Emmanuelle Becker

Yuchen Liu

Aurélie Lardenois

Thomas Walther

Joe Horecka

Igor Stuparevic

Michael Law

Régis Lavigne

Bertrand Evrard

Philippe Demougin

Michael Riffle

Randy Strich

Ronald W Davis

Charles Pineau

Michael Primig
email: michael.primig@inserm.fr

Integrated RNA-and protein profiling of fermentation and respiration in diploid budding yeast provides insight into nutrient control of cell growth and development

Keywords: Proteome, transcriptome, fermentation, respiration, Ume6, Rim4

Diploid budding yeast undergoes rapid mitosis when it ferments glucose, and in the presence of a non-fermentable carbon source and the absence of a nitrogen source it triggers sporulation. Rich medium with acetate is a commonly used pre-sporulation medium, but our understanding of the molecular events underlying the acetate-driven transition from mitosis to meiosis is still incomplete. We identified 263 proteins for which mRNA and protein synthesis are linked or uncoupled in fermenting and respiring cells. Using motif predictions, interaction data and RNA profiling we find among them 28 likely targets for Ume6, a subunit of the conserved Rpd3/Sin3 histone deacetylase-complex regulating genes involved in metabolism, stress response and meiosis. Finally, we identify 14 genes for which both RNA and proteins are detected exclusively in respiring cells but not in fermenting cells in our sample set, including CSM4, SPR1, SPS4 and RIM4, which were thought to be meiosis-specific. Our work reveals intertwined transcriptional and post-transcriptional control mechanisms acting when a MATa/ strain responds to nutritional signals, and provides molecular clues how the carbon source primes yeast cells for entering meiosis.

Significance

Our integrated genomics study provides insight into the interplay between the transcriptome and the proteome in yeast cells undergoing vegetative growth in the presence of glucose (fermentation) or acetate (respiration). Furthermore, it reveals novel target genes involved in these processes for Ume6, the DNA binding subunit of the conserved histone deacetylase Rpd3 and the co-repressor Sin3. We have combined data from an RNA profiling experiment using tiling arrays that cover the entire yeast genome, and a large-scale protein detection analysis based on mass spectrometry in diploid MATa/ cells. This distinguishes our study from most others in the field -which investigate haploid yeast strains -because only diploid cells can undergo meiotic development in the simultaneous absence of a non-fermentable carbon source and nitrogen. Indeed, we report molecular clues how respiration of acetate might prime diploid cells for efficient spore formation, a phenomenon that is well known but poorly understood.

Introduction

Budding yeast is an important model organism for biochemical studies, genetic analyses and genomic profiling of cell growth and division, and cell differentiation [START_REF] Cullen | The regulation of filamentous growth in yeast[END_REF][START_REF] Neiman | Sporulation in the budding yeast Saccharomyces cerevisiae[END_REF][START_REF] Goranov | Growth and division--not a one-way road[END_REF]. In addition, it is useful for the field of toxicogenomics, which investigates the cellular response to environmental stressors and drugs; [START_REF] Giaever | Chemogenomic profiling: identifying the functional interactions of small molecules in yeast[END_REF][START_REF] Schlecht | Cationic amphiphilic drugs are potent inhibitors of yeast sporulation[END_REF], reviewed in [START_REF] Santos | Yeast toxicogenomics: genomewide responses to chemical stresses with impact in environmental health, pharmacology, and biotechnology[END_REF].

S. cerevisiae was the first eukaryote for which the complete genome sequence was determined (see Saccharomyces Genome Database, SGD) [START_REF] Goffeau | Life with 6000 genes[END_REF][START_REF] Cherry | Saccharomyces Genome Database: the genomics resource of budding yeast[END_REF]. The protein-coding part of the yeast genome includes verified genes, which are conserved open reading frames (ORFs) typically associated with a biological process, uncharacterized genes, which play no known roles, and dubious genes, which are assumed not to encode functional proteins [START_REF] Cherry | Saccharomyces Genome Database: the genomics resource of budding yeast[END_REF]. The yeast transcriptome has been studied in numerous conditions using microarrays and RNA-Sequencing (RNA-Seq) [START_REF] Lashkari | Yeast microarrays for genome wide parallel genetic and gene expression analysis[END_REF][START_REF] Wang | RNA-Seq: a revolutionary tool for transcriptomics[END_REF][START_REF] David | A highresolution map of transcription in the yeast genome[END_REF][START_REF] Nagalakshmi | The transcriptional landscape of the yeast genome defined by RNA sequencing[END_REF]. Furthermore, protein-profiling experiments detected nearly all theoretically predicted yeast proteins in pre-fractionated extracts from growing cells [START_REF] De Godoy | Comprehensive mass-spectrometry-based proteome quantification of haploid versus diploid yeast[END_REF][START_REF] Picotti | Full dynamic range proteome analysis of S. cerevisiae by targeted proteomics[END_REF].

Very recent feasibility studies reported vastly improved experimental protocols yielding quantitative information about protein concentrations in total cell extracts [START_REF] Thakur | Deep and highly sensitive proteome coverage by LC-MS/MS without prefractionation[END_REF][START_REF] Nagaraj | System-wide perturbation analysis with nearly complete coverage of the yeast proteome by singleshot ultra HPLC runs on a bench top Orbitrap[END_REF][START_REF] Hebert | The one hour yeast proteome[END_REF].

To produce the metabolite adenosine triphosphate (ATP), which is critical for storing chemical energy, yeast cells process fermentable and non-fermentable carbon sources by activating distinct metabolic pathways. Fermentation of sugars such as glucose and fructose is efficient and down-regulates enzymes required for metabolizing alternative carbon sources via a process called carbon catabolite repression; reviewed in [START_REF] Gancedo | Yeast carbon catabolite repression[END_REF]. Fermentation leads to the accumulation of glycerol and ethanol, which are further processed via oxidation. When yeast cells are cultured in rich medium in the presence of the two-carbon compound acetate, they use the tricarboxylic acid (TCA) cycle, and the glyoxylate cycle for energy production [START_REF] Zaman | How Saccharomyces responds to nutrients[END_REF][START_REF] Lee | TCA cycle-independent acetate metabolism via the glyoxylate cycle in Saccharomyces cerevisiae[END_REF]. Previous analyses of yeast cell growth under highly controlled conditions limited for carbon, nitrogen, phosphorus and sulphur revealed that both transcriptional control and posttranslational regulation mechanisms play roles in the response to nutrient starvation [START_REF] Gutteridge | Nutrient control of eukaryote cell growth: a systems biology study in yeast[END_REF][START_REF] Boer | The genome-wide transcriptional responses of Saccharomyces cerevisiae grown on glucose in aerobic chemostat cultures limited for carbon, nitrogen, phosphorus, or sulfur[END_REF][START_REF] Castrillo | Growth control of the eukaryote cell: a systems biology study in yeast[END_REF].

Fermentation and respiration are not only relevant for understanding yeast growth and development but also for human diseases; for example, the so-called Warburg effect has been observed in tumour cells that excessively ferment glucose into lactate [START_REF] Diaz-Ruiz | The Warburg and Crabtree effects: On the origin of cancer cell energy metabolism and of yeast glucose repression[END_REF].

Ume6 is the DNA binding subunit of two conserved repressor complexes including either the histone deacetylase (HDAC) Rpd3 and the co-repressor Sin3 [START_REF] Kadosh | Targeted recruitment of the Sin3-Rpd3 histone deacetylase complex generates a highly localized domain of repressed chromatin in vivo[END_REF][START_REF] Rundlett | Transcriptional repression by UME6 involves deacetylation of lysine 5 of histone H4 by RPD3[END_REF], or the chromatinremodeling factor Isw2 [START_REF] Goldmark | The Isw2 chromatin remodeling complex represses early meiotic genes upon recruitment by Ume6p[END_REF]. Ume6 was initially identified as a mitotic repressor for early

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M A N U S C R I P T meiosis-specific genes, and later shown to orchestrate metabolic functions, stress response, and the onset of meiotic differentiation [START_REF] Steber | UME6 is a central component of a developmental regulatory switch controlling meiosis-specific gene expression[END_REF][START_REF] Strich | UME6 is a key regulator of nitrogen repression and meiotic development[END_REF][START_REF] Williams | The Ume6 regulon coordinates metabolic and meiotic gene expression in yeast[END_REF]. Among others, Ume6 directly represses SIP4, which encodes a C6 zinc cluster transcriptional activator involved in gluconeogenesis, during growth in the presence of glucose [START_REF] Williams | The Ume6 regulon coordinates metabolic and meiotic gene expression in yeast[END_REF][START_REF] Harbison | Transcriptional regulatory code of a eukaryotic genome[END_REF]. Ume6 becomes unstable when vegetatively growing cells switch from metabolising glucose to processing acetate, and is temporarily degraded during meiosis by the anaphase promoting complex/cyclosome (APC/C), before it accumulates again at later stages of spore formation [START_REF] Mallory | Gcn5p-dependent acetylation induces degradation of the meiotic transcriptional repressor Ume6p[END_REF][START_REF] Strich | Ume6p is required for germination and early colony development of yeast ascospores[END_REF].

In earlier work we have developed Direct Iterative Protein Profiling (DIPP) to comprehensively analyse the proteome of fermenting diploid yeast cells in a cost-effective manner by widely available standard mass spectrometry [START_REF] Lavigne | Direct iterative protein profiling (DIPP) -an innovative method for large-scale protein detection applied to budding yeast mitosis[END_REF]. In the present study we report the changes in the proteome of diploid MATa/ yeast cells undergoing logarithmic growth in rich medium with fermentable glucose (YPD) or non-fermentable acetate (YPA) in the context of corresponding RNA-profiling data from wild-type cells and a ume6 mutant strain.

