1

Assessing the mechanisms responsible for differences in nitrogen 1

requirements between Saccharomyces cerevisiae wine yeasts in 2

alcoholic fermentation 3

Claire Brice1,2,3, Isabelle Sanchez1,2,3, Catherine Tesnière1,2,3, Bruno Blondin1,2,3 4

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1 INRA, UMR1083 Science pour l’Œnologie, 2 Place Viala, F-34060 Montpellier, France. 6

7 2 Montpellier SupAgro, UMR1083 Science pour l’Œnologie, Place Viala, F-34060 8

Montpellier, France. 9

10 3 Université Montpellier 1, UMR1083 Science pour l’Œnologie, 2 Place Viala, F-34060 11

Montpellier, France. 12

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Address correspondence to Bruno Blondin, blondin@supagro.inra.fr 15

Received: 16

Accepted: 17

Number of tables: 1 18

Number of figures: 7 19

Supplemental material: 7 20

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AEM Accepts, published online ahead of print on 13 December 2013Appl. Environ. Microbiol. doi:10.1128/AEM.03856-13Copyright © 2013, American Society for Microbiology. All Rights Reserved.

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Abstract 33

Nitrogen is an essential nutrient for wine yeast during alcoholic fermentation, and its 34

abundance determines the fermentation rate and duration. The capacity to ferment in 35

conditions of nitrogen deficiency differs between yeasts. A characterization of the nitrogen 36

requirements of a set of 23 strains revealed large differences in their fermentative 37

performances under nitrogen deficiency, and these differences reflect the “nitrogen 38

requirements” of the strains. We selected and compared two groups of strains, one with low 39

nitrogen requirements (LNR) and the other with high nitrogen requirements (HNR). A 40

comparison of various physiological traits indicated that the differences are not related to the 41

ability to store nitrogen or the protein content. No differences in protein synthesis activity 42

were detected between strains with different nitrogen requirements. Transcriptomic analysis 43

revealed expression patterns specific to each of the two groups of strains with an 44

overexpression of stress genes in HNR strains and a stronger expression of biosynthetic genes 45

in LNR. Our data suggest that differences in glycolytic flux may originate from variation in 46

sensing and signaling nitrogen in condition of starvation. 47

Introduction 48

Alcoholic fermentation for wine-making is conducted in an environment that is stressful (high 49

ethanol content, nutrient starvation, low pH) for Saccharomyces cerevisiae yeasts. The 50

availability of diverse nutrients in grape musts (vitamins, sterols, unsaturated fatty acids, 51

pantothenic acid, or nitrogen) is often limited, restricting yeast cell growth. Nitrogen 52

limitation is by far the most frequent growth limitation encountered during wine fermentation 53

(1). Various nitrogen sources are available in grape musts, including ammonium ions, amino 54

acids and peptides. Only ammonium ions and the alpha-amino nitrogen (free amino acids 55

except proline) are used by yeasts in fermentation. Grape musts have diverse nitrogen 56

compositions and the concentration of assimilable nitrogen ranges from 60 to 500 mg liter-1 57

(2). The nitrogen content is dependent on many factors including rootstock, climate, vine 58

growing conditions, grape variety and grape processing (3). Nitrogen availability in musts is a 59

major determinant of the maximal fermentation rate (1), and also affects the size of the total 60

yeast cell population; however, the effect on fermentation rate is mainly independent on the 61

population size (2). Indeed, low assimilable nitrogen concentrations can lead to slow 62

fermentations (4-10). In enological conditions musts are considered as nitrogen-limited when 63

the assimilable nitrogen content is below 150 mg liter-1 (2, 11). 64

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The ability to ferment under conditions of low nitrogen availability differs between yeast 65

strains. This variability is manifested by differences in the capacity to maintain the 66

fermentation rate during the stationary phase. Indeed, most alcoholic fermentation occurs 67

during the stationary phase and the ability to ferment strongly during this phase has a large 68

effect on the total fermentation time. These differences of performance between yeast strains 69

have been reported as “nitrogen requirement” for alcoholic fermentation (1, 12). Various 70

approaches have been used to assess nitrogen requirements, for example measuring the 71

nitrogen flux required to support a constant fermentation rate (13, 14). According to these 72

measures, wine yeasts can be classified as high or low nitrogen-requiring strains. 73

The stationary phase, critical for fermentation, results from nitrogen depletion from yeast; 74

yeast cells adjust their metabolism and the fermentation rate drops more or less progressively 75

(14). The molecular mechanisms leading to the slow-down of the fermentation flux during 76

starvation are still unclear. Several studies have reported a link between the drop of the 77

fermentation rate and sugar transporter activity: nitrogen depletion and the shutdown of 78

protein synthesis results in the catabolic inactivation and degradation of sugar transporters 79

(10). However, the details of the consequences for the fermentation rate of such regulation 80

during starvation are not known. The onset of nitrogen starvation triggers massive 81

physiological changes involving in autophagy, nitrogen recycling systems, a decrease of 82

protein synthesis and the reorientation of the carbon flux to favor glycogen and trehalose 83

storage. Substantial transcriptional remodeling associated with the entry into starvation during 84

wine alcoholic fermentations has been described (15, 16), and includes the development of a 85

general stress response. In addition, specific depletion of amino acids induces genes encoding 86

enzymes of amino-acid biosynthesis (17-19). Many of these transcriptional changes are 87

controlled by the TOR pathway which senses the nitrogen status of the cell and adapts the 88

nitrogen metabolism to nutrient availability (20, 21). This control allows transition from the 89

use of rich sources of nitrogen to that of poor nitrogen sources. 90

Entry into stationary phase is also characterized by changes in protein abundance, which 91

generally decreases, despite some neosynthesis of stationary phase-specific or stress-induced 92

proteins (16). This neosynthesis implies mechanisms for recycling nitrogen compounds, such 93

as autophagy and ribophagy. Piggott et al. (22) have shown that autophagy is critical for yeast 94

fitness in alcoholic fermentation, illustrating the role of nitrogen reallocation under starvation. 95

