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Transcriptome profiling of Issatchenkia orientalis under ethanol stress

Transcriptome profiling of Issatchenkia orientalis under ethanol stress Issatchenkia orientalis, a non-Saccharomyces yeast that can resist a wide variety of environmental stresses, has potential use in winemaking and bioethanol production. Little is known about gene expression or the physiology of I. orien- talis under ethanol stress. In this study, high-throughput RNA sequencing was used to investigate the transcriptome profile of I. orientalis in response to ethanol. 502 gene transcripts were differentially expressed, of which 451 were more abundant, and 51 less abundant, in cells subjected to 4 h of ethanol stress (10% v/v). Annotation and statistical analyses suggest that multiple genes involved in ergosterol biosynthesis, trehalose metabolism, and stress response are differentially expressed under these conditions. The up-regulation of molecular chaperones HSP90 and HSP70, and also genes associated with the ubiquitin–proteasome proteolytic pathway suggests that ethanol stress may cause aggregation of misfolded proteins. Finally, ethanol stress in I. orientalis appears to have a nitrogen starvation effect, and many genes involved in nutrient uptake were up-regulated. Keywords: Issatchenkia orientalis, RNA-Seq, Transcriptome, Ethanol stress, Wine fermentation The non-conventional wine yeast Issatchenkia orien- Introduction talis was first described in 1960 but was reclassified to Fruit wines are fermented alcoholic beverages that derive P. kudriavzevii in 1965 (Kurtzman et  al. 2008). Several their flavors from raw materials (fruits, and often flowers I. orientalis strains produce ethanol and have higher and herbs) as well as from the fermentation process. Two thermotolerance, salt tolerance, and acid tolerance than distinct yeasts are usually involved in the production of S. cerevisiae (Isono et  al. 2012; Koutinas et  al. 2016). a savory and pleasant fruit wine. The wine yeast Saccha- Because of its resistance to multiple stress factors, I. ori- romyces cerevisiae is primarily responsible for alcoholic entalis has potential application in bioethanol production fermentation and the synthesis of secondary metabolites, and succinic acid production (Kitagawa et al. 2010; Kwon while non-Saccharomyces yeasts or non-conventional et al. 2011; Xiao et al. 2014). wine yeasts contribute additional flavor, texture, and High-throughput RNA sequencing (RNA-Seq) is now nutritional qualities (Archana et  al. 2015). The role of routinely used to generate global transcription profiles, non-Saccharomyces wine yeasts in fruit wine fermenta- often to compare gene expression under different con - tion has attracted increasing interest (Ciani et  al. 2010). ditions. Many studies have used RNA-Seq to examine Several studies have focused on multi-strain fermenta- transcription in S. cerevisiae and the fission yeast Schizos- tion and mixed yeast culture (Fleet 2003; Giovani et  al. accharomyces pombe in response to environmental shifts 2012; Sadoudi et al. 2012), and some non-Saccharomyces (Kasavi et al. 2016; Lackner et al. 2012; Lewis et al. 2014). yeasts have been suggested for use in mixed starter cul- However, gene expression in I. orientalis has not yet tures with S. cerevisiae (Masneufpomarede et al. 2015). been studied. In particular, the underlying mechanisms that allow I. orientalis to tolerate ethanol have not been explored, nor have they been compared with those in S. cerevisiae. *Correspondence: wuzufang@nbu.edu.cn Yingjie Miao and Guotong Xiong contributed equally to this work In this study we used RNA-Seq to investigate changes Department of Food Science and Engineering, School of Marine Sciences, in the gene expression profile of I. orientalis under Ningbo University, Ningbo 315211, People’s Republic of China © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Miao et al. AMB Expr (2018) 8:39 Page 2 of 13 ethanol stress. We identified a wide variety of differen - trehalose (Sigma-Aldrich) (Kitichantaropas et  al. 2016; tially expressed genes, some of which may play important Mahmud et al. 2009). roles in the stress response. Determination of ergosterol concentration Materials and methods Issatchenkia orientalis was cultured in medium with 10% Yeast strains, media, and growth conditions ethanol for 0, 4, 12, 24, and 48 h. Cells were collected by Issatchenkia orientalis strain CBS 12547 was originally centrifugation, washed with ultrapure water, then frozen isolated from tropical fruit and food sources, and is in liquid nitrogen and freeze-dried at − 20 °C. 100 mg of involved in the fermentation of some traditional Afri- dried cells were resuspended in 3 mL ethanol containing can foods (Greppi et  al. 2013; Pedersen et  al. 2012). The 25% potassium hydroxide (m/v; 25  g KOH dissolved in strain was maintained in the Food Biotechnology Labo- 35 mL pure water, add ethanol to 100 mL) and incubated ratory at Ningbo University. Yeast was initially cultured at 85 °C for 1 h. The entire sample was mixed with 3 mL for 24  h in YPD medium (1% yeast extract, 2% peptone, n-heptane and extracted by vortexing for 3  min. Finally, and 2% glucose) at 30 °C with agitation at 150 rpm. 1 mL absorbance of the supernatant at 282  nm was measured was withdrawn, added to 100  mL fresh YPD medium, and compared with samples containing known con- and incubated as before until the culture reached expo- centrations of ergosterol (Sigma-Aldrich) (Arthington- nential phase (8  h). For the RNA-Seq experiment, etha- Skaggs et al. 1999). nol was added to a final concentration of 10% (v/v), and incubation continued for another 4  h. Three cultures RNA extraction, library construction and sequencing were treated in parallel with ethanol (TE1/TE2/TE3) and As noted earlier, I. orientalis was cultured in medium three untreated cultures were used as negative controls with 10% ethanol for 4  h before cells were harvested for (T1/T2/T3). Yeast cells were harvested by centrifugation RNA extraction. Total RNA from each sample was iso- at 4 °C, 2000×g and stored at − 80 °C. lated using TRIZOL (Aidlab Biotech, Beijing, China). RNA concentration was quantified using a Qubit RNA Scanning electron microscopy (SEM) Assay Kit and a Q ubit 2.0 Fluorometer (Life Tech- Issatchenkia orientalis was cultured in medium with nologies, CA, USA). RNA integrity and purity were 10% ethanol for 24 h. Cells were collected by centrifuga- evaluated using the RNA Nano 6000 Assay Kit and the tion at 1000×g, 4  °C for 10  min and washed three times NanoPhotometer spectrophotometer (IMPLEN, CA, with physiological saline. Cells were then resuspended USA). in 2.5% glutaraldehyde for 4  h at 4  °C and washed three Construction of cDNA libraries and RNA sequencing times with 0.1 M PBS (pH = 7.4) for 15 min per wash. The were performed by Beijing BioMarker Technologies (Bei- cells were transferred through a series of ethanol solu- jing, China). In brief, poly-A mRNA was isolated using tions (30, 50, 70, 80, 90, 95 and 100%; 10 min each), and poly-T oligomer bound to magnetic beads. mRNA was then through a series of tert-butanol-anhydrous ethanol fragmented using divalent cations at elevated tempera- mixtures (ratio 1:3, 1:1, 3:1, 3:0; 10 min each). Finally, the ture. RNA fragments were copied as cDNA using