This approach enabled us to gain insight into molecular events that underlie the switch from fermentation to respiration, and that prime diploid MATa/ cells for the transition from mitotic growth to meiotic differentiation.

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Materials and Methods

Culture conditions for protein profiling

Samples from YPD (yeast extract, peptone and dextrose) were cultured as published [START_REF] Lavigne | Direct iterative protein profiling (DIPP) -an innovative method for large-scale protein detection applied to budding yeast mitosis[END_REF]. In addition, cells grown in YPD were transferred into 100 ml YPA (rich medium with acetate) pre-warmed to 30°C at a cell density of 2x10 6 cells/ml and cultured until they reached 3x10 7 cells/ml, before they were harvested in two 50 ml aliquots and washed with sterile water. The pellets were then snap-frozen in liquid nitrogen and stored at -80°C.

Construction of gene deletion strains

To reduce the chance of unlinked mutations affecting the growth experiments, both copies of target genes were deleted in homozygous diploid cells. Most deletions were made in the BY4743 background using the method as published [START_REF] Winzeler | Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis[END_REF]. Three deletions were made in the related strain JHY222 [START_REF] Lardenois | Execution of the meiotic noncoding RNA expression program and the onset of gametogenesis in yeast require the conserved exosome subunit Rrp6[END_REF].

Plate growth assays

To monitor the growth phenotype of deletion strains we used two methods. Homozygous diploid strains were streaked on YD/G418 and grown at 30ºC for three days. Single colonies from each strain were suspended in water and diluted to approximately 100 cells/ l. 4 l of cell suspension were spotted onto YPD and YPEG (ethanol, glycerol) plates and incubated at 30ºC for two days (YPD) and three days (YPEG). Alternatively, strains were re-streaked for single colonies on YPD, YPA and YPEG plates, and incubated at 30ºC for three days (YPD)

or four days (YPA, YPEG).

Protein detection by immunofluorescence

Rim4 immunostaining was done as described [START_REF] Scherthan | Yeast FISH: delineation of chromosomal targets in vegetative and meiotic yeast cells[END_REF]. Briefly, cells were fixed in 70% ethanol

Chromatin immunoprecipitation (ChIP) assay

Ume6/DNA complexes formed in vivo were precipitated using a standard assay with SK1 cells cultured in rich medium (YPD). A 50 ml mid-log culture was cross-linked with 1% formaldehyde for 15 minutes at room temperature. Cross-linked Ume6/DNA complexes were quenched with 140 mM glycine (Sigma, USA) for five minutes, collected, washed and eluted before reversing crosslinks. Finally, DNA was precipitated, digested with proteinase K and amplified by Q-PCR as published. Relative ChIP signals were calculated using the formula (2^-IP (CT target -CT control)/input (CT target -CT control)). The NUP85 coding region was used as an internal control [START_REF] Mallory | Gcn5p-dependent acetylation induces degradation of the meiotic transcriptional repressor Ume6p[END_REF][START_REF] Meluh | Budding yeast centromere composition and assembly as revealed by in vivo cross-linking[END_REF].

Protein extraction, sample pre-fractionation and peptide analysis by mass spectrometry

Duplicate samples from YPA were pre-fractionated, and the samples were analysed using DIPP as published [START_REF] Lavigne | Direct iterative protein profiling (DIPP) -an innovative method for large-scale protein detection applied to budding yeast mitosis[END_REF]. Data were analysed using the Proteome Discoverer 1.2 software in conjunction with Mascot (Matrixscience) and SEQUEST database search engines to identify peptides and proteins. In the first step the MS/MS spectra were compared to annotation data for budding yeast proteins (Saccharomyces Genome Database release 06/01/2010) [START_REF] Cherry | Saccharomyces Genome Database: the genomics resource of budding yeast[END_REF]. The mass tolerance for MS and MS/MS was set at 10 ppm and 0.5 Dalton. Trypsin selectivity was set to full, with one miss cleavage possible. The protein modifications were fixed carbamidomethylation of cysteines, variable oxidation of methionine, variable acetylation of lysine and variable phosphorylation of serine, threonine and tyrosine. Peptides identified were selected via Xcorr values and the Mascot score to achieve a false discovery rate of 1 % and a false positive rate of 5 %. We used Proteome Discoverer to assemble peptides detected in the first and second run, which are then masked in subsequent LC-MS/MS analyses. Before the third analysis step was executed, we combined peptide exclusion files from the first two runs.

Peptides that were not masked were exported as a text file containing uncharged and accurate mass values (at five decimals) and a retention time window of approximately one minute. The

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instrument was set to function with uncharged masses and to calculate the mass of a peptide based on its exact mass and charge state. A mass tolerance of ±10 ppm was employed to filter out peptides already identified within the specified retention time window.

Minimum Information About a Proteomics Experiment (MIAPE) compliance

The mass spectra and search result data files are available for downloading at the Yeast Resource Center Public Data Repository (YRC PDR) via www.yeastrc.org/pdr/becker2014/ [START_REF] Riffle | The Yeast Resource Center Public Data Repository[END_REF].

Culture conditions for RNA profiling

Triplicate samples of SK1 wild-type and ume6 mutant strains were cultured in YPD, YPA and SPII using standard media and conditions [START_REF] Williams | The Ume6 regulon coordinates metabolic and meiotic gene expression in yeast[END_REF][START_REF] Primig | The core meiotic transcriptome in budding yeasts[END_REF].

Probe synthesis, GeneChip hybridization and scan

RNA profiling with Yeast 2.0 GeneChips was done as published [START_REF] Schlecht | Expression profiling of mammalian male meiosis and gametogenesis identifies novel candidate genes for roles in the regulation of fertility[END_REF].

Differential expression analysis of wild-type versus ume6 mutant strains

To identify up-regulated genes in ume6 mutant we first performed a quantile normalization and then selected genes for which the expression level was above the 25 th percentile (9.006) in at least one condition, and that showed a fold-change >2 between mutant and wild-type. A limma test with a False Discovery Rate (FDR) 5% was done to identify genes differentially expressed and up-regulated in the ume6 mutant. The statistical filtration was done using R [START_REF] Smyth | Limma: linear models for microarray data[END_REF][START_REF] Team | R: A language and environment for statistical computing[END_REF].

Tiling array expression data integration

DNA-strand specific whole-genome expression data obtained with tiling arrays and duplicate samples from diploid SK1 cells cultured in rich medium (YPD) and rich medium with acetate (YPA) were integrated and compared to the mass spectrometry measurements; data processing methods and expression threshold level parameters were as published [START_REF] Lardenois | Execution of the meiotic noncoding RNA expression program and the onset of gametogenesis in yeast require the conserved exosome subunit Rrp6[END_REF]. For each gene annotated in the reference genome, we selected the segments derived from Sc_tiling experiments, which overlapped its coordinates, and computed an average value for

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the gene. Therefore, each segment's contribution to the overall signal is proportional to its overlap with the corresponding gene. Equivalent values were computed for overlapping antisense transcripts for each gene. Genes were organized into 11 clusters, and for each gene (as represented by segments) we extracted the list of the clusters it was associated with.

Microarray expression data

Expression signals were obtained with Affymetrix DNA-strand specific tiling arrays (Sc_tlg)

from reference [START_REF] Lardenois | Execution of the meiotic noncoding RNA expression program and the onset of gametogenesis in yeast require the conserved exosome subunit Rrp6[END_REF].

Regulatory motif prediction

The URS1 motif was predicted using the published Promoter Analysis Protocol (PAP) [START_REF] Trepos-Pouplard | Proteome analysis and genome-wide regulatory motif prediction identify novel potentially sex-hormone regulated proteins in rat efferent ducts[END_REF].

Global genomics data integration

Information about protein and genes and the Gene Ontology annotation data were downloaded from SGD [START_REF] Cherry | Saccharomyces Genome Database: the genomics resource of budding yeast[END_REF]. The relative abundance data of proteins expressed in log-phase growth were extracted from the quantitative Western blot analysis of TAP-tagged strains available via SGD (http://yeastgfp.yeastgenome.org/) [START_REF] Ghaemmaghami | Global analysis of protein expression in yeast[END_REF]. Phosphorylated sites predictions were extracted from [START_REF] Hornbeck | PhosphoSitePlus: a comprehensive resource for investigating the structure and function of experimentally determined post-translational modifications in man and mouse[END_REF]. Genome annotation data for haploid S288c and SK1 strains were used as published [START_REF] Lavigne | Direct iterative protein profiling (DIPP) -an innovative method for large-scale protein detection applied to budding yeast mitosis[END_REF].

Identifying target genes showing a similar pattern as the reference genes

We filtered genes similar to KGD1, MDH1, MDH2, SDH1, FUM1, IDH1, ACO1, CIT1, MLS1, ICL1 whereby (i) the target genes were expressed above the background (BG)=5.99 in YPD and YPA, (ii) the fold change between expression in YPD and YPA was >2.0, (iii) the log-expression in YPA was >12, and (iv) we observed >3-fold more bands to contain the protein in YPA than YPD.

A C C E P T E D M A N U S C R I P T Gene Ontology enrichment analysis

We performed the GO enrichment analysis with the GOToolBox [START_REF] Martin | GOToolBox: functional analysis of gene datasets based on Gene Ontology[END_REF], using an hypergeometric law and a Benjamini and Hochberg correction for multiple testing. Biological

Process GO annotations with corrected p-values <0.01 were selected.