Although the yeast response to nitrogen status in alcoholic fermentation has been described, 96

the molecular mechanisms underlying the differences in nitrogen requirement between strains 97

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are unknown. We report here an investigation of a set of wine yeast strains with different 98

nitrogen requirements with the aim of elucidating the underlying molecular mechanisms. We 99

combined a physiological analysis with a transcriptomic study and found that differences in 100

nitrogen requirement may be linked to differences in signaling in response to nitrogen 101

starvation. 102

103

MATERIALS AND METHODS 104

Yeast strains and inoculation. The eight S. cerevisiae strains used in this study were 105

acquired from various laboratory and industrial sources, and are listed in Table 1. When no 106

published data was available, species identification was checked by ITS restriction with 107

HaeIII (23). Among these strains, EC1118 is a reference wine yeast strain, since its genome 108

has been fully sequenced and characterized (24). The strains were stored and plated on YEPD 109

plates for single colony isolation. To inoculate experimental cultures, a preculture was 110

prepared by transferring a single colony from the agar plate to 50-ml YEPD medium in a 250-111

ml Erlenmeyer flask. This inoculum was grown overnight in an incubator at 28°C, with 112

shaking (280 rpm). One aliquot from this preculture to inoculate fermenters to a density of 113

1.106 cells ml-1. 114

Synthetic culture medium. Yeast was cultured in the synthetic medium (SM) described by 115

Bely et al. (2). It contains 200 g liter-1 glucose and its amino acid composition simulates the 116

nitrogen content of a standard grape juice. For these fermentations, we used a medium 117

containing only glucose to overcome the already known effects of variations in fructose 118

uptake (36) that were out of the scope of the present study. The standard SM, called “normal 119

medium”, contained 425 mg liter-1 assimilable nitrogen (SM425), and another SM with a 120

lower nitrogen content was also used (100 mg liter-1 assimilable nitrogen; SM100). In both 121

SM425 and SM100, the nitrogen source consisted of ammonium salts (30%) and α-amino 122

acids (70%) which, except proline, were considered as assimilable nitrogen. 123

Fermentation conditions, kinetics, and cycloheximide treatment. The fermenters (1.2 liter, 124

containing 1 liter of medium) were closed with fermentation locks (CO2 bubbling outlets 125

filled with water). The filling conditions were controlled and fermentations were carried out 126

under isothermal conditions (28°C), with permanent stirring (300 rpm). All fermentations 127

were run in duplicate or triplicate. 128

The amount of CO2 released during fermentation was calculated from automatic 129

measurements (taken every 20 min) of fermenter weight (25). The CO2 production rate was 130

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calculated by polynomial smoothing of the last 10 measurements of fermenter weight loss. 131

The numerous acquisitions of weight and the precision of weighing (0.1 to 0.01 g) allowed the 132

CO2 production rate to be calculated with accuracy (1). 133

To test the involvement of protein synthesis, cell cultures in SM100 were treated at the 134

beginning of the stationary phase with cycloheximide (Sigma, St Louis MO, USA) (100 µg 135

ml-1). Cycloheximide is an inhibitor of eukaryotic protein synthesis and acts by blocking the 136

elongation-factor-2-mediated translocation of peptidyl-tRNA from the A site to the P site on 137

the 60S ribosomal subunit. The efficiency of cycloheximide inhibition was verified by 138

measuring the rate incorporation rate of L-[35S] methionine in a control strain as a marker of 139

protein synthesis. 140

Quantification of yeast strain nitrogen requirement. One approach to determining the 141

yeast nitrogen requirement is to add, throughout fermentation in nitrogen-limited medium, the 142

amount of assimilable nitrogen necessary to maintain the CO2 production at a constant rate 143

during the stationary phase (13, 14). Fermentations were performed in SM100. When the CO2 144

production rate decreased, diammonium phosphate (DAP; 300 mg liter-1, equivalent to 63 mg 145

liter-1 assimilable nitrogen atoms) was added using a GILSON peristaltic pump. The amount 146

of nitrogen added to maintain the CO2 production rate constant is proportional to the 147

fermentation rate. The effects of the nitrogen additions were quantified by plotting the amount 148

added against CO2 produced and calculating the mean nitrogen consumption between 10 and 149

50 g liter-1 of CO2 produced (P10-50). 150

Biomass, nitrogen and protein determinations. The yeast population was characterized by 151

determining the number of cells after sonication using an electronic particle counter 152

(Multisizer 3 Coulter Counter; Beckman Coulter), and by measuring the dry weight. 153

The total nitrogen concentration was determined by distillation and back-titration according to 154

the Kjeldhal mineralization method (26). The residual ammonium concentration in the 155

medium was measured by spectrophotometry (Enzytec fluid Ammonia, R-Biopharm, 156

Darmstadt, Germany) with glutamate dehydrogenase (GLDH) according to the 157

manufacturer’s instructions. Residual free amino acids in the medium were assayed by cation-158

exchange chromatography followed by post-column derivatization with ninhydrin (Biochrom 159

30, Biochrom, Cambridge, U.K.). Total available nitrogen (TAN) and the residual nitrogen 160