ran- cells were dried and coated with a gold/palladium alloy dom primers, and second strand cDNA synthesis was (40:60) to a thickness of 10–20  nm and observed with a then performed. Double-stranded cDNAs were ligated to Hitachi S3400N scanning electron microscopy system. a single ‘A’ base and the sequencing adapters. Fragments (200 ± 25 bp) were then separated by agarose gel electro- Determination of trehalose concentration phoresis and selected for PCR amplification as sequenc - Issatchenkia orientalis was cultured in medium with 10% ing templates. Finally, the library was constructed for ethanol for 0, 4, 12, 24, and 48 h. Cells were collected by sequencing on the Illumina HiSeq 4000 sequencing centrifugation and washed with ultrapure water. Col- platform. lected cells were frozen in liquid nitrogen and freeze- dried at − 20 °C. 100 mg of dried cells were resuspended Quality control and read mapping in 1  mL ice-cold 0.5  mol/L trichloroacetic acid solution To obtain high-quality data, raw mRNA-Seq reads were by brief treatment with ultrasound, and then maintained processed using in-house Perl scripts. Reads were dis- in the same solution at room temperature for 45  min carded if they were spoiled by adaptor contamination, in order to extract the trehalose from the cells. 250  µL contained ambiguous (N) base calls, or if more than 10% extract was incubated with 1  mL 80% sulfuric acid solu- of bases had quality values < 30. The minimum acceptable tion containing 0.2% anthrone in a boiling water bath for length was 60 bp to avoid sequencing artifacts. All subse- 5  min. Absorbance at 620  nm was measured and com- quent analyses were based on the filtered data set. Reads pared with samples containing known concentrations of were mapped to the I. orientalis reference genome (NCBI Miao et al. AMB Expr (2018) 8:39 Page 3 of 13 Accession Number: GCA_000764455.1) using TopHat2 Table S1). A Tiangen FastQuant RT Kit (with gDNase) (http://ccb.jhu.edu/software/tophat/index.shtml). Gene and a KAPA SYBR FAST Universal qPCR Kit were used names were assigned to sequences based on matches for reverse transcription and qPCR, respectively. All with the highest score. qPCR reactions were performed using a QuantStudio 7 Flex Real-Time PCR system (Applied Biosystems, Functional annotation Thermo Fisher Scientific). The PCR reaction was con - Issatchenkia orientalis genes were aligned to annotated ducted following the manufacturer’s instructions, and sequences using BLAST (http://blast.ncbi.nlm.nih.gov/ three biological replicates were used in all experiments. Blast.cgi) and the following databases: Nr (NCBI non- Negative controls (template consisting of ultrapure redundant protein sequences, Nt (NCBI non-redundant water) were run for each gene. Run-time control of the nucleotide sequences, Pfam (Protein family), KOG/COG PCR instrument, baseline correction, and determina- (Clusters of Orthologous Groups of proteins), Swiss-Prot tion of Cq values were performed using QuantStudio 7 (manually annotated and reviewed protein sequence Flex Real-Time PCR Software v1.2 (Applied Biosystems, database), protein data bank (PDB), KO (KEGG Ortholog Thermo Fisher Scientific). database), and GO (Gene Ontology). The Blast2GO suite (Götz et al. 2008) was used to assign GO terms for molec- Results ular function, biological process, and cellular component. Intracellular trehalose and ergosterol concentration and SEM imaging Analysis of differential expression Intracellular concentrations of trehalose and ergosterol, To compare gene expression level between conditions, measured after 4, 12, 24 and 48  h of ethanol stress, are the transcript level of each expressed gene was calculated shown in Fig. 1. Compared with unstressed controls, eth- and normalized to fragments per kilobases per million anol-stressed yeast cells contained higher levels of both mapped reads (FPKM) using the formula: compounds. Carbohydrates such as trehalose and glyco- gen are compatible solutes that resist osmotic pressure cDNA fragments FPKM = across the cytoplasmic membrane and prevent yeast cells Mapped fragments million × Transcript Length(kb) from dehydration. Ergosterol is an important component Differential expression analysis of data from the two of the yeast cytoplasmic membrane and is also thought to experimental conditions (ethanol stress vs. control) was be involved in stress response. performed using the DESeq R package (1.10.1). P-values SEM images were captured after ethanol stress for 24 h were adjusted using Benjamini and Hochberg’s approach (Fig. 2). Stressed cells formed large flocs containing hun - for controlling the false discovery rate (FDR). Differen - dreds of connected cells. tially expressed genes (DEGs) were defined as those with fold change > 3 (P < 0.05) and FDR < 0.01. Library construction and RNA‑Sequencing Libraries (NCBI Accession: PRJNA413795) were con- KEGG and GO enrichment analyses for DEGs structed for RNA-Seq from three control samples (T1- KEGG (http://www.genome.jp/kegg/) is a database T3; NCBI Accessions: SRX3277329, SRX3277330 and resource for understanding functions and utilities of SRX3277331), and three 10% ethanol-stressed samples the biological system from molecular-level informa- (TE1-TE3; NCBI Accessions: SRX3277326, SRX3277327 tion, especially large-scale molecular datasets generated and SRX3277328). After setting aside reads with adaptor by genome sequencing. KEGG is often used to tenta- contamination, ambiguous base calls, insufficient length, tively assign functions and other properties to genes. We or unacceptable numbers of low quality base scores, used KOBAS (Mao et  al. 2005) to determine if any dif- 27.15  Gb of high-quality data were obtained with aver- ferentially expressed genes were significantly enriched age quality values ≥ 30 for more than 85% of the reads. in KEGG pathways. To determine which Gene Ontology 76.01% of reads from the control libraries, and 77.17% (GO) categories were statistically overrepresented among of reads from the ethanol-stressed libraries, mapped the DEGs, topGO and Cytoscape version 3.4.0 with to the I. orientalis genome, indicating successful library BiNGO plugin version 3.0.3 (Maere et al. 2005) were used construction. to identify significantly enriched biological networks and to output the results as graphs. Identification of differentially expressed genes Gene expression levels were calculated using FPKM val- Quantitative PCR for selected DEGs ues. As shown in Fig.  3, DEGs detected between con- Real time quantitative PCR (qPCR) primers for selected trol and ethanol stressed transcriptomes were required DEGs were designed using Primer 5.0 (Additional file  1: to meet criteria for fold change > 3 (P < 0.05) as well as Miao et al. AMB Expr (2018) 8:39 Page 4 of 13 Fig. 1 Intracellular concentrations of trehalose and ergosterol in ethanol-stressed I. orientalis. The data were obtained at the indicated times (h) after ethanol was introduced into the culture. Values are represented as mean ± S.D. Three biological replicates were used. a Intracellular trehalose. b Intracellular ergosterol Fig. 2 SEM images of control and ethanol-stressed I. orientalis cells. SEM images were captured after ethanol stress for 24 h. A Control cells at a magnification of 10,000×. B Ethanol-stressed cells at a magnification of 10,000×. C Control cells