Identification of proteins detected specifically in YPA

We selected proteins for which peptides were found only in YPA but not YPD classified the genes, which encode them into loci, which are not significantly differentially expressed in YPD and YPA, which are differentially expressed in YPD and YPA, with a fold-change >1.5

as assessed by a linear models for microarray data (LIMMA) statistical test with FDR=5%, and genes, which are expressed <BG=5.99 in YPD and >BG=5.99 in YPA, with a foldchange >1.5 as assessed by a limma statistical test with FDR=5% [START_REF] Smyth | Limma: linear models for microarray data[END_REF][START_REF] Team | R: A language and environment for statistical computing[END_REF].

Results

Experimental design

To determine the proteome in the presence of glucose or acetate we combined previously published DIPP data on rapid growth in rich medium with glucose (YPD) [START_REF] Lavigne | Direct iterative protein profiling (DIPP) -an innovative method for large-scale protein detection applied to budding yeast mitosis[END_REF], with a new dataset from cells cultured in a medium where glucose was replaced by acetate (YPA). We included published RNA profiling data from YPD and YPA samples that were analyzed with tiling arrays (Sc_tlg) [START_REF] Lardenois | Execution of the meiotic noncoding RNA expression program and the onset of gametogenesis in yeast require the conserved exosome subunit Rrp6[END_REF]. We previously reported that SIP4, CAT8 and ADR1 -which encode transcription factors involved in gene expression during the diauxic shift -are strongly derepressed in a ume6 mutant (GeneChip expression data are shown in www.germonline.org; [START_REF] Lardenois | GermOnline 4.0 is a genomics gateway for germline development, meiosis and the mitotic cell cycle. Database : the journal of biological databases and curation[END_REF]) [START_REF] Galdieri | Transcriptional regulation in yeast during diauxic shift and stationary phase[END_REF]. Since a large-scale in vivo protein-DNA interaction study showed that Ume6 binds to their promoters (ChIP-Chip in vivo binding data are shown in www.germonline.org) [START_REF] Harbison | Transcriptional regulatory code of a eukaryotic genome[END_REF],

we investigated the role of the Rpd3/Sin3/Ume6 regulatory complex in respiring diploid cells.

To this end, we used expression data from UME6 wild-type cells and a ume6 mutant strain (Lardenois, Walther, Becker et al., in preparation; the complete dataset will be published elsewhere). The transcriptome and proteome data were then integrated with functional genomics data [START_REF] Giaever | Chemogenomic profiling: identifying the functional interactions of small molecules in yeast[END_REF][START_REF] Cherry | Saccharomyces Genome Database: the genomics resource of budding yeast[END_REF][START_REF] Hillenmeyer | The chemical genomic portrait of yeast: uncovering a phenotype for all genes[END_REF]. To allow for direct comparison with previous studies, we have employed standard flask culture conditions for asynchronous cell division in rich media with

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glucose or acetatethe latter is used as a non-fermentable carbon source in pre-sporulation growth medium [START_REF] Primig | The core meiotic transcriptome in budding yeasts[END_REF][START_REF] Klapholz | The role of the SPO11 gene in meiotic recombination in yeast[END_REF][START_REF] Jambhekar | Control of meiosis by respiration[END_REF] and cell differentiation in sporulation medium.

The proteome of diploid budding yeast during fermentation and respiration

Like in earlier work on the proteome of cells cultured in YPD [START_REF] Lavigne | Direct iterative protein profiling (DIPP) -an innovative method for large-scale protein detection applied to budding yeast mitosis[END_REF], we found that protein size and migration speed in the gel -that is, protein presence in gel slices 1 (top) to 30 (bottom)are correlated similarly across YPA replicates (Figure 1A). We also observed that the number of bands a protein was present in was highly reproducible in both media (correlation coefficient r 2 0.898 in YPD and 0.877 in YPA; Figure 1B).

By combining data obtained with cells cultured in rich media with glucose or acetate we detected 5513 out of 6713 predicted proteins in at least one of the four samples (82%, taking verified, uncharacterized and dubious genes into account). More specifically, we found 4517 of 4877 proteins encoded by verified genes (93%; Table 1, Figure 1C). Among 4952 proteins detected in YPD, 3520 were found in both samples, while 883 and 549 were detected only in YPD1 or YPD2, respectively. Likewise, among 5020 protein scored as present in YPA, 3733

were reproducibly detected, while 655 and 632 were present only in replicates 1 or 2 (Figure 1D). As expected, the vast majority of verified ORFs encode proteins, which are expressed highly enough to be detected in almost all samples, while the output among uncharacterized genes is split into two major groups that are either reproducibly detected, or that are undetectable in the experimental conditions used (Table 2, Figure 1E).

In the case of dubious ORFs we detect few proteins, which is in keeping with the notion that many of these loci are annotation artefacts. However, we note that 20 proteins encoded by dubious ORFs are detected in all four YPD/YPA samples (Figure 1E, Supplemental file S1, Class 1 detection pattern, filter for "dubious"). Among them, Yjr146w was detected in an earlier protein profiling study [START_REF] Ghaemmaghami | Global analysis of protein expression in yeast[END_REF] and two other proteins may be involved in cell aging (Yim2 [START_REF] Burtner | A genomic analysis of chronological longevity factors in budding yeast[END_REF]) or drug-resistance (Imd1 [START_REF] Hyle | Functional distinctions between IMP dehydrogenase genes in providing mycophenolate resistance and guanine prototrophy to yeast[END_REF]). For eight dubious ORFs we also detect Among approximately 1000 proteins known to localise to mitochondria we detect the majority (718) in all samples; notably, 57 proteins are scored as present only in cells grown in YPA (including 31 that are detected in both YPA replicates). To the contrary, only 9 proteins were found exclusively in cells cultured in YPD (Figure 1F).

We conclude that DIPP detects the vast majority of known yeast proteins and that growth in acetate increases the protein content of mitochondria in accordance with their metabolic role in respiration [START_REF] Reinders | Toward the complete yeast mitochondrial proteome: multidimensional separation techniques for mitochondrial proteomics[END_REF][START_REF] Renvoise | Quantitative variations of the mitochondrial proteome and phosphoproteome during fermentative and respiratory growth in Saccharomyces cerevisiae[END_REF][START_REF] Sickmann | The proteome of Saccharomyces cerevisiae mitochondria[END_REF]. Finally, the group of dubious ORFs may represent an opportunity for the discovery of genes involved in vegetative growth.

Comparing expression levels and protein detection patterns in diploid MATa/ cells

We subsequently asked if DIPP's capacity to detect a protein was related to the overall expression level of the corresponding gene, and found that the median intensities of logtransformed microarray expression signals increase with the number of samples in which the proteins are detected by DIPP in cells cultured in YPD or YPA (Figure 2A). Gene expression values are also positively correlated with the number of bands a protein is detected in, and the average number of peptides identified (Figure 2B,C). To further correlate gene expression and protein function, we compared protein profiles in YPD and YPA media with mRNA concentrations during fermentation (YPD), respiration (YPA) and sporulation (SPII hourly time points from 1 to 12) using our published tiling array data from SK1 MATa/ wild-type cells [START_REF] Lardenois | Execution of the meiotic noncoding RNA expression program and the onset of gametogenesis in yeast require the conserved exosome subunit Rrp6[END_REF]. Coherently, we found that proteins encoded by genes showing peak expression in cells undergoing mitotic divisions tend to be reproducibly detected in one or both growth media (Figure 2D, patterns 1-4). On the contrary, proteins encoded by genes for which mRNA synthesis is induced during early, middle and late stages of meiosis and gametogenesis, are not -or at least not reproduciblydetected in mitotic cells (Figure 2D, patterns 5-10). It is unclear why in the case of very late sporulation genes the DIPP pattern is slightly shifted toward protein detection again, perhaps it reflects the fact that certain proteins play roles during mitotic growth and spore formation (Figure 2D, pattern 11).

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Transcriptional regulation versus post-transcriptional control during fermentation and respiration

We first asked if proteins important for metabolizing acetate showed correlated profiles of mRNA induction and protein accumulation. To address this question, we determined the mRNA/protein patterns underlying the switch from processing glucose to metabolizing acetate in the cases of genes essential for the process. We selected 10 reference proteins required for respiration, and found them both in fermenting and respiring cells (Figure S1A) [START_REF] Cherry | Saccharomyces Genome Database: the genomics resource of budding yeast[END_REF]. Subsequently, we selected 60 genes, which show RNA/protein patterns similar to the reference genes (Figure S1B). Gene Ontology terms enriched in this group point to metabolic processes (cellular respiration, p-value 1.45x10 -7 ; carboxylic acid metabolic process,

1.24x10 -24
), and a response to nutritional cues (nitrogen compound biosynthetic process, 3.52x10 -7 ; Figure S1C). We next assayed genes involved in mitochondrial inheritance and function (AIM41, YCP4), the TCA cycle (LSC2), ethanol metabolism (ADH2), iron transport (FTH1), cell structure (CWP1), stress response (CMK2, LSB3, YJR085C), and possibly respiration (Ypr010c-a via interaction with Fcj1 [START_REF] Tkach | Dissecting DNA damage response pathways by analysing protein localization and abundance changes during DNA replication stress[END_REF]) for growth on a non-fermentable carbon source. However, none of them failed to grow on YPA and YEPG media after incubation at 30ºC; FUM1, ACO1, OM14 and PTH1 were used as positive controls (Figure S1D). We conclude that proteins important for growth on acetate are not necessarily present only in respiring cells but already accumulate during fermentation.