(RN) present in the medium at the end of the fermentation were determined as the sums of 161

ammoniacal nitrogen and nitrogen from amino acids as determined before and after 162

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fermentation, respectively. Total yeast assimilable nitrogen (TYAN) was calculated as the 163

difference between TYAN and the RN. 164

Total cell protein was determined with the bicinchoninic acid protein assay (BCA method), 165

which is a modified Biuret method. In brief, proteins were extracted from cells by incubation 166

with 50% (vol/vol) DMSO for one hour at 100°C, and quantified with a BC Assay kit (Sigma-167

Aldrich, France). Bovine Serum Albumin (BSA) was used as the standard for calibration. 168

Protein synthesis determination. Protein synthesis rates were estimated by measurement of 169

the incorporation of L-[35S]methionine into the acid-precipitable protein fraction: 100-µl of 170

culture medium (containing 5.106 cells) was added to 10-µl of L-[35S]methionine (1µCi). 171

Incorporation was stopped either immediately (controls) or after incubation for 30 min at 172

25°C by adding 20-µl of the samples (cells + L-[35S]methionine) to 1-ml of cold 10% 173

trichloroacetic acid and 100-µl of BSA (1mg BSA ml-1) as a carrier protein to precipitate the 174

proteins. The samples were incubated 30 min on ice. The precipitable material was collected 175

by filtration through a 0.2-µm-pore-size Nucleopore filter and washed with 30-ml of cold 176

10% trichloroacetic acid. The radioactivity was measured in a liquid scintillation counter. 177

RNA extraction, labeling and microarray hybridization. The eight were subjected to 178

whole-genome expression analysis. EC1118 is considered as the reference strain. Two 179

biological replicates were performed for each strain. Total RNA was isolated from cultures at 180

45 g liter-1 of CO2 production (50% fermentation progress), by the TRIzol® method 181

according to Chomczynski and Sacchi (27). For each RNA extraction, 1.109 cells were 182

pelleted by centrifugation (3000 rpm, 2 min) and lysed mechanically by vortexing with glass 183

beads (d=0.3 mm) in 400-µl TRIzol® (GIBCO BRL) at 4°C for 8 min. The liquid phase was 184

collected and TRIzol® added to give a final volume of 4-ml; the samples were incubated for 5 185

min at room temperature, and 800-µl chloroform was added. The mixture was vortexed and 186

incubated for 2 min at temperature room, then centrifuged (10000 rpm, 15 min). An equal 187

volume of cold (-20°C) isopropanol was added to the aqueous phase to precipitate the RNA. 188

The samples were incubated for 10 min at room temperature and centrifuged (10000 rpm, 10 189

min). The resulting nucleic acid pellet was washed twice with 2-ml 75% ethanol / DEPC-190

treated water and then dissolved in 150-µl of nuclease-free water (Qiagen, USA). The RNA in 191

100 µg aliquots of these preparations was purified with the RNeasy® mini kit (Qiagen, USA) 192

following the RNA cleanup protocol, including membrane DNase digestion. Fluorescent 193

cDNAs were synthesized from 100 ng of total RNA using the one-color RNA Spike-in kit 194

(Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s 195

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recommendation (indirect labeling of mRNAs with Cyanin 3 dCTP dye). Labeled cDNA was 196

purified with the RNeasy® mini kit (Qiagen, USA). 197

Agilent 8x15k gene expression microarrays with specific designs were obtained from Agilent 198

Technologies (Agilent Technologies, Santa Clara, CA, USA) and used for microarray 199

hybridization. The array design was based on ID 038619 with the addition of 39 genes from 200

the new regions of EC1118 ((24); available on GEO under the accession number GPL17690). 201

600 ng of labeled cRNA was hybridized on the slide for 17 hours in a rotating oven (10 rpm) 202

at 65°C using the Agilent technologies kit, according to the manufacturer’s instruction and 203

then washed. A Genepix 4000B scanner (Axon Instrument Inc.) was used for array 204

digitalization, and data were analyzed with Genepix Pro7 (Axon Instruments). 205

Statistical analyses 206

R.2.14.2 software (28) was used for statistical analyses. To obtain a general overview of the 207

profile of the high and low nitrogen-requiring strains, principal components analysis (PCA) 208

was performed using the FactoMineR package (29) with the following parameters: maximal 209

fermentation rate (Rmax), fermentation rate at 50% sugar consumption (R50), total fermentation 210

duration (Fd) and the quantified nitrogen requirement (QNr). The limma package (30-32) was 211

used to normalize and analyze the microarray data (quantile method for normalization 212

between arrays). To identify differential gene expression between strains with high and low 213

nitrogen requirements, a modified t-test was applied by filtering on confidence at p < 0.05, 214

using the Benjamini and Hochberg false discovery rate as multiple testing correction of the t-215

test p-values (33). Only genes with a log fold difference greater than 1 (positive or negative) 216

were taken into consideration. The genes for which expression differed were grouped 217

according to Gene Ontology (GO) process terms using Funspec with the Bonferroni 218

correction at a p-value cutoff of 0.05 (32). Hierarchical clustering was used to group the 219

selected genes by similarity of expression profiles using cluster version 3.0 (applying centered 220

correlation and complete-linkage clustering) and the results were loaded into JavaTreeView 221

version 1.1.5r2 for data display (34, 35). The complete data set is available through the Gene 222

Expression Omnibus database (accession number GSE50626). 223

Results 224

Yeast nitrogen requirements during the stationary phase. Twenty-three Saccharomyces 225

cerevisiae strains from various origins were screened to determine their abilities to ferment in 226

a must with a low nitrogen content (Fig. S1). Their nitrogen requirements were quantified (by 227

calculating the average slope between 10 and 50 g liter-1 of CO2 released after nitrogen 228