at a magnification of 20,000×. D Ethanol-stressed cells at a magnification of 20,000× Miao et al. AMB Expr (2018) 8:39 Page 5 of 13 Fig. 3 Distribution of DEGs in ethanol-stressed and control I. orientalis. a Volcano plot showing all DEGs. Dashed lines indicate inclusion criteria for false discovery rate (FDR < 0.01; = line at 2 on the y-axis) and Fold Change (FC > 3; line at ~ 1.6 on the x-axis). Red, more abundant in ethanol-stressed cells; green, more abundant in control cells; black, does not meet inclusion criteria and assumed to be unchanged. b MA plot showing FC vs. FPKM for all DEGs. Colors are as in (a). c Heatmap showing all DEGs. Colors indicate expression levels of DEGs FDR < 0.01. Of 502 transcripts with threefold or greater KEGG pathway analysis change, 451 were more abundant and 51 less abundant DEGs were annotated using KEGG to identify ortholo- in ethanol-stressed cells. With four exceptions, all suc- gous genes, and KOBAS was used to test for statistically cessfully matched with entries in the nr (498), Swiss-Prot significant enrichment of DEGs in KEGG pathways. (379), KEGG (205), or GO (224) databases, yielding a Q-values (Storey 2003) were generated by KOBAS, and total of 498 unique and annotated DEGs. are analogous to P-values in the context of our analysis. Transcript levels for a subset of DEGs in several func- As shown in Fig.  5, DEGs were significantly enriched tional groups were determined by real time quantitative in pathways used for protein processing in endoplas- PCR. The results are shown in Fig. 4. mic reticulum (ko04141, q < 0.01) and meiosis (ko04113 Miao et al. AMB Expr (2018) 8:39 Page 6 of 13 Fig. 4 Normalized transcript levels for selected I. orientalis DEGs determined by RT-qPCR. Transcript levels (fold changes) for DEGs are shown relative to levels in I. orientalis before ethanol stress. Bars represent mean values ± S.D. for three biological replicates. DEGs are grouped by function. a DEGs associated with the ergosterol pathway. b DEGs associated with the trehalose pathway. c DEGs associated with responses to stress and stimulus. d DEGs associated with heat shock proteins (HSPs). e DEGs associated with the ubiquitin–proteasome proteolytic pathway. f DEGs associated with meiosis, sporulation, and ascospore cell wall assembly q < 0.05). Pathways involving lipoic acid metabolism GO annotation and analyses (ko00785) and steroid biosynthesis (ko00100) also had DEGs were annotated and classified by GO category, high enrichment scores, but with q values > 0.05. which are divided into three ontologies: molecular func- tion, cellular component, and biological process. TopGO Miao et al. AMB Expr (2018) 8:39 Page 7 of 13 Fig. 5 KEGG pathways enrichment analysis. The diameter of the circle is proportional the number of DEGs enriched in each pathway. The color of circle represents the q-value for enrichment analysis revealed that several biological process catego- A protein–protein interaction (PPI) network was gen- ries (Table  1) were enriched for DEGs, including car- erated to identify key proteins involved in the response bohydrate metabolism, transmembrane transport, ion made by I. orientalis to ethanol stress (Additional file  2: homeostasis, nuclear or cell division, and process in Figure S1). Five proteins in this network (DSK2, HSP82, response to stress or stimuli. HSA1, BiP, and SMK1) are significantly and differentially Figure  6 shows molecular interaction graphs for expressed. These may play important roles in the stress the three GO ontology classifiers, generated using response. Cytoscape-BiNGO. In the biological process ontology (Fig.  6a), statistically overrepresented GO categories Discussion can be divided into five groups (process in response to Issatchenkia orientalis, a non-Saccharomyces yeast stimuli, protein folding and refolding, sugar transport, that can tolerate a variety of stressful environments, is DNA repair and flocculation). In the molecular function potentially useful in winemaking and bioethanol pro- ontology (Fig. 6b), overrepresented GO categories can be duction. However, it is less tolerant to ethanol than S. divided into four clusters. The largest group consists of cerevisiae (Archana et  al. 2015), and can grow and fer- binding functions, specifically nucleotide binding, pro - ment only when ethanol concentrations are under 10%. tein binding, and sugar binding. Other groups involved In S. cerevisiae, a cluster of environmental stress response ATP hydrolase activities, ubiquitin protein ligase activi- (ESR) family genes have coordinated expression under ties, and sugar transmembrane transport activities. In a variety of stress conditions (Gasch et  al. 2001), and 73 the cellular component ontology (Fig. 6c), the overrepre- genes in the ESR family are up-regulated during ethanol sented GO categories included cell wall, ER, and plasmid stress (Alexandre et al. 2001). In contrast, little is known membrane. about gene and protein expression in I. orientalis under Miao et al. AMB Expr (2018) 8:39 Page 8 of 13 Table 1 Enriched biological process terms of the DEGs Table 1 continued after ethanol stress (KS < 0.05) GO:ID Term Annotated DEGs KS GO:ID Term Annotated DEGs KS GO:0031349 Positive regulation of 26 6 0.047 defense response GO:0005991 Trehalose metabolic process 19 7 0.0018 GO:0052510 Positive regulation by organ- 26 6 0.047 GO:0048284 Organelle fusion 13 2 0.0025 ism of defense response of GO:0015833 Peptide transport 14 7 0.0039 other organism involved in symbiotic interaction GO:0005978 Glycogen biosynthetic 7 3 0.0048 process GO:2000241 Regulation of reproductive 13 1 0.0476 process GO:0042981 Regulation of apoptotic 8 2 0.0101 process GO:0055085 Transmembrane transport 129 20 0.0143 environmental stress. In this study, RNA-Seq was used to GO:0005992 Trehalose biosynthetic 7 4 0.0144 process conduct a genome-wide transcriptional survey of I. ori- GO:0075136 Response to host 46 6 0.0175 entalis during a short period of ethanol stress (4  h). 502 GO:0012501 Programmed cell death 11 2 0.0182 genes were identified as differentially expressed under GO:0006875 Cellular metal ion homeo- 21 4 0.0187 these conditions. Among these, 451 and 51 genes were stasis up-regulated and down-regulated, respectively, with fold GO:0042173 Regulation of sporulation 17 3 0.022 change > 3 (P < 0.05) and FDR < 0.01. resulting in formation of a cellular spore Ergosterol biosynthesis GO:0006915 Apoptotic process 9 2 0.0251 KEGG enrichment analysis identified the steroid bio - GO:0008643 Carbohydrate transport 16 6 0.0262 synthesis pathway (ko00100) as highly enriched (Fig.  5) GO:0044003 Modification by symbiont 38 7 0.0265 of host morphology or including many DEGs associated with steroid biosyn- physiology thesis (especially ergosterol biosynthesis). In S. cer- GO:0006879 Cellular iron ion homeostasis 10 4 0.0281 evisiae, ergosterol protects cell membrane integrity and GO:0006566 Threonine metabolic process 16 2 0.0326 enhances membrane fluidity in response to stress (Chi GO:0006139 Nucleobase-containing 823 58 0.0334 and Arneborg 2000; Ren et al. 2014), but genes associated compound metabolic with ergosterol biosynthesis are transcriptionally down- process regulated (Alexandre et al. 2001). GO:0005993 Trehalose catabolic process 13 3 0.0357 We found that ergosterol accumulates after ethanol GO:0043940 