Supplementary Material Supplementary Tables Table S1 – Media Used in this Study Table S2 – Expression Data Table S3 – Quiescent specific and Growth rate dependent genes Table S4 – Metabolite Data Table S5 – List of Mutants Defective in Surviving Starvation Supplementary Figures Figure S1. Method for analyzing gene expression values. Figure S2. Overlap of genes exhibiting quiescent specific expression with those affected by acute heat shock. Figure S3. Singular value decomposition of starvation expression data. Figure S4 Discordance in trehalose intermediates and gene expression levels during starvation. Table S1 – Media Used in this Study Batch culture media (per 1L) Glucose starvation batch media Compound Yeast Nitrogen Base w/o amino acids Glucose Synthetic Defined 6.7 g Glucose starvation 6.7 g 20 g - Nitrogen starvation batch media Compound Yeast Nitrogen Base w/o amino acids and ammonium sulfate Glucose Na2SO4 (NH4)2SO4 Nitrogen limited 1.7 g Nitrogen starvation 1.7 g 20 g 5g 625 mg 20 g 5g - Phosphate starvation batch media Compound Glucose 1000X vitamins 1000X metals 10X salts KH2PO4 Phosphate limited 20 g 1 ml 1 ml 100 ml 20 mg 10X Salts for phosphate starvation batch media 10X salts (per 1L) Ammonium Sulfate Potassium Chloride Magnesium Sulfate Sodium Chloride Calcium Chloride 50 g 10 g 5g 1g 1g Phosphate starvation 20 g 1 ml 1 ml 100 ml - Chemostat media (per 1L) Compound Yeast Nitrogen Base w/o amino acids and ammonium sulfate (NH4)2SO4 Glucose Glucose limitation 1.7 g Nitrogen limitation 1.7 g 5g 8g 0.053 g 20 g Phosphate limitation Compound KH2PO4 CaCl2.2H2O MgSO4.7H2O KCl (NH4)2SO4 NaCl Glucose 1000X vitamins 1000X metals 10 mg 0.1 g 0.5 g 1g 5g 0.1 g 5g 1 ml 1 ml 1000X vitamins and metals (Lu et al. 2009) 1000X vitamins (per 1L) Biotin Calcium pantothenate Folic acid Myo-inositol Nicotinic acid 1000X Metals (per 1L) 1 mg 400 mg 2 mg 2000 mg 400 mg p-aminobenzoic acid 200 mg Pyridoxine HCl Riboflavin Thiamine HCl 400 mg 200 mg 400 mg Boric acid Copper Sulfate.5H2O Potassium Iodide Ferric Chloride.6H2O Manganese Sulfate.H2O Sodium Molybdate.2 H2O Zinc Sulfate.7H2O 500 mg 40 mg 100 mg 200 mg 400 mg 200 mg 400 mg Table S2 – Expression Data (Downloadable Excel Spreadsheet) The prototrophic W303 derivative, Y3358, and the CEN.PK prototroph, Y3840, were grown in liquid SD (prior transfer to glucose starvation), nitrogen limited (prior transfer to nitrogen starvation) or phosphate limited media (prior transfer to phosphate starvation media) to an OD600 of 0.1 and then filtered onto nylon filters. Filter membranes were incubated on agarose plates with respective limited media for 2.5 h and then transferred to starvation plates; afterwards a series of time points was collected. For each of the starvation conditions (glucose, nitrogen or phosphate) time point 0, collected immediately prior to transfer of remaining filters to starvation media, was used as reference sample to which the following time points in respective starvation conditions were compared. For the W303 strain eight time points, besides time point 0 used as a reference, ranging from 30 minutes (30 min) to 8 days (8 d) were collected for glucose (W303 G), nitrogen (W303 N) and phosphate starvation (W303 P) each. For the CEN.PK strain three time points were collected for glucose (CEN.PK G), nitrogen (CEN.PK N) and phosphate starvation (CEN.PK P) ranging from 2 days after transfer to starvation media (2 d) to 4 days after transfer (4 d). Comparison between the reference samples used: time points 0 in SD (W303 G 0 min), nitrogen limited (W303 N 0 min) and phosphate limited media (W303 P 0 min), with glucose time point 0 (W303 G 0 min) used as reference, was also conducted. A comparison between the CEN.PK strain grown in a chemostat in glucose limited medium at dilution rate of 0.25/h (CEN.PK 0.25/h G limited) with CEN.PK grown in SD (CEN.PK G 0 min), nitrogen limited (CEN.PK N 0 min) and phosphate limited media (CEN.PK P 0 min) at time point 0 was also conducted, to make possible a comparison between our CEN.PK data and data generated by Brauer et al. 2008. Table S3 – Quiescent specific and Growth rate dependent genes (Downloadable Excel Spreadsheet) Gene lists with effect size and FDR corrected p-values for testing significant growth-rate or quiescent specific components. The lists are divided into two sheets: the overall model tests across all the conditions, and the nutrient specific model tests on a per nutrient basis. Data are provided for both CEN.PK and W303 strains. The “Saturated?” column denotes whether or not the probe intensity on the array has been saturated. Lastly, the group column identifies whether there is “Both” a significant growth-rate and quiescent specific component, “Gr-Dn” or “Gr-Up” a significant growth-rate component, “Q-Dn” or “Q-Up” a significant quiescent-specific component, or “None” no significant component identified. The nutrient specific model bins the results into “CQ-Up/CQ-Dn”, “NQ-Up/NQ-Dn”, and “PQ-Up/PQ-Dn” for quiescent-specific effects in glucose, nitrogen, and phosphate starvations, respectively. Table S4 – Metabolite Data (Downloadable Excel Spreadsheet) This table lists log-2 transformed metabolite concentrations relative to yeasts growing exponentially in SDMin. The first three rows denote the nutrient perturbation, whether the condition was nutrient starvation or nutrient limitation, and the stringency of the perturbation, where starvation units are time in minutes and limitation units are in dilution rate (mL/mL*h). Values reported for starvation are mean averages of biological replicates (n=3) normalized for cell count and size. Values for limitation were calculated as in Boer (2010) and re-normalized by subtracting the ratio of concentrations of an exponentially growing culture and the phosphate limited reference (D=0.05 h-1). Table S5 – List of Mutants Defective in Surviving Starvation (Downloadable Excel Spreadsheet) Sheet 1: Genes by Screen. Genes whose deletion resulted in loss of viability following starvation of cells for the indicated nutrient are listed and subdivided into those that were identified in the plate based screen (“plate screen only”) or that showed at least a 10 fold drop in relative abundance in the liquid based screen (“liquid only”) or that were identified in both screens (“both”). Sheet 2: Gene overlap. The genes listed in Sheet 1 are organized by those that appeared in only one screen (“G only”, “N only” or “P only”), or only in two of the three screens (“G+N only”, “G+P only” or “N+P only”) or in all three (“G+N+P”). Figure S1. Method for analyzing gene expression values. Data for ALD4 expression as a function of growth rate and starvation are used to illustrate the components of the method used to determine the extent to which a gene exhibits growth rate specific or quiescence specific gene expression. The figure defines the growth-rate slope, nutrient-specific effects for carbon, nitrogen, and phosphorus, and a quiescence specific effect that are applied in the equation shown in Materials and Methods. Figure S2. Overlap of genes exhibiting quiescent specific expression with those affected by acute heat shock. Histogram showing the fraction of genes from a total of 5448 that exhibit a given change in mRNA levels (log2) 15 min following transfer from 23° to 37° (blue line), taken from Gasch et al. (2000). Green line: histogram of the fraction of genes identified as repressed in a quiescence specific manner in this current study (218 total) that exhibit a given change in the Gasch et al acute heat shock experiment. Red line: histogram of the fraction of genes identified as induced in a quiescence specific manner in this current study (93 total) that exhibit a given change in the Gasch et al acute heat shock experiment. Figure S3. Singular value decomposition of starvation expression data. Transcription data displayed in Figure 2 was subjected to singular value decomposition and the major eigengenes as well as the extent to which each eigengene contributes to the variation in the data are shown. Singular value decomposition (SVD) is a matrix decomposition that is commonly used as an unsupervised technique for finding the major patterns in a data set. SVD takes a matrix X as input and returns i) a matrix V of linearly independent vectors of length 1, here referred to as “eigengenes,” that capture the major signals in the data; ii) a diagonal matrix of constants or “singular values” S that scale the magnitudes of these eigengenes; and iii) a matrix U that gives each original row (in this case, the expression data for each gene) in terms of its contributions from S and V , such that X = USV T . SVD is related to principal-components analysis or PCA (Wall et al. 2003). A more thorough treatment can be found in Alter, 2000. Percent signal explained by each eigengene was calculated by taking 100·si / ∑nj=1 sj, where n is the number of eigengenes (equal to the number of experimental conditions) and si is the singular value for eigengene i (equivalent to Si,i above). SVD was performed in the R software environment (R_Development_Core_Team 2009). Figure S4 Discordance in trehalose intermediates and gene expression levels during starvation. On a diagram of the trehalose synthesis and degradation pathways, metabolic intermediates (ovals) and the genes encoding enzymes (rounded rectangles) catalyzing interconversion of those intermediates are indicated the change in levels of the metabolites and mRNAs at four days starvation for glucose (upper panel), nitrogen (middle panel) or phosphate (lower panel) relative to those in cells growing exponentially on SDmin. Supplementary References Boer VM, Crutchfield CA, Bradley PH, Botstein D, Rabinowitz JD. 2010. Growth-limiting intracellular metabolites in yeast growing under diverse nutrient limitations. Molecular biology of the cell 21: 198-211. R_Development_Core_Team. 2009. R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. Wall ME, Rechtsteiner A, Rocha LM. 2003. Singular value decomposition and principal component analysis. in A practical approach to microarry data analysis (eds. DP Berrar, W Dubitzky, M Granzow), pp. 91-109. Kluwer, Norwell, MA.