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addition) according to the constant rate fermentation procedure (CRF) described by Manginot 229

et al (14) (see the Materials and Methods). The distribution of the phenotype “nitrogen 230

requirement” shows substantial diversity between the strains; the distribution of the strains 231

according to nitrogen requirement was a continuum, indicating that it is a quantitative trait 232

involving several genes. These results are consistent with the findings of Manginot et al (14). 233

Eight strains of the 23 strains were selected for their extreme nitrogen requirements (low and 234

high): five strains (4CAR1, L2868, EC1118, Fermiflor, Zymasil) exhibited a low nitrogen 235

requirement (P10-50 close to 1 mg N g-1 CO2; Fig. 1), and three strains (K1M, 7013, MTF1782) 236

showed a high nitrogen requirement (P10-50 between 2.2 mg and 2.5 mg N g-1 CO2; Fig. 1). 237

These selected strains were thus representatives of strains with a low (LNR) or a high nitrogen 238

requirements (HNR). The fermentation kinetics for the eight strains on a nitrogen-limited 239

medium (SM100) were studied (Fig. 2). The results showed that all the strains exhibited low 240

fermentation rates and long durations of fermentation. The Rmax did not differ greatly between 241

the strains and did not correlate with nitrogen requirement; similarly, the cell population size 242

was not correlated with the nitrogen requirement of the strains (Fig. S2). However, during the 243

stationary phase, after the growth had stopped, the fermentation profiles of the strains 244

diverged substantially, such that by 70 hours of fermentation the two groups of strains were 245

clearly segregated: the strains with-low nitrogen requirements exhibited a higher fermentation 246

rate than strains with high nitrogen requirements. This is consistent with the classification of 247

the strains based on the measurement of nitrogen requirements by ammonium addition. We 248

verified that the HNR strains did not exhibit any intrinsic fermentation capacity defect by 249

examining their behavior in nitrogen-rich must (SM425): in these conditions, there were only 250

small differences in the fermentation profiles between the strains (Fig. S3). 251

Analysis of the relationships between traits. We investigated the relationships between the 252

phenotypic traits of the eight yeasts, by principal components analysis (PCA) (Fig. 3A, 3B). 253

We considered four measures to build the variable factor map: Rmax, R50, Tf, QNr. The 254

projection on the two principal axes preserves 95% of the information and explains 255

respectively 88% and 7% of this variation. The PCA of kinetic measures allowed the 256

discrimination of strains according to their nitrogen requirement. Also, there was a strong 257

correlation between the strain nitrogen requirement and total fermentation duration, in 258

agreement with the observations of Bely et al (2). PCA also indicated that nitrogen 259

requirement and total fermentation duration were not correlated with the R50 and Rmax. These 260

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results are consistent with notion that the ability of cells to maintain a high fermentation rate 261

in stationary phase is linked to their nitrogen requirement. 262

Comparisons of physiological traits. We examined whether differences in nitrogen 263

requirement were associated with variations in the cellular content of yeast cells. We 264

measured total nitrogen, protein content and dry mass of yeast cells at various stages of the 265

fermentation process (Fig. S4). For all the measures, differences between the LNR and HNR 266

groups are small and they are not statistically different. There was no clear segregation of the 267

nitrogen content between LNR and HNR strains (Fig. S4A). There are also, no significant 268

differences between strains in the protein content at mid-fermentation (Fig. S4B) or at 80% 269

fermentation progression (Fig. S4C). At the end of fermentation there were small differences, 270

and although several LNR strains displayed a slightly greater protein content, this was not the 271

case for the Fermiflor strain. Higher protein content was thus not a general feature of LNR 272

strains. For all strains, dry mass values increase between mid-fermentation and the end of 273

fermentation, in agreement with previous reports (Fig. S4D, S4E) (37). However, HNR and 274

LNR strains showed slightly different behaviors, with a larger increase in dry mass for HNR 275

strains than LNR strains during the stationary phase. The increase in dry mass was consistent 276

with the storage of carbohydrates (glycogen, trehalose) at this stage, as previously 277

demonstrated by Manginot et al (14). We also checked the utilization of the nitrogen sources 278

by the strains. All the nitrogen sources (except proline) were metabolized by all the strains 279

during the first 20 hours of fermentation (data not shown). This excludes the possibility that 280

the differences in nitrogen requirements were consequences of differences in the uptake of 281

nitrogen sources. 282

Assessment of the role of protein synthesis. The ability of yeast strains to maintain a high 283

fermentation rate during the stationary phase can be dependent on their capacity to synthesize 284

proteins. We therefore examined the effects of inhibiting protein synthesis on the fermentation 285

kinetics. We added the protein synthesis inhibitor cycloheximide to cultures of the seven 286

strains on SM100 at the beginning stationary phase (47 hours of fermentation); as cell growth 287

had already stopped by this time, any effects of the drug could not be due to inhibition of 288

growth. Cycloheximide modified the fermentation profiles confirming the involvement of 289

protein synthesis. Cycloheximide triggered a large and rapid decrease of the fermentation rate 290

of the LNR strain L2868 immediately after its addition (Fig. 4). Its effect on the fermentation 291

rate of HNR strain MTF1782 was smaller, and the kinetic profile remained similar to that of 292

the control (Fig. 4). Furthermore, the kinetic profiles of the strains L2868 and MTF1782 293