Regulation of sexual sporula- 9 1 0.0358 tion resulting in formation stress (Fig.  1). Transcripts for the ergosterol biosynthe- of a cellular spore sis genes ERG2, ERG3, and ERG27 are significantly more GO:0040020 Regulation of meiosis 9 1 0.0358 abundant in ethanol-stressed cells, in contrast to results GO:0055082 cellular chemical homeo- 27 4 0.0397 reported for these genes in S. cerevisiae. ECM22, which stasis encodes a sterol element-binding transcription factor that GO:0006540 glutamate decarboxylation 7 1 0.0398 regulates sterol uptake and sterol biosynthesis (Woods to succinate and Höfken 2016), is also more abundant. ERG25 is an GO:0009068 Aspartate family amino acid 11 2 0.0429 catabolic process exception, and is less abundant under ethanol stress. The GO:0046187 Acetaldehyde catabolic 11 2 0.0429 results confirm the role of ergosterol in I. orientalis as an process important cytoplasmic membrane protectant in response GO:0006567 Threonine catabolic process 11 2 0.0429 to ethanol stress. GO:0006117 Acetaldehyde metabolic 11 2 0.0429 process Trehalose metabolism GO:0090304 Nucleic acid metabolic 621 50 0.0429 Analyses (Table 1, Fig. 4) show that genes involved in tre- process halose and glycogen metabolism are up-regulated during GO:0043650 Dicarboxylic acid biosyn- 6 1 0.0435 ethanol stress. The intracellular carbohydrates trehalose thetic process and glycogen are compatible solutes that resist osmotic GO:0006457 protein folding 38 10 0.0439 pressure across the cytoplasmic membrane. Trehalose GO:0030003 Cellular cation homeostasis 25 4 0.0453 is involved in ethanol tolerance in S. cerevisiae (Mah- GO:0051701 Interaction with host 88 11 0.0454 mud et  al. 2009; Wang et  al. 2013; Yi et  al. 2016). The GO:0052173 Response to defenses of 56 6 0.0468 other organism involved in up-regulation of trehalose and glycogen synthesis genes, symbiotic interaction and the accumulation of trehalose (Fig. 1), are consistent Miao et al. AMB Expr (2018) 8:39 Page 9 of 13 Fig. 6 Gene Ontology enrichment analysis using Cytoscape-BiNGO. The number of enriched DEGs in each GO category is proportional to node diameter. Darker nodes are associated with lower P-values. a Biological process. b Cellular component. c Molecular function Miao et al. AMB Expr (2018) 8:39 Page 10 of 13 with this role. Stress tolerance in yeast may rely on tre- and assembly of client proteins, and also work in concert halose-6p synthase (TPS1), the first enzyme in treha - with the ubiquitin–proteasome system  (UPS), direct- lose biosynthetic pathway, rather than on trehalose itself ing misfolded proteins for degradation (Li et  al. 2012). (Petitjean et al. 2015). In fact, we found that several genes HSP42, HSP78, and HSP104, which were mentioned in trehalose biosynthetic pathway, including TPS1, are above, also help process aggregations of unfolded or mis- up-regulated during ethanol stress. We conclude that folded proteins (Glover and Lindquist 1998). the regulation of the trehalose pathway plays an impor- Cytoscape-BiNGO analysis suggests that proteins tant role in protecting cells against ethanol stress in I. with ubiquitin-protein ligase activity are up-regulated, orientalis. including genes encoding ubiquitin-associated proteins (UBP16, BUL2, TOM1, HUL4, BRE1, and CUE2). The Response to stress and stimulus UPS degrades proteins that have exceeded their func- Genes involved in the response to biotic and abiotic stim- tional lifetime and destroys most unfolded and misfolded ulus, including heat and pH, were also enriched (Table 1, proteins (Amm et  al. 2014). Proteins with ubiquitin- Fig. 6). Up-regulation of heat stress response genes, such protein ligase activity, mainly E3 ligases, often work with as LRE1, WSC1, SGT2, and a variety of heat shock pro- HSP90/HSP70 chaperone systems and recognize mis- teins, was observed in all samples in response to etha- folded proteins (Berndsen and Wolberger 2014; Petrucelli nol. In stress-tolerant S. cerevisiae strains, intracellular et  al. 2004). The gene encoding ubiquitin domain-con - trehalose accumulates and heat shock protein genes are taining protein DSK2, which involved in the ubiquitin– continuously induced in response to stresses that damage proteasome proteolytic pathway and in spindle pole body proteins, including heat, ethanol, osmotic, and oxidative duplication, was identified by PPI analysis as a key factor stress (Kitichantaropas et al. 2016). in the response to ethanol stress (Additional file  2: Figure Expression of RIM101, a pH-response transcription S1). factor, was up-regulated in response to ethanol. The The up-regulation of genes encoding HSP proteins homologous gene in S. cerevisiae regulates response and and E3 ubiquitin ligases suggests that protein misfold- resistance to low pH and acidic conditions (Mira et  al. ing occurs under ethanol stress, possibly affecting pro - 2009). In S. cerevisiae, high concentrations of ethanol teins that help maintain plasma membrane integrity and affect the integrity of the cell membrane, changing pro - function. Since the accumulation of improperly folded ton permeability and causing intracellular acidification proteins is toxic, the HSP90/HSP70 based chaperone (Rosa and Sá-Correia 1996; Teixeira et  al. 2009). Vacu- machinery and the ubiquitin–proteasome proteolytic olar acidification is a potential mechanism to recover pathway may be essential in the response to ethanol cytosolic homeostasis after ethanol-induced intracellular stress. acidification in S. cerevisiae (Martínez-Muñoz and Kane 2008). Similar mechanisms in I. orientalis may help I. ori- Starvation effect and transport entalis maintain pH stability in the presence of ethanol. Genes associated with meiosis, reproduction, sporula- tion, ascospore cell wall assembly, and membrane bio- HSP90, HSP70, and ubiquitin genesis were up-regulated (Fig.  6, Table  1). For example, Genes associated with protein folding and refolding RRT12 encodes a spore wall-localized subtilisin-family (Fig.  6) are up-regulated under ethanol stress, such as protease required for spore wall assembly (Suda et  al. HSP42, HSP78, and HSP104 (Fig. 4). PPI analysis suggests 2009). GAS4 encodes a 1,3-beta-glucanosyltransferase an important role for HSP82 (homolog of yeast HSP90) that elongates 1,3-beta-glucan chains during spore wall and HSA1 (HSP70 1) in protein folding and refolding assembly (Ragni et  al. 2007). FLO1 encodes a cell wall (Additional file  2: Figure S1). Based on our RNA-Seq protein that participates directly in adhesive cell–cell results, other genes encoding HSP binding proteins and interactions during yeast flocculation (Fichtner et  al. co-chaperones such as STI1, AHA1, SSE1, MAS5, FES1, 2007). IFF6 encodes a GPI-anchored cell wall protein and SIS1 are also up-regulated. involved in cell wall organization and hyphal growth. In eukaryotes, HSP90 proteins are conserved, abundant Finally, CZF1 is a transcription factor involved in the molecular chaperones involved in many essential cellular regulation of filamentous growth in yeasts that responds processes (Li et al. 2012). Two cytosolic HSP90 isoforms to temperature and carbon source (Brown et  al. 1999; exist in yeast: an inducible form HSP82, and a constitu- Vinces et  al. 2006). It is possible that CZF1 is involved tive form HSC82. The association of HSP90 with HSP70 in the flocculation of