Ume6 coordinates the transition between cell growth and development, and both its concentration and DNA binding activity decrease when cells are cultured in acetate [START_REF] Williams | The Ume6 regulon coordinates metabolic and meiotic gene expression in yeast[END_REF][START_REF] Mallory | Gcn5p-dependent acetylation induces degradation of the meiotic transcriptional repressor Ume6p[END_REF][START_REF] Mallory | Meiosis-Specific Destruction of the Ume6p Repressor by the Cdc20-Directed APC/C[END_REF][START_REF] Law | Acetylation of the transcriptional repressor Ume6p allows efficient promoter release and timely induction of the meiotic transient transcription program in yeast[END_REF]. To learn more about the transcriptional control mechanisms that contribute to the mitotic regulation of genes differentially expressed during fermentation and respiration we combined URS1 motif predictions with genome-wide expression data from wild-type cells and ume6 mutant cells. We find that likely direct Ume6 targets are induced in YPA. This comprises six genes for which promoters contain known URS1 motifs (ACH1, ACS1, CAT2, GDH2, PRX1, UBI4), and three genes that have promoters containing predicted URS1 elements (ADY2, PRB1, OM14; Figure S1B and Supplemental file S1). ADY2 encodes a membrane bound acetate permease essential for the packaging of meiotic nuclei into four spores [START_REF] Rabitsch | A screen for genes required for meiosis and spore formation based on whole-genome expression[END_REF][START_REF] Paiva | Ady2p is essential for the acetate permease activity in the yeast Saccharomyces cerevisiae[END_REF]. The latter function is apparently specific for Ady2 since no meiotic phenotype was reported for its paralog Ato2 [START_REF] Gentsch | Mutations at different sites in members of the Gpr1/Fun34/YaaH protein family cause hypersensitivity to acetic acid in Saccharomyces cerevisiae as well as in Yarrowia lipolytica[END_REF]. To validate our motif predictions and the RNA profiling data we confirmed that Ume6 binds to the promoters of ACH1 and ADY2 in vivo using chromatin immunoprecipitation assays. The binding signal intensities were found to be

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in the same range as the Ume6 targets CAR1 and HOP1 ( [START_REF] Steber | UME6 is a central component of a developmental regulatory switch controlling meiosis-specific gene expression[END_REF][START_REF] Park | The yeast UME6 gene product is required for transcriptional repression mediated by the CAR1 URS1 repressor binding site[END_REF][START_REF] Vershon | Meiotic induction of the yeast HOP1 gene is controlled by positive and negative regulatory sites[END_REF]; Figure S1E).

Furthermore, ume6 mutants in three different genetic backgrounds fail to grow normally on YPA and -in the case of W303display increased cell size (Figure S1F, G) [START_REF] Strich | UME6 is a key regulator of nitrogen repression and meiotic development[END_REF]. These findings extend the metabolic role of the Rpd3/Sin3/Ume6 regulator as diploid cells switch from fermentation to respiration.

Protein profiling reveals that carbon-source specificity is biased toward acetate

We next sought to distinguish genes that are transcriptionally controlled by Rpd3/Ume6, from those that are regulated at the post-transcriptional level when diploid cells switch from fermentation to respiration. We therefore assembled a list of 94 proteins detected only in YPD and 169 proteins found only in YPA and then used tiling array data to filter three groups of YPD/YPA mRNA patterns in each medium.

The first YPD-specific group includes 56 genes for which we detect a gradient of similar concentrations (that is, the differences are not statistically significant) in both YPD and YPA (Figure 3A). This group contains Spo23 (a Spo1 interactor [START_REF] Tevzadze | Genetic evidence for a SPO1-dependent signaling pathway controlling meiotic progression in yeast[END_REF]) and Mum3 (spore wall formation [START_REF] Engebrecht | Yeast meiotic mutants proficient for the induction of ectopic recombination[END_REF]), for which weak mRNA signals are detected in YPD [START_REF] Lardenois | Execution of the meiotic noncoding RNA expression program and the onset of gametogenesis in yeast require the conserved exosome subunit Rrp6[END_REF], and five genes upregulated in the ume6 mutant (BSC2, MCH1, SDT1, YNL155W, ZPS1).

The second YPD-specific group comprises 17 genes for which mRNAs coherently peak in rich medium (Figure 3B). We note the presence of supposedly sporulation-specific Smk1

(MAP kinase required or spore wall assembly [START_REF] Krisak | SMK1, a developmentally regulated MAP kinase, is required for spore wall assembly in Saccharomyces cerevisiae[END_REF]) in this group. We also find another gene showing elevated levels during growth in the absence of Ume6 (DIF1).

The third YPD-specific group covers 21 genes for which mRNAs peak in YPA (Figure 3C).

This pattern clearly indicates that transcription and translation and protein stability are uncoupled for these loci. Again, we detect a protein considered to be sporulation-specific (Sps100, involved in spore wall formation [START_REF] Law | The SPS100 gene of Saccharomyces cerevisiae is activated late in the sporulation process and contributes to spore wall maturation[END_REF]), for which tiling array data show clear expression in dividing cells [START_REF] Lardenois | Execution of the meiotic noncoding RNA expression program and the onset of gametogenesis in yeast require the conserved exosome subunit Rrp6[END_REF].

The first YPA-specific group includes 58 genes for which we observe similar mRNA levels across media (Figure 4A). Among the proteins that accumulate in YPA we found Spo21, which is mainly involved in meiosis specific modification of the spindle pole body, but for which a function in nutrient utilization was also reported [START_REF] Bajgier | SPO21 is required for meiosisspecific modification of the spindle pole body in yeast[END_REF][START_REF] Cai | Genomewide screen reveals a wide regulatory network for di/tripeptide utilization in Saccharomyces cerevisiae[END_REF], and Sae2, a protein involved in DNA-damage response and meiotic double strand break repair [START_REF] Prinz | Isolation of COM1, a new gene required to complete meiotic double-strand break-induced recombination in Saccharomyces cerevisiae[END_REF][START_REF] Mckee | A general method for identifying recessive diploid-specific mutations in Saccharomyces cerevisiae, its application to the isolation of mutants blocked at intermediate stages of meiotic prophase and characterization of a new gene SAE2[END_REF]. This

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group also contains MET30, STS1 and YLR194C, which harbour predicted URS1 motifs in their promoters and are partially de-repressed in acetate in the absence of Ume6 (Figure 4A).

The second YPA-specific group comprises 97 loci for which mRNA is detected at various levels in YPD but its concentration increases in YPA (Figure 4B). URS1 motif predictions and RNA profiling data indicate that these genes include eleven new potential Ume6 targets involved in acetate transport and spore packaging (ADY2), respiration (RSF1), energy metabolism (BNA2), cell wall integrity (PUN1), protein transport (VPS73), protein turnover (DAS1), protein modification (PIG1), thermotolerance (NTH2), DNA repair (PDC1), multidrug transport (PDR10), and stress response (YLR446W). Interestingly, we also find RIM8

and RIM13, which control RIM1, a positive regulator of sporulation and invasive growth [START_REF] Su | Identification of functionally related genes that stimulate early meiotic gene expression in yeast[END_REF][START_REF] Li | Proteolytic activation of Rim1p, a positive regulator of yeast sporulation and invasive growth[END_REF], and IMP2, a gene required for respiration and sporulation [START_REF] Donnini | IMP2, a nuclear gene controlling the mitochondrial dependence of galactose, maltose and raffinose utilization in Saccharomyces cerevisiae[END_REF].

The third YPA-specific group consists of 14 genes for which mRNAs and proteins are detected only in YPA (Figure 5A). Some gene functions are coherent with growth in YPA, such as energy metabolism controlled by glucose and ethanol (ICL2), stress-induced protein transport (YPT53), stress response (SPL2), carbohydrate transport (MPH3), glycerol transport (STL1), sulphur catabolism important for efficient anaerobic growth (JLP1), amino acid biosynthesis (STR3), and fatty-acid oxidation (POT1). Furthermore, we identified a dubious gene (YDR510C-A), a gene involved in cell fusion during mating (FUS1), and another four genes previously considered to be meiosis-specific (CSM4, RIM4, SPR1 and SPS4 [START_REF] Soushko | An RNA-binding protein homologue that promotes sporulation-specific gene expression in Saccharomyces cerevisiae[END_REF][START_REF] Deng | RIM4 encodes a meiotic activator required for early events of meiosis in Saccharomyces cerevisiae[END_REF][START_REF] Berchowitz | A developmentally regulated translational control pathway establishes the meiotic chromosome segregation pattern[END_REF][START_REF] Conrad | Rapid telomere movement in meiotic prophase is promoted by NDJ1, MPS3, and CSM4 and is modulated by recombination[END_REF][START_REF] Wanat | Csm4, in collaboration with Ndj1, mediates telomere-led chromosome dynamics and recombination during yeast meiosis[END_REF][START_REF] Garber | The SPS4 gene of Saccharomyces cerevisiae encodes a major sporulation-specific mRNA[END_REF]).