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become very similar after cycloheximide addition. It was thus possible that the differences in 294

behavior were due to more protein synthesis in LNR than in HNR strains. We therefore 295

compared the protein synthesis activities by measuring the rate of L-[35S] methionine 296

incorporation into L2868 and MTF1782 (Fig. S5). Protein synthesis was very similar in the 297

two strains (difference of 5%). The differences in kinetic profile are therefore not caused by 298

differences in overall protein synthesis activity. We also confirm that cycloheximide inhibited 299

protein synthesis in these conditions. These results suggested the involvement of other 300

mechanisms in the differences of the glycolytic flux between strains during the stationary 301

phase of alcoholic fermentation. 302

Transcriptomic analysis 303

Gene expression is a major determinant of phenotypic diversity (17) and may contribute to 304

differences in strain fermentation properties. The analysis of gene expression patterns can 305

provide insights into the molecular and physiological mechanisms associated with differences 306

in fermentation capacities. We therefore examined the gene expression profiles of the eight 307

selected strains. The transcriptomes were analyzed in cells fermenting in a nitrogen-limited 308

medium (SM100) and harvested at stationary phase (45g liter-1 of CO2 produced and 309

corresponding to 6% ethanol). At this stage, the strains exhibited differences in fermentation 310

rates. In addition, the transcriptome has been shown to be stable at this stage when growth has 311

stopped for all cells and nutrients have been depleted (15). We compared the global 312

expression patterns by microarray, using two biological replicates for each strain. Intraclass 313

coefficients of correlation (ICC) were 94.6% to 99.1% (data not shown), indicating good 314

reproducibility of data. 315

Assessing variations in gene expression in the yeast population. Four hundred and fifty 316

four genes were differentially expressed between the two groups of strains (HNR and LNR), 317

with an absolute logFC greater than 1; 248 genes were overexpressed in LNR strains and 206 318

genes in HNR strains. The set of genes overexpressed in HNR strains was enriched in genes 319

related to the stress response and encoding proteins involved in the production of reserve 320

carbohydrates including trehalose and glycogen (TPS1, TPS2, TPS3, UGP1, TLS1, PGM2, 321

GLG1, GLG2) (Fig. 5A); in response to nitrogen starvation, these strains overexpressed genes 322

involved in the metabolism of amino acids such as arginine (ARG1), serine (CHA1), alanine 323

(ALD2), glutathione (GLO1), and also genes involved in glutamate biosynthesis (GAD1, 324

GDH2) and sulfate metabolism (MET5, SUL2). Conversely, the set of genes overexpressed in 325

LNR strains included many encoding proteins involved in protein synthesis (Fig. 5B). This 326

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suggests that in LNR strains, genes encoding rRNA processing, were less susceptible to stress 327

caused by nitrogen starvation. 328

Hierarchical clustering analysis. A global comparison of the expression patterns of all the 329

strains revealed 2484 genes that were differentially expressed (adjP < 0.05). The aim of this 330

analysis was to observe a tendency of gene expression in a strain group. Hierarchical 331

clustering of gene expression profiles identified eight clusters of expression profiles for which 332

there was a tendency in segregation between LNR and HNR strains (Fig. S6). In fact, the 333

segregation is not totally perfect between the two groups of strains. For example, Fermiflor 334

LNR strain showed similarity to HNR strain in cluster II and a part of cluster III (Fig. 6), and 335

with a neutral position for other clusters. Interestingly, EC1118 strain also classified as LNR 336

strain, presented a HNR transcriptomic response for all the clusters. For the others strains, 337

observations showed generally four clusters (clusters I, II, III, IV) including groups of genes 338

overexpressed in HNR strains (Fig. 6). Cluster I contains genes involved in proteasome 339

degradation (PRE2, PRE3, PRE6) and cluster II is enriched in genes involved in the ubiquitin 340

protein degradation pathway. Cluster III is composed of two small sub-clusters with similar 341

profiles, and includes genes involved in storage of reserves, such as trehalose and glycogen 342

(TPS1, TPS2, TSL1, PGM2, GLC8). Cluster IV contains genes associated with autophagy. 343

Thus, protein degradation and nitrogen recycling both appear to be more active in HNR than 344

LNR strains. Indeed, the four clusters contain genes controlled by NCR (Nitrogen Catabolite 345

Repression) and the TOR pathway (VID30 in cluster I, GDH3 in cluster II, PUT1 in cluster 346

III, ATG14 in cluster IV). Four clusters (V, VI, VII, VIII) included genes overexpressed in 347

LNR strains. They contain genes involved in ribosome biogenesis, for example of the RPS 348

and RPL families, and families of genes involved in RNA processing (UTP, NOP, IMP, RRP, 349

RPF family genes). These data indicate that there is a very common mechanism: a stronger 350

expression of genes involved in protein and ribosome synthesis, transcription and RNA 351

metabolism in the LNR than HNR strains. But the expression data obtained for EC1118 strain 352

reveal a different behavior and suggest that additional mechanisms are involved. EC1118 353

transcriptomic data present an exception since it has a physiological profile of a LNR strain, 354

but a transcriptomic profile similar to HNR strains. This suggest that the EC1118 355

fermentation ability in nitrogen-deficient condition origins from a mechanism different from 356

the other strains. There is also evidence that the fermentation rate does not determine the 357

transcriptomic profile of the strain. 358

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The genes overexpressed in LNR strains include 25 of the 39 new genes identified in three 359

regions (A, B and C) of the EC1118 strain genome, originating from horizontal transfer (27). 360