I. orientalis cells that we observed and a variety of co-chaperones generates large dynamic under ethanol stress (Fig. 2). multi-chaperone complexes known as HSP90/HSP70 Genes with transporter activities were also up-reg- machinery. These play critical roles in the recruitment ulated. These include genes involved in amino acid and Miao et al. AMB Expr (2018) 8:39 Page 11 of 13 peptide transport (transporter specific for methio - Additional files nine, cysteine and oligopeptide), carbohydrate trans- port (transporter specific for hexose such as mannose, Additional file 1: Table S1. RT-qPCR Primers used in this study. fructose and glucose) and transmembrane transport. In Additional file 2: Figure S1. Protein–Protein Interaction network. addition, genes involved in protein transport, coenzyme transport, lipid transport, a-factor pheromone transport, and genes in the major transporter facilitator superfamily Abbreviations DEGs: differentially expressed genes; FC: fold change; FDR: false discovery rate; (MFS) were up-regulated. GO: gene ontology; NGS: next generation sequencing; PDB: protein data bank; Nitrogen starvation in S. cerevisiae induces meiosis, PIR: protein information resource; PPI: protein–protein interaction; PRF: Protein pseudohyphal growth, and sporulation. The presence Research Foundation; RNA-seq: RNA-sequencing; FPKM: fragments per kilo- bases per million mapped reads; RT-qPCR: reverse transcription quantitative of ethanol may affect the transmembrane transport of PCR; SEM: scanning electron microscopy. nutrients, leading to a pseudo-starvation state that elicits a nitrogen starvation response by the cell (Chandler et al. Authors’ contributions Corresponding author ZW conceived and designed the study, and was the 2004; Kasavi et  al. 2016; Stanley et  al. 2010). Consistent guarantor of integrity for the entire project. YM and GX contributed equally with this hypothesis, up-regulation of meiosis, sporula- to the work. YM contributed to experimental design, data analysis/interpreta- tion, and transportation-associated genes suggests that tion, manuscript preparation, and manuscript editing. GX conducted literature research, experimental studies, data acquisition, and statistical analysis. RL I. orientalis responds to ethanol stress as if it were expe- worked primarily on experimental studies and data acquisition. XZ and PW riencing nitrogen starvation. In effect, I. orientalis cells reviewed and edited the manuscript. All authors read and approved the mistakenly perceive that they are growing in a nutrient- manuscript. deficient environment, rather than in a nutrient-complete culture medium. The up-regulation of transmembrane Competing interests transport genes is thus an attempt by the cell to cope with The authors declare that they have no competing interests. the pseudo-starvation state caused by ethanol stress. Availability of data and materials The pseudo-starvation state may be due to the lack RNA-Seq raw data for the six libraries (NCBI Accession: PRJNA413795), repre- of coenzymes such as NAD + and coenzyme A (CoA). senting reads from the control samples T1-T3 (NCBI Accessions: SRX3277329, SRX3277330 and SRX3277331) and the 10% ethanol-stressed samples TE1-TE3 NAD + is an important cofactor for the glycolysis enzyme (NCBI Accessions: SRX3277326, SRX3277327 and SRX3277328) are available glyceraldehyde 3-phosphate dehydrogenase (GAPDH), online. while CoA is required for fatty acid metabolism and the Consent for publication oxidation of pyruvate in the citric acid cycle. We found Not applicable. that several genes encoding NAD(P) + -dependent enzymes were up-regulated, which implies that demand Ethics approval and consent to participate Not applicable. for NAD(P) + had increased. This is consistent with the transcriptional activation of Liz1 (Stolz et  al. 2004), Funding which encodes a plasma membrane-localized transport This study was funded by National Natural Science Foundation, China (NNSF No. 31471709) and the K.C. Wong Magna Fund at Ningbo University. protein for the uptake of pantothenate, the precursor of coenzyme A (CoA). A lack of pantothenate would result Publisher’s Note in slow growth, delayed septation, and mitotic defects. Springer Nature remains neutral with regard to jurisdictional claims in pub- In conclusion, our data provide a global view of tran- lished maps and institutional affiliations. scriptional changes in I. orientalis under ethanol stress. Received: 4 November 2017 Accepted: 8 March 2018 The changes are likely to reflect adaptation to stressful conditions at multiple levels. We observed modifications in the trehalose and ergosterol biosynthetic pathways, and also activation of various genes related to stress. Examples include heat shock proteins and their co-chap- References Alexandre H, Ansanaygaleote V, Dequin S, Blondin B (2001) Global gene erones, which refold aggregated and misfolded proteins, expression during short-term ethanol stress in Saccharomyces cerevisiae. and the ubiquitin–proteasome system, which targets FEBS Lett 498(1):98–103. https://doi.org/10.1016/S0014-5793(01)02503-0 misfolded proteins for degradation. Finally, ethanol stress Amm I, Sommer T, Wolf DH (2014) Protein quality control and elimination of protein waste: the role of the ubiquitin–proteasome system. BBA Mol Cell appears to induce a nutrition starvation effect, which is Res 1843(1):182–196. https://doi.org/10.1016/j.bbamcr.2013.06.031 associated with changes in cellular uptake, pseudohyphal Archana KM, Ravi R, Anu-Appaiah KA (2015) Correlation between etha- growth, and sporulation. These results provide a basis nol stress and cellular fatty acid composition of alcohol producing non-Saccharomyces in comparison with Saccharomyces cerevisiae by for future investigations of the mechanisms that regulate multivariate techniques. J Food Sci Technol 52(10):6770–6776. https://doi. ethanol stress in I. orientalis. org/10.1007/s13197-015-1762-y Miao et al. AMB Expr (2018) 8:39 Page 12 of 13 Arthington-Skaggs BA, Jradi H, Desai T, Morrison CJ (1999) Quantitation of nov., Lindnera gen. nov. and Wickerhamomyces gen. nov. FEMS Yeast Res ergosterol content: novel method for determination of fluconazole sus- 8(6):939–954. https://doi.org/10.1111/j.1567-1364.2008.00419.x ceptibility of Candida albicans. J Clin Microbiol 37(10):3332–3337 Kwon Y-J, Wang F, Liu C-Z (2011) Deep-bed solid state fermentation of sweet Berndsen CE, Wolberger C (2014) New insights into ubiquitin E3 ligase sorghum stalk to ethanol by thermotolerant Issatchenkia orientalis IPE mechanism. Nat Struct Mol Biol 21(4):301–307. https://doi.org/10.1038/ 100. Bioresour Technol 102(24):11262–11265. https://doi.org/10.1016/j. nsmb.2780 biortech.2011.09.103 Brown DH Jr, Giusani AD, Chen X, Kumamoto CA (1999) Filamentous growth Lackner DH, Schmidt MW, Wu S, Wolf DA, Bähler J (2012) Regulation of of Candida albicans in response to physical environmental cues and transcriptome, translation, and proteome in response to environmental its regulation by the unique CZF1 gene. Mol Microbiol 34(4):651–662. stress in fission yeast. Genome Biol 13(4):R25. https://doi.org/10.1186/ https://doi.org/10.1046/j.1365-2958.1999.01619.x gb-2012-13-4-r25 Chandler M, Stanley G, Rogers P, Chambers P (2004) A genomic approach to Lewis JA, Broman AT, Will J, Gasch AP (2014) Genetic architecture of ethanol- defining