To find out if genes showing such patterns played new roles during respiration, we used a plate growth assay to analyse diploid homozygous csm4, rim4, sps4, stl1 and str3 mutants in the presence of fermentable-or non-fermentable carbon sources. None of the mutants appeared to be respiration deficient. HO and FAR3 were used as negative controls, and ACO1

and MRM1 were positive controls (Figure 5B). The third group includes three genes with known URS1 motifs (CSM4, SPl2, STR3) and two new targets that contain predicted Ume6 binding sites in their upstream regions (POT1, RIM4). Rim4 is an RNA-binding protein important for mRNA translation during meiosis and spore formation [START_REF] Deng | RIM4 encodes a meiotic activator required for early events of meiosis in Saccharomyces cerevisiae[END_REF][START_REF] Berchowitz | A developmentally regulated translational control pathway establishes the meiotic chromosome segregation pattern[END_REF]. Since the Ume6 level decreases and its ability to bind DNA diminishes when cells enter respiration, it is possible that RIM4 is partially de-repressed in YPA [START_REF] Mallory | Gcn5p-dependent acetylation induces degradation of the meiotic transcriptional repressor Ume6p[END_REF][START_REF] Mallory | Meiosis-Specific Destruction of the Ume6p Repressor by the Cdc20-Directed APC/C[END_REF][START_REF] Law | Acetylation of the transcriptional repressor Ume6p allows efficient promoter release and timely induction of the meiotic transient transcription program in yeast[END_REF]. To rule out that Rim4 accumulates in YPA because the protein is strongly induced in a small sub-population of cells undergoing premature sporulation [START_REF] Soushko | An RNA-binding protein homologue that promotes sporulation-specific gene expression in Saccharomyces cerevisiae[END_REF][START_REF] Deng | RIM4 encodes a meiotic activator required for early events of meiosis in Saccharomyces cerevisiae[END_REF], we analysed a strain expressing C-terminally tagged Rim4 (which showed normal growth and sporulation properties), and found that all cells cultured in YPA expressed the protein (Figure 5C).

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Discussion

We integrated transcriptome and proteome data from growing diploid MATa/ yeast cells cultured in rich medium, and pre-sporulation medium where glucose was replaced by the nonfermentable carbon source acetate. We interpreted the output in the context of functional information. Our results contribute to yeast genome annotation, and they help gain insight into the molecular processes, which coordinate energy metabolism with stress response during cell growth and differentiation.

Why are proteins absent although the cells contain their cognate mRNAs?

For 1630 ORFs we find evidence for mRNAs but not for proteins in at least one medium (some proteins in this category also fall into the class of 5513 proteins detected in at least one medium, which is why their sum is greater than the total number of 6713 predicted proteins).

The proteins in this group tend to be somewhat smaller than the global average (median of 231 amino acids versus 358 for all proteins; p-value of Wilcoxon rank sum test with continuity correction < 2.2x10 -16 ; Figure S2A). 598 proteins are less than 150 amino acids long, and may therefore have been lost during the pre-fractionation procedure, but for 1032 cases other explanations such as annotation artefacts, special biological roles or inherent instability during growth seem more likely. Indeed, among 1032 ORFs we find 47 dubious genes and 200 uncharacterized genes. Furthermore, significantly enriched terms from the Biological Process Gene Ontology reveal functions likely not important during fast mitotic growth in rich media: this includes processes related to retroviruses and gametogenesis such as transposition, RNA-mediated and transposition (p-value: 6.28x10 -32 ), reproduction (7.38x10 -5 ), reproductive process (3.85x10 -5 ), and DNA recombination (2.08x10 -8 ). Other pathways are DNA metabolic process (0.00141), protein catabolic process (0.00264), proteolysis (0.00400), regulation of biological quality (0.00868), and notably, response to chemical stimulus (0.00343), reflecting in part the activity of stress-response genes likely repressed during growth under perfect conditions (Figure S2B).

Gene expression and protein function

We identified two genes as potentially important for fermentation (YJR085C, YPR010C-A), which were not among the loci studied by systematic gene deletion experiments and chemoprofiling [START_REF] Hillenmeyer | The chemical genomic portrait of yeast: uncovering a phenotype for all genes[END_REF][START_REF] Giaever | Functional profiling of the Saccharomyces cerevisiae genome[END_REF]. Strain lacking these genes did, however, not display any discernible

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growth phenotype in plate assays using ethanol/glycerol or acetate as non-fermentable carbon sources (Figure S1C). Since the genes have no obvious paralog, genetic redundancy cannot explain this result. Given that Yjr085c is a mitochondrial protein induced by chemically induced DNA damage, it is possible that its role is limited to a stress response other than processing a non-fermentable carbon course [START_REF] Reinders | Toward the complete yeast mitochondrial proteome: multidimensional separation techniques for mitochondrial proteomics[END_REF][START_REF] Sickmann | The proteome of Saccharomyces cerevisiae mitochondria[END_REF][START_REF] Tkach | Dissecting DNA damage response pathways by analysing protein localization and abundance changes during DNA replication stress[END_REF][START_REF] Lee | Global protein expression profiling of budding yeast in response to DNA damage[END_REF]. Likewise, Ypr010c-a interacts with, among others, Ubi4 (protein degradation) and Nab2 (mRNA export), which are involved in DNA replication stress [START_REF] Tkach | Dissecting DNA damage response pathways by analysing protein localization and abundance changes during DNA replication stress[END_REF][START_REF] Finley | The yeast polyubiquitin gene is essential for resistance to high temperatures, starvation, and other stresses[END_REF][START_REF] Kuhlmann | Structural basis for the molecular recognition of polyadenosine RNA by Nab2 Zn fingers[END_REF]. Yjr085c and Ypr010c-a may therefore exert their activities only under experimental conditions unrelated to carbon metabolism.

Nutritional control of cell fate involves the HDAC Rpd3/Sin3/Ume6 complex

We identified 14 loci for which we detected both mRNA and proteins exclusively in YPA.

This group includes four cases -CSM4, SPR1, SPS4, and RIM4 -thought to be active only in sporulating cells. It is unclear why previous studies did not observe these proteins in dividing cells as well, but it might be due to cell culture protocols and strain backgrounds. Csm4 localizes to the nuclear envelope and is required for the mobility of telomeres during meiosis, and Sps4 was initially reported to be a meiosis-specific gene induced during middle sporulation [START_REF] Conrad | Rapid telomere movement in meiotic prophase is promoted by NDJ1, MPS3, and CSM4 and is modulated by recombination[END_REF][START_REF] Garber | The SPS4 gene of Saccharomyces cerevisiae encodes a major sporulation-specific mRNA[END_REF]. In SK1, we find that the CSM4 and SPS4 are weakly transcribed in YPA and strongly induced when cells have initiated meiosis (SGV; www.germonline.org). This low level of expression might be enough for the proteins to accumulate. The detection of Spr1 in YPA is unexpected because the protein acts during late sporulation where it renders spores thermoresistant [START_REF] Muthukumar | The Saccharomyces cerevisiae SPR1 gene encodes a sporulation-specific exo-1,3-beta-glucanase which contributes to ascospore thermoresistance[END_REF]. Moreover, tiling array data show that SPR1 is only partially expressed in

YPA from what appears to be an internal transcription start site (SGV via www.germonline.org), and it is possible that the overlapping antisense lncRNA SUT785 contributes to SPR1's mitotic repression. Perhaps the fortuitous expression of a shorter mRNA or mRNA levels below our threshold level of detection are sufficient to allow for the accumulation of a truncated or a full-length version of Spr1 in YPA. RIM4 is induced in YPA as opposed to an earlier report where neither mRNA nor protein were detected in this medium [START_REF] Soushko | An RNA-binding protein homologue that promotes sporulation-specific gene expression in Saccharomyces cerevisiae[END_REF]. Both studies used the SK1 strain and YPA pre-sporulation medium but work by Soushko and Mitchell was done in an SK1-derived background lacking the GAL80 regulator, which somehow may influence RIM4 expression. The upstream region of RIM4 contains URS1 motifs and was found to interact with Ume6 in vivo [START_REF] Harbison | Transcriptional regulatory code of a eukaryotic genome[END_REF][START_REF] Soushko | An RNA-binding protein homologue that promotes sporulation-specific gene expression in Saccharomyces cerevisiae[END_REF]. Moreover, we observe that the RIM4 mRNA is de-repressed in ume6 mutant cells cultured in YPD and YPA. While the data imply that RIM4 is transcriptionally controlled like a typical early meiotic gene [START_REF] Mitchell | Control of meiotic gene expression in Saccharomyces cerevisiae[END_REF],

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our results beg the question why RIM4 is induced in YPA when the Rpd3/Sin3/Ume6 repressor complex is still active. It is known that acetate triggers Ume6's degradation and negatively influences its ability to bind DNA, which perhaps enables Rim4 to accumulate during vegetative growth in respiring cells (at least in a genetic background that sporulates efficiently) [START_REF] Mallory | Gcn5p-dependent acetylation induces degradation of the meiotic transcriptional repressor Ume6p[END_REF][START_REF] Mallory | Meiosis-Specific Destruction of the Ume6p Repressor by the Cdc20-Directed APC/C[END_REF][START_REF] Law | Acetylation of the transcriptional repressor Ume6p allows efficient promoter release and timely induction of the meiotic transient transcription program in yeast[END_REF]. It is not clear why Rim4 accumulates in YPA, because it does not seem to be involved in producing energy with a non-fermentable carbon source (see Figure 4B). However, the protein is required for the complex and highly stage-specific translational control of many proteins during meiosis via binding to 5'-UTRs, including some that are important for M-phase [START_REF] Berchowitz | A developmentally regulated translational control pathway establishes the meiotic chromosome segregation pattern[END_REF]. We therefore speculate that Rim4's early accumulation in respiring cells during late-log phase enables the SK1 background to rapidly progress through the meiotic differentiation pathway once it is triggered by the onset of Ime1 expression [START_REF] Neiman | Sporulation in the budding yeast Saccharomyces cerevisiae[END_REF].