The A region is not differentially expressed between LNR and HNR strains (Fig. S7). The B 361

region is present in four strains, three LNR strains (L2868, 4CAR1, EC1118) and a HNR 362

strain (K1M). The presence/absence of the introgressions was verified by PCR on multiple 363

genes in the region (data not shown). Expression data show that only LNR strains expressed 364

genes containing in this region (Fig. S7). Concerning the C region, three strains containing the 365

totality of the introgression (L2868, 4CAR1, EC1118) and for two strains, we have controlled 366

he absence of these regions (7013, MTF1782). Three strains contain only three genes of the C 367

region (Fermiflor, Zymail, K1M). Expression data show that these three genes are expressed 368

in all the LNR strains, but also in the HNR strain K1M (Fig. S7). These are the genes FOT1 369

and FOT2 corresponding to two fungal oligopeptide transporters (38) and the SEO1 gene 370

corresponding by sequence homology to a putative permease, member of the allantoate 371

transporter subfamily (27). Although the expression of these three genes in all LNR strains 372

might suggest a role in the nitrogen requirement, a similar behavior in the HNR strain K1M 373

contradicts this hypothesis. 374

Correlation between gene expression and fermentation rates. The expression of a total of 375

282 genes was found to correlate with fermentation rate (R50): 129 positively and 153 376

negatively. Only the set of positively correlated genes was enriched in particular functions 377

(Fig. 7). From these data, high fermentation rates on nitrogen-deficient medium were 378

associated with the overexpression of genes involved in protein synthesis, transcription and 379

rRNA processing. These genes belong to different clusters with genes overexpressed in LNR 380

strains. There was no detectable functional enrichment among the set of genes negatively 381

correlated with R50; nevertheless, various genes involved in the metabolism of energy reserves 382

(GLC2, GLC3, YPI1) are member of this group. 383

Discussion 384

The fermentation capacities of yeast strains in media with low nitrogen content differ (12, 14, 385

38). We screened 23 strains of different origins for their nitrogen requirements and confirmed 386

here the variability of this character. The nitrogen requirements of the strains varied more than 387

2.5 fold and were associated with diversity in fermentation duration on a nitrogen-limited 388

medium. Through the characterization of fermentation properties of eight strains with 389

contrasted nitrogen requirements, we confirm that the differences are linked to how well the 390

strains maintain a high fermentation rate after the entry into stationary phase in conditions of 391

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nitrogen starvation. We checked that the differences in fermentation performances were not 392

triggered by differences in the utilization of the available nitrogen sources: all the strains 393

consumed the assimilable nitrogen sources during the first hours of the fermentation, in 394

agreement with previous observations (39). There were only small differences between high 395

nitrogen-requiring (HNR) and low nitrogen-requiring (LNR) strains in term of cellular 396

contents: we did not find any correlations between strain fermentation capacity and abilities to 397

store nitrogen, consistent with the report by Julien et al (38); and we could not differentiate 398

HNR and LNR strains on the basis of protein content. The total dry mass during starvation at 399

the end of the fermentation was higher for HNR than LNR strains, consistent with greater 400

accumulation of storage carbohydrate. This observation is in agreement with Manginot et al 401

(13) that reported that the amount of glycogen was directly correlated to the nitrogen 402

requirement of the strains and thus higher in HNR strains. Therefore, the only difference in 403

cellular contents we could find to be related to the nitrogen requirements was greater storage 404

of carbohydrates in HNR than LNR. 405

Protein synthesis can be a determinant of yeast fermentation capacity. We confirmed that 406

protein biosynthesis was required to maintain the fermentation rate in stationary phase. 407

However, the rates of protein synthesis in representative HNR and LNR strains were similar, 408

and could not explain the differences in fermentation rate between these two strains. 409

The transcriptomic study revealed expression patterns that were common to the strains of each 410

group (HNR and LNR), (except for EC1118 strain), indicating that their member strains 411

display similar physiological responses. However, there were large differences between the 412

two groups of strains: genes involved in synthetic processes (protein synthesis, RNA 413

processing, etc) were overexpressed in LNR strains whereas genes involved in protein 414

degradation, nitrogen recycling and the stress response were more strongly expressed in HNR 415

strains. Except for the EC1118 strain, it seems that a common mechanism allows 416

distinguishing LNR and HNR strains. The HNR strains exhibit a pattern characteristic of 417

nutrient-starved and stressed cells whereas LNR strains exhibit a clearly weaker starvation 418

response. The higher expression of genes involved in ribosome biogenesis in LNR strains 419

indicates that the TOR pathway is more active than it is in HNR strains, and therefore that 420

nitrogen starvation is less severe. The stress response genes controlled by Msn2/Msn4, 421

including genes for the synthesis of the storage carbohydrates glycogen and trehalose, were 422

expressed more strongly in the HNR than LNR strains, and there was an increase in storage 423

carbohydrates. This is consistent with the previous observation that the dry mass for HNR 424

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strains was greater than that for LNR strains, presumably due to the accumulation of glycogen 425

and trehalose as demonstrated in a previous study (14). In nitrogen limited conditions, the 426

HNR yeasts were more sensitive to nitrogen-starvation, resulting in a stronger stress response, 427

weaker expression of biosynthetic genes and lower fermentation rate. The susceptibility to 428

nitrogen-starvation could correspond to a sensitivity threshold, which would be more 429

important in HNR strains. This sensitivity to nitrogen could also be the result of a nitrogen 430

dose effect in the medium. Ethanol production at this stage was identical for the two groups of 431

strains, ruling out a possible role in the differences in stress response. 432

These differences may be consequences of differences in the nitrogen sensing signaling 433

systems. The differences in gene expression and the physiological responses are reminiscent 434

of the observations of Watanabe et al (41) who analyzed the effects of a mutated form of the 435