the ethanol stress response in the yeast Saccharomyces cerevi- responsive transcriptome variation in Saccharomyces cerevisiae strains. siae. Ann Microbiol 54(4):427–454 Genetics 198(1):369–382. https://doi.org/10.1534/genetics.114.167429 Chi Z, Arneborg N (2000) Saccharomyces cerevisiae strains with different Li J, Soroka J, Buchner J (2012) The Hsp90 chaperone machinery: conforma- degrees of ethanol tolerance exhibit different adaptive responses to tional dynamics and regulation by co-chaperones. Biochim et Biophys produced ethanol. J Ind Microbiol Biotechnol 24(1):75–78. https://doi. Acta (BBA) Mol Cell Res 1823(3):624–635. https://doi.org/10.1016/j. org/10.1038/sj.jim.2900769 bbamcr.2011.09.003 Ciani M, Comitini F, Mannazzu I, Domizio P (2010) Controlled mixed culture Maere S, Heymans K, Kuiper M (2005) BiNGO: a Cytoscape plugin to assess fermentation: a new perspective on the use of non-Saccharomyces overrepresentation of gene ontology categories in biological networks. yeasts in winemaking. FEMS Yeast Res 10(2):123–133. https://doi. Bioinformatics 21(16):3448–3449. https://doi.org/10.1093/bioinformatics/ org/10.1111/j.1567-1364.2009.00579.x bti551 Fichtner L, Schulze F, Braus GH (2007) Differential Flo8p-dependent regula- Mahmud SA, Nagahisa K, Hirasawa T, Yoshikawa K, Ashitani K, Shimizu H (2009) tion of FLO1 and FLO11 for cell–cell and cell–substrate adherence Eec ff t of trehalose accumulation on response to saline stress in Saccharo - of S. cerevisiae S288c. Mol Microbiol 66(5):1276–1289. https://doi. myces cerevisiae. Yeast 26(1):17–30. https://doi.org/10.1002/yea.1646 org/10.1111/j.1365-2958.2007.06014.x Mao X, Cai T, Olyarchuk JG, Wei L (2005) Automated genome annotation and Fleet GH (2003) Yeast interactions and wine flavour. Int J Food Microbiol pathway identification using the KEGG Orthology (KO) as a controlled 86(1–2):11–22. https://doi.org/10.1016/S0168-1605(03)00245-9 vocabulary. Bioinformatics 21(19):3787–3793. https://doi.org/10.1093/ Gasch AP, Spellman PT, Kao CM, Carmelharel O, Eisen MB, Storz G, Botstein D, bioinformatics/bti430 Brown PO (2001) Genomic expression programs in the response of yeast Martínez-Muñoz GA, Kane P (2008) Vacuolar and plasma membrane proton cells to environmental changes. Mol Biol Cell 11(12):4241–4257. https:// pumps collaborate to achieve cytosolic pH homeostasis in yeast. J Biol doi.org/10.1091/mbc.11.12.4241 Chem 283(29):20309–20319. https://doi.org/10.1074/jbc.M710470200 Giovani G, Rosi I, Bertuccioli M (2012) Quantification and characterization of Masneufpomarede I, Bely M, Marullo P, Albertin W (2015) The genetics of cell wall polysaccharides released by non-Saccharomyces yeast strains non-conventional wine yeasts: current knowledge and future challenges. during alcoholic fermentation. Int J Food Microbiol 160(2):113. https:// Front Microbiol. 6:1563. https://doi.org/10.3389/fmicb.2015.01563 doi.org/10.1016/j.ijfoodmicro.2012.10.007 Mira NP, Lourenço AB, Fernandes AR, Becker JD, Sa-Correia I (2009) The RIM101 Glover JR, Lindquist S (1998) Hsp104, Hsp70, and Hsp40: a novel chaperone pathway has a role in Saccharomyces cerevisiae adaptive response and system that rescues previously aggregated proteins. Cell 94(1):73–82. resistance to propionic acid and other weak acids. FEMS Yeast Res https://doi.org/10.1016/S0092-8674(00)81223-4 9(2):202–216. https://doi.org/10.1111/j.1567-1364.2008.00473.x Götz S, García-Gómez JM, Terol J, Williams TD, Nagaraj SH, Nueda MJ, Robles Pedersen LL, Owusukwarteng J, Thorsen L, Jespersen L (2012) Biodiversity and M, Talón M, Dopazo J, Conesa A (2008) High-throughput functional probiotic potential of yeasts isolated from Fura, a West African spontane- annotation and data mining with the Blast2GO suite. Nucleic Acids Res ously fermented cereal. Int J Food Microbiol 159(2):144–151. https://doi. 36(10):3420–3435. https://doi.org/10.1093/nar/gkn176 org/10.1016/j.ijfoodmicro.2012.08.016 Greppi A, Rantisou K, Padonou W, Hounhouigan J, Jespersen L, Jakobsen Petitjean M, Teste M-A, François JM, Parrou J-L (2015) Yeast tolerance to vari- M, Cocolin L (2013) Yeast dynamics during spontaneous fermenta- ous stresses relies on the trehalose-6P synthase ( Tps1) protein, not on tion of mawè and tchoukoutou, two traditional products from trehalose. J Biol Chem 290(26):16177–16190. https://doi.org/10.1074/jbc. Benin. Int J Food Microbiol 165(2):200–207. https://doi.org/10.1016/j. M115.653899 ijfoodmicro.2013.05.004 Petrucelli L, Dickson D, Kehoe K, Taylor J, Snyder H, Grover A, De Lucia M, Isono N, Hayakawa H, Usami A, Mishima T, Hisamatsu M (2012) A compara- McGowan E, Lewis J, Prihar G (2004) CHIP and Hsp70 regulate tau ubiq- tive study of ethanol production by Issatchenkia orientalis strains under uitination, degradation and aggregation. Hum Mol Genet 13(7):703–714. stress conditions. J Biosci Bioeng 113(1):76–78. https://doi.org/10.1016/j. https://doi.org/10.1093/hmg/ddh083 jbiosc.2011.09.004 Ragni E, Coluccio A, Rolli E, Rodriguez-Peña JM, Colasante G, Arroyo J, Neiman Kasavi C, Eraslan S, Oner ET, Kirdar B (2016) An integrative analysis of tran- AM, Popolo L (2007) GAS2 and GAS4, a pair of developmentally regulated scriptomic response of ethanol tolerant strains to ethanol in Saccha- genes required for spore wall assembly in Saccharomyces cerevisiae. romyces cerevisiae. Mol BioSyst 12(2):464–476. https://doi.org/10.1039/ Eukaryot Cell 6(2):302–316. https://doi.org/10.1128/ec.00321-06 c5mb00622h Ren B, Dai H-Q, Pei G, Tong Y-J, Zhuo Y, Yang N, Su M-Y, Huang P, Yang Y-Z, Kitagawa T, Tokuhiro K, Sugiyama H, Kohda K, Isono N, Hisamatsu M, Takahashi Zhang L-X (2014) ABC transporters coupled with the elevated ergos- H, Imaeda T (2010) Construction of a β-glucosidase expression system terol contents contribute to the azole resistance and amphotericin B using the multistress-tolerant yeast Issatchenkia orientalis. Appl Microbiol susceptibility. Appl Microbiol Biotechnol 98(6):2609–2616. https://doi. Biotechnol 87(5):1841–1853. https://doi.org/10.1007/s00253-010-2629-9 org/10.1007/s00253-013-5425-5 Kitichantaropas Y, Boonchird C, Sugiyama M, Kaneko Y, Harashima S, Auesu- Rosa MF, Sá-Correia I (1996) Intracellular acidification does not account karee C (2016) Cellular mechanisms contributing to multiple stress for inhibition of Saccharomyces cerevisiae growth in the presence tolerance in Saccharomyces cerevisiae strains with potential use in high- of ethanol. FEMS Microbiol Lett 135(2–3):271–274. https://doi. temperature ethanol fermentation. AMB Express 6(1):107. https://doi. org/10.1016/0378-1097(95)00465-3 org/10.1186/s13568-016-0285-x Sadoudi M, Tourdotmaréchal R, Rousseaux S, Steyer D, Gallardochacón JJ, Bal- Koutinas M, Patsalou M, Stavrinou S, Vyrides I (2016) High temperature alco- lester J, Vichi S, Guérinschneider R, Caixach J, Alexandre H (2012) Yeast– holic fermentation of orange peel by the newly isolated thermotolerant yeast interactions revealed by aromatic profile analysis of Sauvignon Pichia kudriavzevii KVMP10. Lett Appl Microbiol 62(1):75–83. https://doi. Blanc wine fermented by single or co-culture of non-Saccharomyces org/10.1111/lam.12514 and Saccharomyces yeasts. Food Microbiol 32(2):243–253. https://doi. Kurtzman CP, Robnett CJ, Basehoar-Powers E (2008) Phylogenetic relationships org/10.1016/j.fm.2012.06.006 among species of Pichia, Issatchenkia and Williopsis