 2.9 RPL11B large subunit ribosomal protein, 60S YEL054c 2.1 RPL12A large subunit ribosomal protein, 60S, L12.e YMR142c 2.7 RPL13B large subunit ribosomal protein, 60S YLR029c 2.5 RPL15A large subunit ribosomal protein, 60S, L15.e.c12 YNL069c 2.8 RP23/RPL16B large subunit ribosomal protein, 60S YBR084c-a 4.4 RPL19A large subunit ribosomal protein, 60S YBL027w 2.4 RPL19B large subunit ribosomal protein, 60S, L19.e YMR242c 2.4 RPL20A large subunit ribosomal protein, 60S, L19.e YPL079w 2.4 RPL21B large subunit ribosomal protein, 60S, L21 YBL087c 3.4 RPL23A large subunit ribosomal protein, 60S, L23.e YOL127w 3.5 RPL25 large subunit ribosomal protein, 60S, L23a.e YBL092w 2.3 RPL32 large subunit ribosomal protein, 60S, L32.e YOR234c 2.5 RPL33B large subunit ribosomal protein, 60S, L35a.e.c16 YER056c-a 3.6 RPL34A strong similarity to mammalian ribosomal L34 proteins YIL052c 3.7 RPL34B large subunit ribosomal protein, 60S, L34.e YDL191w 5.4 RPL35A large subunit ribosomal protein, 60S YLR325c 3.5 RPL38 large subunit ribosomal protein, 60S YIL189w 2.3 RPL39 large subunit ribosomal protein, 60S, L39.e YPR043w 3 RPL43A large subunit ribosomal protein, 60S, L37a.e YNL306w 2.7 MRPS18 mitochondrial, small subunit ribosomal protein, S18; upregulated at 90 only YKL142w 2.2/3.6 MRP8 mitochondrial ribosomal protein; upregulated at 30 & 90. YGL125w 2.5 MET13 putative methylene tetrahydrofolate reductase, predicted mitochondrial ribosmal protein, large subunit; upregulated at 90. *All RPO are upregulated at 30 ; several RPO are also upregulated at 10. Three mitochondrial RPO are upregulated at

25 Table 2: Listing of upregulated ORFs in the Stress Response Categories "Cell Rescue, Defense, Cell Death and Aging". Accession Gene Foldinduction Description induced after 10,30 & 90 minutes S YDL022w GPD glycerol-3-phosphate dehydrogenase S YNL160w YGP secreted glycoprotein S YER062c GPD DL-glycerol phosphatase D YHR053c CUP1A 14.7 metallothionein D YHR055c CUP1B 13.4 metallothionein SD YDR513w TTR1 5.2 glutaredoxin SD YCL035c GRX1 4 glutaredoxin D YKR066c CCP1 2.5 cytochrome-c peroxidase precursor induced after 10 & 30 minutes S YDR155c CPH1 3 cyclophilin (peptidylprolyl isomerase) S YER057c HIG1 2.8 Heat-shock inducible Inhibitor of cell Growth induced after 10 minutes S YAL005c SSA1 3.1 heat shock protein of HSP70 family, cytosolic S YMR186w HSC heat shock protein S YDR033w 2.7 strong similarity to putative heat shock protein YRO2 S YGL128c 2.1 putative heat shock protein induced after 30 minutes D YLR043c TRX1 2.6 thioredoxin I S YER012w PRE S proteasome subunit C11(b4) S YBR082c UBC4 2.2 E2 ubiquitin-conjugating enzyme D YBR244w 2.2 strong similarity to glutathione peroxidases D YGR209c TRX2 2.2 thioredoxin II S YER042w MSRA 2.1 responsible for the reduction of methionine sulfoxide induced after 30 & 90 minutes S YFL014w HSP heat shock protein S YMR251w-a HOR7 7.7 hyperosmolarity-responsive protein D YHR008c SOD2 3.1 superoxide dismutase (Mn) precursor, mitochondrial D YBL064c 2.6 strong similarity to thiol-specific antioxidant enzyme S YEL039c CYC7 2.1 cytochrome-c isoform 2 induced after 90 minutes D YOR273c 13.8 similarity to resistance proteins S YML070w DAK dihydroxyacetone kinase, induced in high salt SD YGR088w CTT1 9 catalase T, cytosolic S YBR072w HSP heat shock protein S YDR074w TPS2 7 alpha,alpha-trehalose-phosphate synthase S YLL026w HSP heat shock protein S YHR104w GRE3 6 aldose reductase S YDR258c HSP heat shock protein of clpb family S YBR169c SSE2 4.9 heat shock protein of the HSP70 family D YLL028w 4.7 similarity to multidrug resistance proteins S YBR126c TPS1 4.7 a,a-trehalose-phosphate synthase, 56 KD S YMR173w DDR heat shock protein S YBR054w YRO2 4.3 similarity to HSP30 heat shock protein Yro1p D YMR015c ERG5 4.2 C-22 sterol desaturase 25