PAS kinase RIM15 in sake yeast. They showed that the altered form of RIM15 reduced the 436

yeast stress response and the amount of storage carbohydrate, and enhanced the fermentation 437

rate. RIM15 is involved in nitrogen signaling downstream from TOR, which senses the 438

nitrogen status of the cell (42, 43). Nitrogen starvation leads to activation of Rim15p resulting 439

in induction of the quiescent program and a stress response (44, 45). This suggests that 440

differences in sensing and nitrogen signaling between HNR and LNR strains could contribute 441

to differences in fermentation rates during starvation. Such a mechanism is consistent with the 442

absence of differences in the amount and synthesis of protein between the two groups of 443

strains. How diversity in nitrogen sensing/signaling modulates the glycolytic flux is unclear, 444

although reducing the stress response in sake yeast could increase the fermentation rate (39, 445

45, 46). This implies a link between nitrogen signaling and the fermentation rate that could 446

explain differences in behavior between the two groups of strains. The data obtained from 447

EC1118 strain suggest that other mechanisms may also be involved in the nitrogen 448

requirement. Moreover, this strain possesses several introgressed non-Saccharomyces 449

cerevisiae regions. We examined whether they could play a role in fermentative capacity in 450

nitrogen-limited conditions. We observed that only three out the five LNR strains expressed 451

genes of these regions indicating that these regions are not necessary to expression of the 452

phenotype “low nitrogen requirement”. However we cannot rule out the contribution of these 453

regions in a given strain. 454

Thus, HNR yeasts may be more sensitive to nitrogen-starvation stress, limiting protein 455

synthesis and decreasing their fermentation rate. In conclusion, the nitrogen requirements of a 456

wine strain may correspond to a common mechanism that could be its ability to sense nitrogen 457

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starvation and develop a quiescent program that reduces the flow of energy and increases its 458

adaptation to stress. Further work is required to identify the genes and mechanisms underlying 459

these phenomena. The nitrogen requirement of a strain is a quantitative trait, so it may be 460

possible to identify the genes responsible for the phenotype by a genetic approach. 461

ACKNOWLEDGMENTS 462

This work was supported by the ANR project ALIA 2009. We are grateful to Christian Brion 463

for assistance with microarray preparation. We thank Jean-Marie Sablayrolles for critical 464

reading of this manuscript. 465

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Figures legends and supplemental material legend 594

Figure 1: Comparison of nitrogen requirements of 8 wine yeast strains. The measure is 595

expressed in mg nitrogen g-1 CO2 based on the amount of nitrogen necessary to support a 596

constant fermentation rate between 10 and 50 g L -1 CO2 (P10-50). LNR: strains with low 597

nitrogen requirements; HNR: strains with high nitrogen requirements. Reproducibility of 598

this technique was tested for two strains (L2868 and MTF1782) and variations are 599

indicated on the graph with error bars. 600

Figure 2: Fermentation profiles of eight different wine yeast strains with contrasted 601

nitrogen requirement in a nitrogen limited medium (SM100). 602

Figure 3: Principal component analysis (PCA) of (A) kinetic traits (Tf: Total fermentation 603

duration; NQr: Quantified nitrogen requirement; Rmax: Maximun fermentation rate; R50: 604

Fermentation rate at 50% sugar consumption) and (B) individuals strains. The values were 605

obtained from fermentation profiles performed on SM100 at 24°C. 606

Figure 4: Effects of cycloheximide addition at 10 g L-1 h-1 of CO2 produced on the 607

fermentation in SM100. Comparison between a strain with low nitrogen requirement 608

(L2868) and a strain with high nitrogen requirement (MTF1782). 609

Figure 5: Analysis of transcriptomic data (mean of two repetitions): enrichment of gene 610

functions among strains with high nitrogen requirement (A) and strains with low nitrogen 611

requirement (B). The values correspond to the number of genes in the Funspec functional 612

category (Bonferroni correction and p-value 0.05). The p-values are indicated for each 613

category. 614

Figure 6: Cluster extracts of genes overexpressed in HNR strains. Cluster I is composed 615

by genes involved in proteome degradation. Cluster II is enriched in genes associate to 616

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ubiquitin protein degradation. Cluster III is composed by genes involved in storage of 617

reserve. Cluster IV is related to the autophagy pathway. 618

Figure 7: Analysis of transcriptomic data (mean of two repetitions): enrichment of gene 619

functions among genes positively correlated with the fermentation rate (R50). The values 620

correspond to the number of genes in the Funspec functional category (Bonferroni 621

correction and p-value 0.05). The p-values are indicated for each category. 622

Figure S1: Nitrogen requirement (NQr) of different yeast strains. The NQr was mesured 623

by constant fermentation rate method, by the amount of nitrogen necessary to regulate the 624

fermentation rate between 10-50g L-1 CO2 (P10-50) in mg nitrogen g-1 CO2. 625

Figure S2: Comparison between cell populations of yeasts strains with contrasted nitrogen 626

requirements, during fermentation on SM100. 627

Figure S3: Comparison between fermentation profiles of eight yeasts strains with 628

contrasted nitrogen requirements. Specific CO2 production rate according to time of 629

fermentation on SM425. 630

Figure S4: Comparison of physiological traits between strains with contrasted nitrogen 631

requirements. (A) Nitrogen content of the yeast cells at 50% fermentation progression on 632

SM100 (B) Protein content of the yeast cells at 50% fermentation progression on SM100 633