We have recently reported that the Rpd3/Sin3/Ume6 complex represses early meiotic transcript isoforms with extended 5'-UTRs during vegetative growth [START_REF] Lardenois | The conserved histone deacetylase Rpd3 and its DNA binding subunit Ume6 control dynamic transcript architecture during mitotic growth and meiotic development[END_REF]. This, together with the observations reported here, raises the interesting possibility that the progressive degradation of Ume6 during the switch from fermentation to respiration and sporulation, simultaneously de-represses the 5'-UTR interacting protein Rim4 and long transcript isoforms that are bound by the protein [START_REF] Berchowitz | A developmentally regulated translational control pathway establishes the meiotic chromosome segregation pattern[END_REF].

Conclusion

The present study reveals complex regulatory processes affecting mRNA and protein stability that underlie the transition from fermentation to respiration in sporulation-competent diploid yeast cells. Moreover, it provides further insight into the role of the conserved HDAC complex Ume6/Rpd3/Sin3 in coordinating metabolic functions with stress response and cellular differentiation in a simple eukaryote. Given the recent major advances in proteomics [START_REF] Hebert | The one hour yeast proteome[END_REF] the next goal will be to determine the comprehensive proteome of the entire diploid yeast life cycle, and to integrate this information with the transcriptome, functional annotation data and protein networks.

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  for one hour at room temperature, washed with PBS and incubated for 60 minutes at 37°C with a mouse anti-V5 monoclonal antibody (Invitrogen, USA) diluted 1:200 in 1x PBS 0.1% Tween 20. Cells were washed three times for 3 minutes each with 1x PBS 0.1% Tween 20 and incubated with a secondary Alexa Fluor 488 Goat Anti-Mouse antibody (Invitrogen)A C C E P T E D M A N U S CR I P T diluted 1:10000 in 1x PBS. Cells were again washed and mounted in Antifade solution (Vector Laboratories) containing 4′,6-diamidino-2-phenylindole (DAPI; 1 μg/ml) to stain DNA. Acquisition of images was done using a Zeiss AxioImager M1 microscope equipped with an AxioCam MRC5 camera controlled by AxioVision 4.7.1 software using standard settings (Zeiss, Le Pecq, France).

  corresponding mRNAs: YMR173W-A almost entirely overlaps DDR48; weak expression of mRNA was observed for YBL071C, YCR013C, YHR056W-A, YLR339C, YMR075C-A and YPR197C and YMR304C-A appears to be co-expressed with the partially overlapping SCW10 locus. In the cases of YAL016C-B, YBR191W-A, YKL165C-A, YLR163W-A, YMR052C-A, YNL319W and YPR136C no expression signals were observed with tiling arrays; these cases A C C E P T E D M A N U S C R I P T are either false positive peptide scores or the transcript levels are below the threshold level of detection of tiling arrays.

Figure 1 .

 1 Figure 1. The proteome of fermentation and respiration. (A) Protein length in amino acids (y-axis) is plotted against the gel slice number (x-axis) for the color codes samples as shown (B) The number of proteins reproducibly found in gel slices 1-30 are plotted. (C) Pie charts show the percentages of proteins encoded by all open reading frames (ORFs) or verified ORFs for riche medium with glucose (YPD) in grey, for rich medium with acetate (YPA) in red, and for the total number of proteins in violet. (D) Venn diagrams show the numbers of proteins detected in sample replicates. (E) Color coded bubble charts show the patterns of detection (0 means not detected, 1 signifies that the protein was detected in one replicate, 2means that it was detected in both replicates) in YPD (y-axis) versus YPA (x-axis). Data are given in grey for all ORFs, in green for verified ORFs, in blue for uncharacterized ORFs, and in red for dubious ORFs. (F) A bubble chart shows the detection pattern in YPD replicates (xaxis) versus YPA samples (y-axis) for proteins known to localize to mitochondria like in panel E.

Figure 2 .

 2 Figure 2. The correlation between gene expression and protein detection. (A) A colorcoded bar diagram shows median log microarray signal intensities (y-axis) for proteins that were not detected (0), detected in one replicate (1), or detected in both replicates (2) in YPD and YPA (x-axis) in grey and red, respectively. An orange line shows the background (BG) threshold signal. (B) log2-transformed intensity values (y-axis) are plotted against the number of gel slices (x-axis) proteins were detected in for YPD (red) and YPA (blue). (C) A bar diagram shows the average log intensity (y-axis) versus the average number of peptides identified (x-axis) in YPD (grey) and YPA (red). The standard deviation is indicated. (D) A heatmap shows the microarray gene expression profiles (1-11) ordered over peak signals.

Figure 3 .Figure 4 .

 34 Figure 3. Ume6-target gene profiling and YPD protein/mRNA detection patterns. (A) A color-coded heatmap indicates predicted URS1 motifs (grey squares), and de-repression in a vegetatively growing ume6 mutant (up in ume6 ). A green to pink color scale indicates gene expression measured by Yeast 2.0 in rich media (YPD, YPA), and three time points in sporulation medium (SPII 4 hours, 8 hours and 10 hours) in wild-type (SK1 MATa/ ) versus ume6 mutant strains (SK1 MATa/ ume6). The color change was set to be consistent with the tiling array expression background. A blue to red color scale indicates the expression level determined with tiling arrays in YPD and YPA. For proteomic data, the number of bands (from 0 to 30) within which a given protein was detected is summarized in bar diagrams given in light grey. GeneChip data (Yeast Genome 2.0), tiling array data (Sc_tlg), and mass spectrometry data (DIPP) are indicated at the bottom. Scales for Yeast Genome 2.0 (left) and Sc_tlg data (right) are given at the bottom. (B) Data are shown for selected genes that show similar patterns as the reference genes. (C)

Figure 5 .Figure S1 .

 5S1 Figure 5. Correlating gene expression, protein detection and protein localization patterns. (A) A color-coded heatmap like in Figure 3 is shown for 14 target genes with an expression and a proteomic pattern specific for YPA. (B) The outcome of a plate patch assay on YPD (fermentation) versus YPEG (respiration) is shown for positive and negative controls in red and green, respectively and new targets given in black. (C) Immunofluorescence data are shown for tagged Rim4 (Rim4 3V5 ) in cells cultured in YPD and YPA. Cells were visualized using differential interference contrast microscopy (DIC). DNA and proteins were visualized using a fluorophor (DAPI) and an antibody against the tag (Rim4 3V5 ), respectively, and both are displayed together to detect nuclear signals and cytoplasmic signals (Merge).

Figure S2 .

 S2 Figure S2. (A) A color-coded bar diagram shows the number of amino acids in the primary sequence of proteins (y-axis) encoded by all ORFs (violet) or by ORFs not identified by proteomics for which we observe expression values greater than the background (BG=5.99, xaxis). The inter-quartile range (IQR, 25 th and 75 th percentiles) is indicated in the graphs in all panels. Wilcoxon rank sum test with continuity correction was used to assess median differences. (B) A bar diagram shows the percentage of genes associated with annotation terms for ORFs above expression background not identified by proteomics (dark grey) and all ORFs (light grey). p-values computed with hypergeometric law and corrected for multiple testing with B&H are indicated above the bars. Only Biological Process annotations with a pvalue < 1% were selected.

Table 1 .

 1

	Annotation		YPD1	YPD2		YPA1	YPA2
	All yeast ORFs		4416	4079		4379	4400
	Verified ORFs		3833	3525		3793	3804
	Uncharacterized ORFs	444	396		441	437
	Dubious ORFs		117	139		126	142
	Table 2
	YPD	YPA	all ORFs	verified	uncharacterized	dubious
	0	0	1090			356		266	491
	0	1	368			198		74	90
	0	2	175			131		30	13
	1	0	381			207		77	85
	1	1	504			335		85	59
	1	2	547			446		82	16
	2	0	94			67		20	7
	2	1	415			331		61	20
	2	2	3011			2769		215	20
	Table 3
	YPD	YPA	average length (all ORFs)	average length (verified ORFs)	median length (all ORFs)	median length (verified ORFs)
	0	0	234		309			126	247
	0	1	308		376			212	335
	0	2	416		443			370	412
	1	0	327		393			229	346
	1	1	388		434			349	417
	1	2	482		502			416	447
	2	0	361		389			338	353
	2	1	471		496			422	451
	2	2	576		581			468	476
	Table 4
	ORF	length (aa)	YPD (log2)		YPA (log2)	2 |YPA-YPD|	N o slices in YPA	N o slices in YPA
	MDH2	377	8.14		13.37	37.5	1.5	29
	ICL1	557	8.19		13.81	49.2	17	29
	MLS1	554	6.63		13.46	113.8	12	29.5
	SDH1	640	10.07		12.83	6.8	17.5	26
	FUM1	488	11.19		13.07	3.7	18	26
	ACO1	778	12.01		13.17	2.2	27.5	30
	MDH1	334	11.29		12.77	2.8	15.5	23
	CIT1	479	9.76		13.2	10.8	20.5	28
	IDH1	360	11.56		13.14	3.0	16.5	21
	KGD1 1014	10.4		12.57	4.5	21	27.5

ACCEPTED MANUSCRIPT

Acknowledgments

We thank Olivier Collin for providing us with access to the GenOuest bioinformatics infrastructure, Olivier Sallou for system administration, Emmanuelle Com for uploading the proteome data to the PRIDE repository, and Angelika Amon and Luke Edwin Berchowitz for tagged Rim4. Yuchen Liu was funded by an Fondation pour la Recherche Médicale 4 th year PhD fellowship. This work was supported by the grants National Institute of General Medical Studies (P41 GM103533) at the US National Institutes of Health to M. R. and Inserm Avenir R07216NS, and Région Bretagne CREATE R11016NN awarded to M. P.