26 S YNL241c ZWF1 3.2 glucose-6-phosphate dehydrogenase S YJL116c NCA3 3 involved in regulation of synthesis of Atp6p and Atp8p D YEL065w SIT1 2.9 probable multidrug resistance protein D YOR153w PDR5 2.8 pleiotropic drug resistance protein D YHL040c 2.8 similarity to C.carbonum toxin pump S YER103w SSA4 2.7 heat shock protein of HSP70 family, cytosolic S YFL016c MDJ1 2.7 heat shock protein - chaperone R YGR144w THI4 2.7 involved in thiamine biosynthesis and DNA repair D YIL121w 2.6 similarity to antibiotic resistance proteins S YOR027w STI1 2.5 stress-induced protein S YER143w DDI1 2.4 induced in response to DNA alkylation damage S YML016c PPZ1 2.4 ser/thr phosphatase required for normal osmoregulation S YJR090c GRR1 2.3 required for glucose repression and cation transport R YGR180c RNR4 2.3 ribonucleotide reductase small subunit R YAL019w FUN similarity to helicases of the Snf2/Rad54 family R YER176w HEL1 2 DNA dependent ATPase/DNA helicase B S YMR273c ZDS1 2 involved in negative regulation of cell polarity S - stress response SD - Stress response & detoxification D - detoxification R - DNA repair 26

27 Table 3. ORFs induced more than two-fold during 90 of Salinity Stress in the Categories "MFS, Nitrogen/Sulfur Metabolism and in Lipid, fatty Acid, and Isoprenoid Biosynthesis". Accession Gene Foldinduction name Description MAJOR FACILITATOR SUPERFAMILY YDR536w STL strong similarity to members of the sugar permease family YOR273c 13.8 similar to resistance proteins YOR348c PUT proline and gamma-aminobutyrate permease YHR094c HXT1 8.6 low-affinity hexose transporter YLL028w 4.7 similar to multidrug resistance proteins YBR021w FUR4 4.5 uracil permease YOL119c 4 similar to monocarboxylate transporter proteins YBR068c BAP2 3.4 leucine permease, high-affinity (S1) YHR096c HXT5 3.1 strong similarity to hexose transporters YMR058w FET3 3.1 cell surface ferroxidase, high affinity YKL039w PTM1 3 member of the major facilitator superfamily YEL065w SIT1 2.9 probable multidrug resistance protein YHL040c 2.8 similar to C.carbonum toxin pump YIL121w 2.6 similar to antibiotic resistance proteins YDR387c 2.5 similar to Itr1p and Itr2p and E.coli arae YIL056w 2.4 similar to YER064c YNR013c 2.3 similar to Pho87p and YJL198w NITROGEN AND SULPHUR METABOLISM YPL111w CAR1 8 arginase YLR438w CAR2 5.5 ornithine aminotransferase YJL172w CPS1 4.4 Gly-X carboxypeptidase YSCS precursor YPL135w 3.5 strong similarity to nitrogen fixation protein (nifu) YGR019w UGA aminobutyrate aminotransferase (GABA transaminase) YFL030w 3.2 similar to several transaminases YJL060w 2.7 similar to kynurenine aminotransferase & glutaminephenylpyruvate transaminase YNL183c NPR1 2.4 ser/thr protein kinase YOR226c 2.3 strong similarity to nitrogen fixation proteins LIPID, FATTY ACID AND ISPRENOID BIOSYNTHESIS YFL014w HSP heat shock protein, induced by free fatty acids & other stresses YML131w 7.3 similar to human leukotriene b4 12-hydroxydehydrogenase YNR019w ARE2 4.8 acyl-coa sterol acyltransferase YOR317w FAA1 4.5 long-chain-fatty-acid--coa ligase YMR015c ERG5 4.2 C-22 sterol desaturase YNL231c 3.4 weak similarity to Sec14p YOL011w 3.2 strong similarity to phospholipases YGL055w OLE1 3 stearoyl-coa desaturase YOR153w PDR5 2.8 pleiotropic drug resistance protein YBR036c CSG2 2.5 calcium dependent regulatory protein YHR190w ERG9 2.5 farnesyl-diphosphate farnesyltransferase YPL057c SUR1 2.5 required for mannosylation of sphingolipids YKL091c 2.5 strong similarity to Sec14p YER061c CEM1 2.3 b-keto-acyl-acp synthase, mitochondrial YFL031w HAC1 2.2 transcription factor YBR222c FAT2 2 AMP-binding protein, peroxisomal 27