determined from multigene sequence analysis, and the proposal of Barnettozyma gen. Miao et al. AMB Expr (2018) 8:39 Page 13 of 13 Stanley D, Bandara A, Fraser S, Chambers PJ, Stanley GA (2010) The ethanol Efg1p and Czf1p. Eukaryot Cell 5(5):825–835. https://doi.org/10.1128/ stress response and ethanol tolerance of Saccharomyces cerevisiae. J Appl EC.5.5.825-835.2006 Microbiol 109(1):13–24. https://doi.org/10.1111/j.1365-2672.2009.04657.x Wang PM, Zheng DQ, Chi XQ, Li O, Qian CD, Liu TZ, Zhang XY, Du FG, Sun PY, Stolz J, Caspari T, Carr AM, Sauer N (2004) Cell division defects of Schizosac- Qu AM (2013) Relationship of trehalose accumulation with ethanol fer- charomyces pombe liz1 − mutants are caused by defects in pantoth- mentation in industrial Saccharomyces cerevisiae yeast strains. Bioresour enate uptake. Eukaryot Cell 3(2):406–412. https://doi.org/10.1128/ Technol 152C(1):371–376. https://doi.org/10.1016/j.biortech.2013.11.033 ec.3.2.406-412.2004 Woods K, Höfken T (2016) The zinc cluster proteins Upc2 and Ecm22 promote Storey JD (2003) The positive false discovery rate: a Bayesian interpretation filamentation in Saccharomyces cerevisiae by sterol biosynthesis- and the q-value. Ann Stat 31(6):2013–2035. https://doi.org/10.1214/ dependent and-independent pathways. Mol Microbiol 99(3):512–527. aos/1074290335 https://doi.org/10.1111/mmi.13244 Suda Y, Rodriguez RK, Coluccio AE, Neiman AM (2009) A screen for spore wall Xiao H, Shao Z, Jiang Y, Dole S, Zhao H (2014) Exploiting Issatchenkia orientalis permeability mutants identifies a secreted protease required for proper SD108 for succinic acid production. Microb Cell Fact 13:121. https://doi. spore wall assembly. PLoS ONE 4(9):e7184. https://doi.org/10.1371/jour- org/10.1186/s12934-014-0121-4 nal.pone.0007184 Yi C, Wang F, Dong S, Li H (2016) Changes of trehalose content and expression Teixeira MC, Raposo LR, Mira NP, Lourenço AB, Sá-Correia I (2009) Genome- of relative genes during the bioethanol fermentation by Saccharo- wide identification of Saccharomyces cerevisiae genes required for myces cerevisiae. Can J Microbiol 1:827–835. https://doi.org/10.1139/ maximal tolerance to ethanol. Appl Environ Microbiol 75(18):5761–5772. cjm-2015-0832 https://doi.org/10.1128/AEM.00845-09 Vinces MD, Haas C, Kumamoto CA (2006) Expression of the Candida albicans morphogenesis regulator gene CZF1 and its regulation by http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png AMB Express Springer Journals

Transcriptome profiling of Issatchenkia orientalis under ethanol stress

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Abstract

Issatchenkia orientalis, a non-Saccharomyces yeast that can resist a wide variety of environmental stresses, has potential use in winemaking and bioethanol production. Little is known about gene expression or the physiology of I. orien- talis under ethanol stress. In this study, high-throughput RNA sequencing was used to investigate the transcriptome profile of I. orientalis in response to ethanol. 502 gene transcripts were differentially expressed, of which 451 were more abundant, and 51 less abundant, in cells subjected to 4 h of ethanol stress (10% v/v). Annotation and statistical analyses suggest that multiple genes involved in ergosterol biosynthesis, trehalose metabolism, and stress response are differentially expressed under these conditions. The up-regulation of molecular chaperones HSP90 and HSP70, and also genes associated with the ubiquitin–proteasome proteolytic pathway suggests that ethanol stress may cause aggregation of misfolded proteins. Finally, ethanol stress in I. orientalis appears to have a nitrogen starvation effect, and many genes involved in nutrient uptake were up-regulated. Keywords: Issatchenkia orientalis, RNA-Seq, Transcriptome, Ethanol stress, Wine fermentation The non-conventional wine yeast Issatchenkia orien- Introduction talis was first described in 1960 but was reclassified to Fruit wines are fermented alcoholic beverages that derive P. kudriavzevii in 1965 (Kurtzman et  al. 2008). Several their flavors from raw materials (fruits, and often flowers I. orientalis strains produce ethanol and have higher and herbs) as well as from the fermentation process. Two thermotolerance, salt tolerance, and acid tolerance than distinct yeasts are usually involved in the production of S. cerevisiae (Isono et  al. 2012; Koutinas et  al. 2016). a savory and pleasant fruit wine. The wine yeast Saccha- Because of its resistance to multiple stress factors, I. ori- romyces cerevisiae is primarily responsible for alcoholic entalis has potential application in bioethanol production fermentation and the synthesis of secondary metabolites, and succinic acid production (Kitagawa et al. 2010; Kwon while non-Saccharomyces yeasts or non-conventional et al. 2011; Xiao et al. 2014). wine yeasts contribute additional flavor, texture, and High-throughput RNA sequencing (RNA-Seq) is now nutritional qualities (Archana et  al. 2015). The role of routinely used to generate global transcription profiles, non-Saccharomyces wine yeasts in fruit wine fermenta- often to compare gene expression under different con - tion has attracted increasing interest (Ciani et  al. 2010). ditions. Many studies have used RNA-Seq to examine Several studies have focused on multi-strain fermenta- transcription in S. cerevisiae and the fission yeast Schizos- tion and mixed yeast culture (Fleet 2003; Giovani et  al. accharomyces pombe in response to environmental shifts 2012; Sadoudi et al. 2012), and some non-Saccharomyces (Kasavi et al. 2016; Lackner et al. 2012; Lewis et al. 2014). yeasts have been suggested for use in mixed starter cul- However, gene expression in I. orientalis has not yet tures with S. cerevisiae (Masneufpomarede et al. 2015). been studied. In particular, the underlying mechanisms that allow I. orientalis to tolerate ethanol have not been explored, nor have they been compared with those in S. cerevisiae. *Correspondence: wuzufang@nbu.edu.cn Yingjie Miao and Guotong Xiong contributed equally to this work In this study we used RNA-Seq to investigate changes Department of Food Science and Engineering, School of Marine Sciences, in the gene expression profile of I. orientalis under Ningbo University, Ningbo 315211, People’s Republic of China © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Miao et al. AMB Expr (2018) 8:39 Page 2 of 13 ethanol stress. We identified a wide variety of differen - trehalose (Sigma-Aldrich) (Kitichantaropas et  al. 2016; tially expressed genes, some of which may play important Mahmud et al. 2009). roles in the stress response. Determination of ergosterol concentration Materials and methods Issatchenkia orientalis was cultured in medium with 10% Yeast strains, media, and growth conditions ethanol for 0, 4, 12, 24, and 48 h. Cells were collected by Issatchenkia orientalis strain CBS 12547 was originally centrifugation, washed with ultrapure water, then frozen isolated from tropical fruit and food sources, and is in liquid nitrogen and freeze-dried at − 20 °C. 100 mg of involved in the fermentation of some traditional Afri- dried cells were resuspended in 3 mL ethanol containing can foods (Greppi et  al. 2013; Pedersen et  al. 2012). The 25% potassium hydroxide (m/v; 25  g KOH dissolved in strain was maintained in the Food Biotechnology Labo- 35 mL pure water, add