Highlights

We determine the yeast proteome of fermentation and respiration in diploid cells We integrate genome-wide RNA profiling data and large-scale protein detection data We identify genes likely regulated at the post-transcriptional level We report novel Rpd3/Ume6 target genes We show how acetate primes cells to enter sporulation by activation of meiotic genes


Baptiste Polteau

Franck Tessier

François Cheviré

Laurent Cario

Fabrice Odobel

Stéphane Jobic

Synthesis of Ni-poor NiO nanoparticles for p-DSSC applications

Keywords: Synthesis, Ni-poor NiO, Nitrogen doping, Nanoparticles

To improve the performances of p-Dye Sensitized Solar Cell (p-DSSC) for the future, the synthesis of modified p-type nickel oxide semiconductor, commonly used as photocathode in such devices, was initiated with Ni 3 O 2 (OH) 4 as precursor. This specific nickel oxyhydroxide was first characterized by X-ray photo-electron spectroscopy and magnetic susceptibility measurements.

Then its thermal decomposition was thoroughly studied in order to control the particles size of the as-prepared NiO nanopowders. Low temperature decomposition in air of this precursor allows the formation of Ni 1-x O nanoparticles with a large amount of Ni vacancies and specific surface areas up to 250 m 2 .g -1 . Its ammonolysis at 250°C leads to nanostructured N-doped NiO (NiO:N) materials.

Introduction

Over the past decade, p-type semiconductors (p-SC) have known a renewed interest for applications for light-emitting diodes, transistors, solar cells, etc. In particular, since the achievement of the first Dye Sensitized Solar Cells (DSSC) by Grätzel in 1991 [1], a new generation of solar cells has been developed where the n-type SC is replaced by a p-type one [2]. Such devices are based on the photoinjection of holes instead of electrons in the external circuit. To date nickel oxide (NiO) is the reference p-type semiconductor for p-DSSC applications [3]. However yields are still far from those of n-DSSC and many studies aim to replace NiO by nanoparticles of CuAlO 2 , CuGaO 2 , CuCrO 2 or NiCo 2 O 4 to achieve higher photovoltaic performances [4][5][6][7]. Following our recent synthesis of Ndoped ZnO nanoparticles with large amount of Zn vacancies (up to 20%) and stabilization of p-type charge carriers [8], we focus on the preparation of Ni-poor NiO and N-doped NiO nanoparticles to improve the p-type semiconductivity of NiO. Its origin is still not totally elucidated but is commonly attributed to a nickel non-stoichiometry inducing a Ni 3+ /Ni 2+ mixed valence in Ni 1-x O.

The nanostructuration will also be a key factor to optimize the p-SC/Dye contact surface. Indeed, to integrate perfectly a p-SC in a p-DSSC, a high specific surface area is necessary to coat the largest amount of dye molecules on NiO nanoparticles to favor the exchange of charge carriers and high photogenerated current. The nanostructuration could also promote a change in the chemical formulas with variation of the Ni 3+ /Ni 2+ ratio that governs the conducting behavior. Moreover, the substitution of one nitrogen atom for one oxygen atom should allow the increase of the concentration and the mobility of positive charge carriers (O 2- N 3-+ h + ) in order to optimize the injection of holes in the electrical circuit of the p-type cell, and thus its performances. We discuss in this work the thermal decomposition of a nickel oxyhydroxide precursor under air and ammonia that leads to nanostructured Ni-poor NiO and N-doped NiO respectively with very high specific surface areas. Plus software and all data refinements were carried out with the Fullprof suite software [9].

Chemical analysis.

Nitrogen and oxygen contents were determined with a Leco TC-600 analyzer using the inert gas fusion method. Nitrogen was detected as N 2 by thermal conductivity and oxygen as CO 2 by infrared detection.

Density measurements.

The Micromeritics AccuPyc 1330 system was used for density measurements by pycnometry under He pressure.

Specific Surface Area measurements. The Brunauer-Emmet-Teller (BET) specific surface area measurements were carried out with a Micromeritics FlowSorb II 2300 instrument using a mixture of N 2 /He (30% / 70%) as gas analyzer.

X-ray Photoelectron Spectroscopy. XPS spectra (Mg K-L3 = 1253.6 eV) were collected in a Leybold-Heraeus ultrahigh vacuum environment with an analyzer operating in the constant pass energy mode (31.5 eV). All spectra were calibrated in energy using C 1s = 284.7 eV as a reference.

Transmission electron microscopy. Transmission electron microscopy (TEM) analyses were realized on a Hitachi H9000NAR (300 kV, Scherzer resolution 0.18 nm) microscope.

Magnetic measurements. Magnetic measurements were performed on a Quantum Design SQUID magnetometer (MPMS XLS 5). The data were collected in the temperatures range from 2 to 300 K under a magnetic field of 5000 Oe.

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Synthesis of nickel oxyhydroxide nanoparticles

The nickel oxyhydroxide precursor (Ni 3 O 2 (OH) 4 ) was prepared as previously reported [10]. A strong alkaline solution was prepared by dissolving 5.1 g of sodium hydroxide (NaOH, 98 % Sigma-Aldrich) in 36 mL of a sodium hypochlorite solution (NaClO, 5 % available chlorine Acros) used as an oxidizing agent [11] The resulting black suspension was kept under stirring for 90 min at room temperature. The precipitate was separated from the solution by centrifugation at 2500 rpm for 5 min, dispersed in distilled water and separated again by centrifugation. These dispersion/separation cycles were repeated 6 times in order to remove any impurities and the resulting precipitate was dried in an oven at 70 °C overnight. Finally, the product was crushed in an agate mortar to obtain the nickel oxyhydroxide powder precursor which was immediately stored in a desiccator to avoid any surface hydration. ). Then, the temperature was raised in a the 200-350°C range with a heating rate of 10°C.min -1 and dwelled for 30 minutes. Finally, the tubular furnace was cooled to room temperature by turning it off and the product was stored in a desiccator. Ni 1-x O materials prepared at X °C are hereafter labelled as NiO-N-X.

Synthesis of nickel-poor nickel oxide

Results and discussion

Characterization of nickel oxyhydroxide nanoparticles

In order to prepare undoped and nitrogen-doped nickel oxide nanoparticules with a large amount of nickel vacancies, we have proceeded as in our previous work on N-doped ZnO (ZnO:N) [8] and highly Zn-deficient ZnO nanoparticles [12] with zinc peroxide as precursor. Indeed, like ZnO 1) suitable for the synthesis of nickel oxide nanoparticles [18]. This agrees with particles size estimated to be around 2-3 nm from a TEM analysis (Fig. 3a), that explains the overall feature of the X-ray diffraction pattern. becomes green at temperatures typically above 800°C as expected for stoichiometric NiO. Figure 5 shows the evolution of the density of prepared "NiO" samples versus the decomposition temperature of Ni 3 O 2 (OH) 4 . The densities measured for samples prepared up to 600 °C deviates from the theoretical density of stoichiometric bulk NiO (6.81). For samples prepared below 350°C the density is drastically lower than that expected and rises strongly with increasing temperature.

Thermal decomposition of nickel oxyhydroxide nanoparticles

Decomposition of nickel oxyhydroxide in air

For samples prepared at higher temperature ( 400 °C), the density is still lower than expected but (Table 1). TEM images confirm the nanostructuration of NiO-250 (similar case to Ni 3 O 2 (OH) 4 ) as agglomerates of nanoparticles (2-3 nm). As observed by XRD, the increase in temperature allows the further crystallization of NiO samples with larger particles sizes. In fact, NiO-500 is still nanostructured with dispersed particles sizes around 10-15 nm. According to Table 1 the crystallites sizes are very close to the particles sizes determined by TEM analyses. In order to optimize the performances of NiO in p-DSSCs, high specific surface area is necessary to coat a maximum number of dye molecules on NiO nanoparticles. Thus, we have also measured the specific surface areas of Ni-poor NiO samples. The results demonstrate for NiO-250 a very high specific surface area close to 250 m 2 .g -1 that is 3 times higher than that of Inframat NiO ( 80 m 2 .g -1 ) commonly used as a reference in p-DSSCs. The thermal treatment at 250 °C maintains the high specific surface area of the nickel precursor despite the structural change from Ni 3 O 2 (OH) 4 to NiO. Note that the specific surface area drops to 42 m 2 .g -1 for NiO-500, due to the increase in particles sizes.

Decomposition of nickel oxyhydroxide under ammonia

To our knowledge, the stabilization of a nitrogen-doped nickel oxide powder has not been evidenced yet. Only theoretical study [20] or N-doped NiO film [21] have been recently reported.

However, the study of the decomposition of lattice [8]. The nitrogen content in NiO-N-250 was determined to be 0.8 wt. % whereas the nitrogen amount in NiO-250 and Ni 3 O 2 (OH) 4 was measured to 0.11 and 0.19 wt. % respectively. The morphology of NiO-N-250 was investigated by TEM analysis (Fig. 3d), XRD and BET specific surface area (Table 1). The results are quite similar to those of NiO-250, i.e. nanoparticles of 2-3 nm in diameter are observed in agreement with the calculated crystallites sizes. An important result is the conservation under nitridation of the high specific surface area that shifts from 250 to 200 m 2 .g -1 from NiO-250 to NiO-N-250.