28 Table 4: Transcripts in yeast gpd1/gpd2 induced higher than in wild type* Accession Gene Description Induction in wild type, 90 NaCl Rank in wild type 90 NaCl** Induction in gdp1/2 YPL187W MF(a)1 mating factor a YLR160C ASP3 nitrogen catabolite-regul. cell wall L-asparaginase II YDR007W TRP1 n-(5 -phosphoribosyl)-anthranilate isomerase YLR155C ASP3 nitrogen catabolite-regul. cell wall L-asparaginase II YLR158C ASP3 nitrogen catabolite-regul. cell wall L-asparaginase II YLR157C ASP3 nitrogen catabolite-regul. cell wall L-asparaginase II YOR135C IDH2 questionable ORF YJR026W TY1A protein YER172C BRR2 putative ATP-dependent RNA helicase YML045W TY1A protein YLR040C weak similartity to hypothetical protein YIL011w YJR028W TY1A protein YMR050C TY1B protein YMR051C TY1A protein YML039W TY1B protein YLR304C ACO1 aconitase, mitochondrial YLR041W questionable ORF YKL109W HAP4 transcriptional activator protein of CYC1 (component of HAP2/HAP3 heteromer) YOL106W identified by SAGE YKL178C STE3 a-factor receptor YDR008C questionable ORF YJR004C SAG1 a-agglutinin YJR073C OPI3 methylene-fatty-acyl-phospholipid synthase (unsat phospholipid N-methyltransferase) YOR302W CPA1 leader peptide YML040W TY1A protein YJR027W TY1B protein YBL005W-A MIPS cannot find YHR218W putative pseudogene YER160C TY1B protein YIL070C mitochondrial acidic matrix protein YNR042W hypothetical protein YJR029W TY1B protein YGR260W similar to allantoate transport protein YGL089C MF(a)2 mating pheromone a-2 factor YAR009C Ty1B protein YAR008W 34kDa subunit, tetrameric trna splicing endonuclease YMR046C TY1A protein YBR012W-B TY1B protein YKL177W questionable ORF YKL157W APE2 aminopeptidase yscii YDL174C DLD1 mitochondrial enzyme D-lactate ferricytochrome c oxidoreductase YDR340W questionable ORF YPL280W strong similarity to YMR322c & YDR533c