(C) Protein content of the yeast cells at 80% fermentation progression on SM100 (D) Dry 634

mass of the yeast calls at 50% fermentation progression on SM100 (E) Dry mass content 635

of yeast cells at 80% fermentation progression on MS100. ( ) Mean values for LNR 636

strains; (- - -) mean values for HNR strains. 637

Figure S5: Mesure of the rate of L-[35S] methionine incorporation in two strains (L2868 638

and MTF1782) with contrasted nitrogen requirements. The average compound to absolute 639

mean values (c.p.m) of three replicates with or without (control) cycloheximide treatment. 640

Figure S6: Global clustering analysis 641

Figure S7: Genes recently identified in EC1118 strain. Genes in grey are differentially 642

expressed in LNR and HNR strains. 643

644

645

646

647

648

649

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650

651

652

653

654

655

656

657

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Figure 1: Comparison of nitrogen requirements of 8 wine yeast strains. The measure is expressed in mg nitrogen g-1 CO2 based on the amount of nitrogen necessary to support a constant fermentation rate between 10 and 50 g L -1 CO2 (P10-50). LNR: strains with low nitrogen requirements; HNR: strains with high nitrogen requirements. Reproducibility of this technique was tested for two strains (L2868 and MTF1782) and variations are indicated on the graph with error bars.

Strains with low nitrogen requirements (LNR) Strains with high nitrogen

requirements (HNR)

0

0,5

1

1,5

2

2,5

3

L2868 4CAR1 EC1118 Fermiflor Zymasil K1M 7013 MTF1782

Nit

roge

n r

eq

uir

em

en

t (m

g N

g-1

CO

2)

0.5

1.5

2.5

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Figure 2: Fermentation profiles of eight different wine yeast strains with contrasted nitrogen requirement in a nitrogen limited medium (SM100).

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0 50 100 150 200 250 300 350 400

dC

O2

dt-1

(gL-1

h-1

)

Time (h)

L2868

4CAR1

EC1118

Fermiflor

Zymasil

K1M

7013

MTF1782

0.7

0.6

0.5

0.4

0.3

0.2

0.1

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Figure 3: Principal component analysis (PCA) of (A) kinetic traits (Tf: Total fermentation duration; NQr: Quantified nitrogen requirement; Rmax: Maximun fermentation rate; R50: Fermentation rate at 50% sugar consumption) and (B) individuals strains. The values were obtained from fermentation profiles performed on SM100 at 24°C.

A B

Tf

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Figure 4: Effects of cycloheximide addition at 10 g L-1 h-1 of CO2 produced on the fermentation in SM100. Comparison between a strain with low nitrogen requirement (L2868) and a strain with high nitrogen requirement (MTF1782).

Cycloheximide added

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0 50 100 150 200 250 300 350 400 450

dC

O2

/dt

(g/l

/h)

Time (h)

MTF1782 + cycloheximide

MTF1782

L2868 + cycloheximide

L2868

0.1

0.7

0.6

0.5

0.4

0.3

0.2

dC

O2 d

t-1 (g

L -

1 h

-1)

Cycloheximide addition

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0 10 20 30 40 50

oxidation-reduction process

response to stress

trehalose biosynthetic process

metabolic process

carbohydrate metabolic process

glycogen biosynthetic process

cellular response to oxidative stress

glucose metabolic process

0 10 20 30 40 50 60

ribosomal large subunit biogenesis

ribosome biogenesis

rRNA processing

maturation of 18S rRNA

endonucleolytic cleavage at A-prime

endonucleolytic cleavage at A1

endonucleolytic cleavage at A2

ribosomal large subunit assembly

ribosomal small subunit biogenesis

positive regulation of transcription from Pol I promoter

GMP biosynthetic process

maturation of 5.8S rRNA from tricistronic rRNA transcript

maturation of LSU-rRNA from tricistronic rRNA transcript

A

B

Figure 5: Analysis of transcriptomic data (mean of two repetitions): enrichment of gene functions among strains with high nitrogen requirement (A) and strains with low nitrogen requirement (B). The values correspond to the number of genes in the Funspec functional category (Bonferroni correction and p-value 0.05). The p-values are indicated for each category.

1.668e-08

5.079e-06

1.309e-05

2.894e-08

6.064e-12

1.668e-08

2.726e-07

5.84e-07

1.077e-06

1.061e-13

<1e-14

<1e-14

<1e-14

6.063e-06

3.471e-07

1.568e-07

3.648e-09

1.306e-10

5.165e-11

4.333e-10

1.088e-14

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Cluster I Cluster II Cluster III Cluster IV

Figure 6: Cluster extracts of genes overexpressed in HNR strains. Cluster I is composed by genes involved in proteome degradation. Cluster II is enriched in genes associate to ubiquitin protein degradation. Cluster III is composed by genes involved in storage of reserve. Cluster IV is related to the autophagy pathway.

Cluster I Cluster III

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Figure 7: Analysis of transcriptomic data (mean of two repetitions): enrichment of gene functions among genes positively correlated with the fermentation rate (R50). The values correspond to the number of genes in the Funspec functional category (Bonferroni correction and p-value 0.05). The p-values are indicated for each category.

0 5 10 15 20 25 30

ribosome biogenesis

rRNA processing

ribosomal large subunit biogenesis

maturation of 18S rRNA

ribosomal large subunit export from cell nucleus

5.723e-11

<1e-14

3.804e-06

1.997e-06

<1e-14

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Table 1: Saccharomyces cerevisiae strains used in this study.

Strain Origin

L2868 IFV

4CAR1 IFV

Fermiflor BNIC

Zymasil BNIC

MTF1782 Wine isolate

K1M Lallemand SA

7013 BNIC

EC1118 Lallemand SA

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