Conclusion

The thermal decomposition of nickel oxyhydroxide nanoparticles was thoroughly studied in order to

Figures & table captions

  diffraction. X-Ray diffraction patterns were recorded in the 5-120° 2 range on a Panalytical X'PERT Powder (Cu K , 40 kV, 40 mA) diffractometer. In situ temperature (30-700 °C range) X-Ray diffraction patterns were recorded in the 10-90 ° 2 range on a Bruker D8 Advance diffractometer using Cu K-L2,3 radiation. All phase analyses were performed using the HighScore

600 mg of Ni 3 O 2 ( 2 . 4

 3224 OH)4 precursor placed in an alumina crucible was decomposed in a muffle furnace in air in the temperature range from 250 to 800°C. These temperatures were dwelled for 2 hours with a heating rate of 10 °C.min -1 . The samples were cool down to room temperature after turning off the furnace and stored in a desiccator. Ni 1-x O materials prepared at X °C are hereafter labelled as NiO-X. Synthesis of nitrogen doped nickel oxide 200 mg of Ni 3 O 2 (OH) 4 precursor powder was placed in a tubular furnace under N 2 flow. The furnace was purged during 10 minutes before introducing NH 3 flow (10 L.h -1

Figure 4

 4 Figure 4 represents the temperature evolution of in situ XRD patterns recorded during the

  evolves linearly (see the dashed line) until reaching the theoretical density of stoichiometric nickel oxide at 750°C. The low density measured for samples prepared below 600°C could indicate the formation of nickel oxide nanoparticles deficient in nickel (i.e. Ni 1-x O) similarly as what was observed during the preparation of zinc deficient zinc oxide [12]. To estimate the amount of nickel vacancies the following equations were used (2, 3): with d exp the measured density; Z the atomic number; M comp the experimental molar weight of the compound; N A the Avogadro's number; V ref the refined cell volume; M O and M Ni the oxygen and nickel molar weights respectively. The density value measured for Ni 1-x O prepared at 250 °C (NiO-250) would almost correspond to a 50 % nickel deficient Ni 1-x O material. For NiO-500, the amount of nickel vacancy is estimated to be 20%. Such low densities are correlated to a high specific surface area and low particles size (see below) and the strong propensity of the material to exhibit oxygen or hydroxide terminated surfaces. Due to the nanoscale of the particles, surface moisturization and carbonatation are likely to happen leading to lower measured densities, thus the estimated vacancy rate is overestimated. A Rietveld refinement on NiO-500 leads to a lower value ( 10%) taking more account of the "bulk" feature of the nanoparticles. The effective vacancy rate is probably intermediate between those M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT values. Nickel oxyhydroxide seems to be an ideal candidate to prepare Ni-poor NiO samples with tunable Ni deficiency via the control of the decomposition temperature of the precursor. The characterization of the nickel oxides morphology was investigated by TEM microscopy (Fig. 3b, 3c), BET specific surface areas and XRD to calculate the crystallites size by Rietveld refinement

  Ni 3 O 2 (OH) 4 under ammonia flow allows the stabilization of N-doped NiO at low temperatures (see NiO-N-250). Indeed, Figure 6 represents the evolution of XRD patterns of Ni 3 O 2 (OH) 4 nitridation at temperatures ranging from 200 to 350 °C. The investigated temperatures range is narrower than the one for previous Ni 1-x O materials because of the rapid reduction of nickel under ammonia at T 350°C. At temperatures below 250 °C a M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT NiO-type cubic phase is formed with very broad peaks. When the nitridation temperature is over 300 °C, a shoulder on the (200) reflection of NiO appears at ~ 44.5° 2 that corresponds to the most expected intense peak of Ni 3 N (111 reflection) (JCPDS file 89-5144). Over 350 °C, Ni 3 N is unambiguously formed. For higher temperatures, nickel cations are definitely reduced into elemental nickel. To assert the insertion of N in NiO-N-250, N(1s) XPS experiments were carried out. Figure 7 displays for NiO-250 no characteristic signal of nitrogen, whereas NiO-N-250 exhibits a N 1s BE peak at 398.2 eV. It proves unambiguously the presence of nitrogen into the NiO host

  control the particles size of the as-prepared NiO nanopowders. The determination of the Ni 3+ /Ni 2+ ratio by XPS and magnetism in the nickel oxyhydroxide confirms the Ni 3 O 2 (OH) 4 formulation. The decomposition of this compound in air at temperatures lower than 600 °C leads to nonstoichiometric NiO nanoparticles with tunable nickel vacancies concentration and high specific surface areas. Additionally, the stabilization of nitrogen-doped NiO is possible by low temperature nitridation of Ni 3 O 2 (OH) 4. All these modified-NiO materials aim to be tested soon in a p-DSSC.

Table 1 .

 1 Summary of the specific surface areas, crystallites and particles sizes for Ni 3 O 2 (OH) 4 , NiO-250, NiO-500 and NiO-N-250 samples.

Fig. 1 .

 1 Fig. 1. Powder X-ray diffraction pattern of nickel oxyhydroxide synthesized by precipitation route.

Fig 2 .Fig 3 .

 23 Fig 2. a) Ni 2p XPS spectrum of Ni 3 O 2 (OH) 4 ; b) Magnetic properties of Ni 3 O 2 (OH) 4 .

Fig 4 .

 4 Fig 4. In situ XRD study patterns of the decomposition of Ni 3 O 2 (OH) 4 in air. The interval of

Fig 5 .

 5 Fig 5. Evolution of the density and Ni vacancies amount in prepared Ni 1-x O samples versus

Fig 6 .

 6 Fig 6. X-ray diffraction patterns of N-doped NiO materials issued from the ammonolysis of Ni 3 O 2 (OH) 4 at 200, 250, 300 and 350°C .

Fig 7 .

 7 Fig 7. N 1s XPS spectra of NiO-250 and NiO-N-250 samples.

Fig. 1 .Fig 2 .Fig 3 .Fig 4 .Fig 5 .Fig 6 .Fig 7 .

 1234567 Fig. 1. Powder X-ray diffraction pattern of nickel oxyhydroxide synthesized by precipitation route.

  All the very broad diffraction peaks can be attributed to the Ni 3 O 2 (OH) 4 phase (JCPDS file 06-0144) that crystallizes in a structure type related to that of lamellar Ni(OH) 2[16]. To determine the Ni 3+ /Ni 2+ ratio and thus to confirm the chemical composition of Ni 3 O 2 (OH) 4, X-ray photoelectron spectroscopy experiments at the Ni threshold and magnetic susceptibility measurements were carried out. Figure2depicts the Ni 2p XPS spectrum of Ni 3 O 2 (OH) .1 eV are assigned to Ni 0 , Ni 2+ and Ni 3+ species, respectively[17]. The peak deconvolution of Ni 2p 3/2 state (Fig.2a) clearly indicates two oxidation states of nickel; Ni(II) and Ni(III). Ni 3+ 2p 3/2 exhibits a BE at 855.8 eV with a peak surface area of 63 %, while Ni 2+ 2p 3/2 exhibits a BE at 854.3 eV with a peak surface area of 37 %. The resulting Ni 3+ /Ni 2+ ratio equals 2. This ratio can be also estimated from magnetism measurements (Fig.2b) with the value of the Curie constant (C) calculated from the curve 1/ = f(T). Using the slope coefficient represented by a grey dashed line, we have determined a Curie constant of 0.63 (SI units) and a magnetic moment eff = (8*C) = 2.24 B . Considering the experimental magnetic moments ( eff ) of Ni 2+ and Ni 3+ (3.2 and 1.7 B respectively), we calculate the ratio Ni 3+ /Ni2+ 2. According to the nickel oxyhydroxide formulation NiO x (OH) y , the formula is well balanced with two Ni 3+ for one Ni 2+ in agreement with the Ni 3 O 2 (OH) 4 chemical composition identified from the JCDPS database (Fig.1). The Ni 3 O 2 (OH) 4 powder has also a high specific surface area of 234 m 2 .g -1 (Table

	ACCEPTED MANUSCRIPT
	nickel (oxy)hydroxide compounds (Ni 2+	1-x Ni 3+ x O M A N U S C R I P T	2 ,
	A C C E P T E D

x (OH) 2-x with 0 x 1), are very unstable oxygenrich precursors which decompose at low temperature ( 400°C) into the cubic-type NiO structure

[13][14][15]

. Consequently, the thermal decomposition of Ni 3+ -rich precursors such Ni 3 O 2 (OH) 4 is expected to favor the presence of Ni 3+ and thus to promote the stabilization of nickel vacancies in the resulting nickel oxide. The observed XRD pattern of as-prepared oxyhydroxide is displayed in Figure 1. 4 and its inverse susceptibility versus temperature. The oxidation states of nickel have been determined from the position in energy of Ni 2p 3/2 XPS peak. Namely, binding energies (BE) of 852.6, 854.6 and 856

Table 1 .

 1 Summary of the specific surface areas, crystallites and particles sizes for Ni 3 O 2 (OH) 4 , NiO-250, NiO-500 and NiO-N-250 samples.

	Sample	Specific surface area (m 2 .g -1 )	Crystallite size (nm)	Particle size (nm)
	Ni 3 O 2 (OH) 4	234	-	2-3
	NiO-250	247	2.0	2-3
	NiO-500	42	9.1	10-15
	NiO-N-250	198	1.8	2-3

Acknowledgement

We are grateful to Eric Gautron, Jonathan Hamon (IMN) and Thierry Guizouarn (ISCR) for their help in TEM, XPS and magnetic studies respectively.

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