29 YCR005C CIT2 non-mitochondrial citrate synthase YER138C transposon Ty putative peptide with frame shift YJL066C hypothetical protein YAR010C TY1A protein YPL118W component small subunit of mitochondrial ribosome YJR113C similar to bacterial, chloroplast & mitochondrial ribosomal protein S7 YOR316C COT1 protein involved in cobalt accumulation; dosage dependent supp of cobalt toxicity YFL068W weak similarity to hypothetical E.coli protein YNL190W hypothetical protein YLR202C questionable ORF YBL005W-B MIPS cannot find YMR011W HXT2 high affinity hexose transporter YNL180C similar to S.pombe Cdc42p & GTP-binding proteins YNL015W PBI2 proteinase inhibitor I2B, inhibits protease Prb1p YDR366C similarity to YOL106w & YER181c YBR012W-A TY1A protein YGL121C hypothetical protein YGL069C questionable ORF YLR382C NAM2 mitochondrial leucyl trna synthetase YBL112C strong similarity to subtelomeric encoded proteins YLL041C SDH2 succinate DH (ubiquinone) iron-sulfur protein subunit YKL148C SDH1 flavoprotein subunit of succinate dehydrogenase YLR256W zinc-finger transcription factor of the Zn(2)-Cys(6) binuclear cluster domain type YMR272C desaturase/ hydroxylase enzyme YBL049W hypothetical protein YOR215C similar to M.xanthus hypothetical protein YOR289W similar to C.elegans hypothetical protein YNL045W strong similarity to human leukotriene-a4 hydrolase YOR356W strong similarity to human electron transfer flavoproteinubiquinone oxidoreductase YPL262W FUM1 mitoch. & cytoplasmic fumarase (fumarate hydrolase) YOL082W unknown YGR255C COQ6 monooxygenase YFL-TYA MIPS cannot find YFL-TYB MIPS cannot find YPL172C COX10 putative farnesyl transferase - heme A synthesis YKL161C strong similarity to ser/thr-specific PK Slt2p YKR013W PRY2 similar to plant PR1 pathogen-related proteins YMR226C similar to ketoreductases YKR006C MRPL13 mitochondrial ribosomal protein YmL YLR168C MSF1 probably involved in intramitochondrial protein sorting YDR077W SED1 putative cell surface glycoprotein YNR036C strong similarity to ribosomal protein S YOR045W TOM6 mitochondrial outer membrane import receptor subunit, 6 kd * Transcripts upregulated in Dgdp1/2 after 60 min in 0.5 M NaCl, including the ~200 most highly induced ORFs, in comparison to transcripts upregulated at 10, 30, and 90 in wild type. ** Rank of induction in wild type out of 6,144 ORFs with #1 being most highly expressed. ORFs upregulated in Dgdp1/2 and in wild type can be found at 29

30 Table 5: Salinity stress-upregulated yeast ORFs with Homology to Mesembryanthemum cdnas* Accession Ice Plant Accession Yeast Homology scores** [l] e-val. Ice Plant cdna library, stress treatment and age*** SGD description 10 minute time point ML05D05 YDR417C 108 8e-27 Flower/Fruit, 500 mm NaCl, 6w questionable ORF MO25F05 YDR544C 34 4e-04 Meristem, no stress, 5w strong similarity to subtelomerically encoded proteins 30 minute time point MO18H07 YDL046W 48 6e-08 Meristem, no stress, 5w hypothetical protein ML05D05 YDR417C 108 8e-27 Flower/Fruit, 500 mm NaCl, 6w questionable ORF MO20H03 YDR417C 99 6e-24 Meristem, no stress, 5w questionable ORF ML07F03# YGR052W 46 6e-08 Flower/Fruit, 500 mm NaCl, 6w similarity to ser/thr protein kinases MP04C05 YGR052W 49 1e-08 Meristem, 500 mm NaCl, 3d similarity to ser/thr protein kinases MO23C02 YGR052W 39 8e-08 Meristem, no stress, 5w similarity to ser/thr protein kinases MP04C05 YGR052W 49 3e-08 Meristem, 500 mm NaCl, 3d similarity to ser/thr protein kinases MO19G08# YGR142W 37 8e-05 Meristem, no stress, 5w BTN2, expression elevated in btn1/btn1p lacking strains ML07G07# YHR217C 33 7e-04 Flower/Fruit, 500 mm NaCl, 6w strong similarity to subtelomeric encoded YDR544c MO25F05 YHR217C 41 3e-06 Meristem, no stress, 5w strong similarity to subtelomerically encoded YDR544c MF06A12# YMR173W-A 44 7e-07 Root, 400 mm NaCl, 12h questionable ORF ML04A12 YMR173W-A 44 7e-07 Flower/Fruit, 500 mm NaCl, 6w questionable ORF MO26G01# YMR291W 52 3e-09 Meristem, no stress, 5w similarity to ser/thr protein kinase 90 minute time point MO18H06 YDL046W 41 3e-07 Meristem, no stress, 5w hypothetical protein MO18H07 YDL046W 48 6e-08 Meristem, no stress, 5w hypothetical protein ML07F03 YGR052W 46 1e-07 Flower/Fruit, 500 mm NaCl, 6w similarity to ser/thr protein kinases MO26G10 YGR052W 41 5e-06 Meristem, no stress, 5w similarity to ser/thr protein kinases MP04C05 YGR052W 49 3e-08 Meristem, 500 mm NaCl, 3d similarity to ser/thr protein kinases MO19G08# YGR142W 37 8e-05 Meristem, no stress, 5w BTN2, elevated in Dbtn1/Btn1p strains MF06A12# YMR173W-A 44 7e-07 Root, 400 mm NaCl, 12h questionable ORF ML04A12 YMR173W-A 44 7e-07 Flower/Fruit, 500 mm NaCl, 6w questionable ORF MO26G01# YMR291W 52 3e-09 Meristem, no stress, 5w similarity to ser/thr protein kinase *Mesembryanthemum ESTs have been deposited into the dbest database (see also Ongoing microarray analyses indicate that several of the plant ORFs are upregulated following salt stress (Michalowski CB, Deyholos M, Bohnert HJ, unpublished). **Only ORFs with the highest homology scores are included, homology to additional ORFs is predicted. ***Homologous sequences seem to be more frequent in plant cdna libraries containing dividing cells. #Ice plant ESTs with >2-fold upregulation during salt stress. Expression changes in plants occur over a wider range of time points compared to yeast (Kawasaki et al., 2001). 30

31 Figure Legends Figure 1. Intensity of 33 P incorporation for 6,144 Saccharomyces cerevisiae ORFs. The Pathways software "single filter analysis application" was used to obtain intensity values for each ORF. Intensity values in the range from 3-72,000 dpm are representative of 12 individual filters used in the experiments. Figure 2. Gene expression profiles in functional categories. The classification is based on the MIPS database ( Figure 3. Comparison of RNA blot hybridizations and microarray expression data. RNA blots (12 are shown) for 39 ORFs were done in triplicate using RNA isolated from control (-) and salt-stressed yeast (+) exposed for 90 min. to 1M NaCl. The RNA blot data (N) were generated using Imagequant software and represent average changes in signals comparing stress to control. The probes were generated by PCR amplification of entire ORFs from genomic DNA and by random primer labeling with α 33 P-dCTP. The average microarray data (M) represent the average of 3 experiments (no salt vs. 90 salt stress) and standard deviation (S) calculated using the "nonbiased" or "n-1" method. The results for 3 of 39 ORFs did not agree (YDR387c [ITR1-like], YHR048w [unknown], YFR017c [unknown]), because expression at one of the time points was at background level and could not be measured accurately. Figure 4: Regulation of transcripts in trehalose and glycerol biosynthesis, acetate production and the TCA cycle. 31

32 Gene names are presented in boxes with fold upregulation indicated. For three of the four upregulated coding regions for TCA cycle enzymes the fold regulation is shown for all three time points. 32

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