ethanol to 100 mL) and incubated ratory at Ningbo University. Yeast was initially cultured at 85 °C for 1 h. The entire sample was mixed with 3 mL for 24  h in YPD medium (1% yeast extract, 2% peptone, n-heptane and extracted by vortexing for 3  min. Finally, and 2% glucose) at 30 °C with agitation at 150 rpm. 1 mL absorbance of the supernatant at 282  nm was measured was withdrawn, added to 100  mL fresh YPD medium, and compared with samples containing known con- and incubated as before until the culture reached expo- centrations of ergosterol (Sigma-Aldrich) (Arthington- nential phase (8  h). For the RNA-Seq experiment, etha- Skaggs et al. 1999). nol was added to a final concentration of 10% (v/v), and incubation continued for another 4  h. Three cultures RNA extraction, library construction and sequencing were treated in parallel with ethanol (TE1/TE2/TE3) and As noted earlier, I. orientalis was cultured in medium three untreated cultures were used as negative controls with 10% ethanol for 4  h before cells were harvested for (T1/T2/T3). Yeast cells were harvested by centrifugation RNA extraction. Total RNA from each sample was iso- at 4 °C, 2000×g and stored at − 80 °C. lated using TRIZOL (Aidlab Biotech, Beijing, China). RNA concentration was quantified using a Qubit RNA Scanning electron microscopy (SEM) Assay Kit and a Q ubit 2.0 Fluorometer (Life Tech- Issatchenkia orientalis was cultured in medium with nologies, CA, USA). RNA integrity and purity were 10% ethanol for 24 h. Cells were collected by centrifuga- evaluated using the RNA Nano 6000 Assay Kit and the tion at 1000×g, 4  °C for 10  min and washed three times NanoPhotometer spectrophotometer (IMPLEN, CA, with physiological saline. Cells were then resuspended USA). in 2.5% glutaraldehyde for 4  h at 4  °C and washed three Construction of cDNA libraries and RNA sequencing times with 0.1 M PBS (pH = 7.4) for 15 min per wash. The were performed by Beijing BioMarker Technologies (Bei- cells were transferred through a series of ethanol solu- jing, China). In brief, poly-A mRNA was isolated using tions (30, 50, 70, 80, 90, 95 and 100%; 10 min each), and poly-T oligomer bound to magnetic beads. mRNA was then through a series of tert-butanol-anhydrous ethanol fragmented using divalent cations at elevated tempera- mixtures (ratio 1:3, 1:1, 3:1, 3:0; 10 min each). Finally, the ture. RNA fragments were copied as cDNA using ran- cells were dried and coated with a gold/palladium alloy dom primers, and second strand cDNA synthesis was (40:60) to a thickness of 10–20  nm and observed with a then performed. Double-stranded cDNAs were ligated to Hitachi S3400N scanning electron microscopy system. a single ‘A’ base and the sequencing adapters. Fragments (200 ± 25 bp) were then separated by agarose gel electro- Determination of trehalose concentration phoresis and selected for PCR amplification as sequenc - Issatchenkia orientalis was cultured in medium with 10% ing templates. Finally, the library was constructed for ethanol for 0, 4, 12, 24, and 48 h. Cells were collected by sequencing on the Illumina HiSeq 4000 sequencing centrifugation and washed with ultrapure water. Col- platform. lected cells were frozen in liquid nitrogen and freeze- dried at − 20 °C. 100 mg of dried cells were resuspended Quality control and read mapping in 1  mL ice-cold 0.5  mol/L trichloroacetic acid solution To obtain high-quality data, raw mRNA-Seq reads were by brief treatment with ultrasound, and then maintained processed using in-house Perl scripts. Reads were dis- in the same solution at room temperature for 45  min carded if they were spoiled by adaptor contamination, in order to extract the trehalose from the cells. 250  µL contained ambiguous (N) base calls, or if more than 10% extract was incubated with 1  mL 80% sulfuric acid solu- of bases had quality values < 30. The minimum acceptable tion containing 0.2% anthrone in a boiling water bath for length was 60 bp to avoid sequencing artifacts. All subse- 5  min. Absorbance at 620  nm was measured and com- quent analyses were based on the filtered data set. Reads pared with samples containing known concentrations of were mapped to the I. orientalis reference genome (NCBI Miao et al. AMB Expr (2018) 8:39 Page 3 of 13 Accession Number: GCA_000764455.1) using TopHat2 Table S1). A Tiangen FastQuant RT Kit (with gDNase) (http://ccb.jhu.edu/software/tophat/index.shtml). Gene and a KAPA SYBR FAST Universal qPCR Kit were used names were assigned to sequences based on matches for reverse transcription and qPCR, respectively. All with the highest score. qPCR reactions were performed using a QuantStudio 7 Flex Real-Time PCR system (Applied Biosystems, Functional annotation Thermo Fisher Scientific). The PCR reaction was con - Issatchenkia orientalis genes were aligned to annotated ducted following the manufacturer’s instructions, and sequences using BLAST (http://blast.ncbi.nlm.nih.gov/ three biological replicates were used in all experiments. Blast.cgi) and the following databases: Nr (NCBI non- Negative controls (template consisting of ultrapure redundant protein sequences, Nt (NCBI non-redundant water) were run for each gene. Run-time control of the nucleotide sequences, Pfam (Protein family), KOG/COG PCR instrument, baseline correction, and determina- (Clusters of Orthologous Groups of proteins), Swiss-Prot tion of Cq values were performed using QuantStudio 7 (manually annotated and reviewed protein sequence Flex Real-Time PCR Software v1.2 (Applied Biosystems, database), protein data bank (PDB), KO (KEGG Ortholog Thermo Fisher Scientific). database), and GO (Gene Ontology). The Blast2GO suite (Götz et al. 2008) was used to assign GO terms for molec- Results ular function, biological process, and cellular component. Intracellular trehalose and ergosterol concentration and SEM imaging Analysis of differential expression Intracellular concentrations of trehalose and ergosterol, To compare gene expression level between conditions, measured after 4, 12, 24 and 48  h of ethanol stress, are the transcript level of each expressed gene was calculated shown in Fig. 1. Compared with unstressed controls, eth- and normalized to fragments per kilobases per million anol-stressed yeast cells contained higher levels of both mapped reads (FPKM) using the formula: compounds. Carbohydrates such as trehalose and glyco- gen are compatible solutes that resist osmotic pressure cDNA fragments FPKM = across the cytoplasmic membrane and prevent yeast cells Mapped fragments million × Transcript Length(kb) from dehydration. Ergosterol is an important component Differential expression analysis of data from the two of the yeast cytoplasmic membrane and is also thought to experimental conditions (ethanol stress vs. control) was be involved in stress response. performed using the DESeq R package (1.10.1). P-values SEM images were captured after ethanol stress for 24 h were adjusted using Benjamini and Hochberg’s approach (Fig. 2). Stressed cells formed large flocs containing hun - for controlling the false discovery rate (FDR). Differen - dreds of connected cells. tially expressed genes (DEGs) were defined as those with fold change > 3 (P < 0.05) and FDR < 0.01. Library construction and RNA‑Sequencing Libraries (NCBI Accession: PRJNA413795) were con- KEGG and GO enrichment analyses for DEGs structed for RNA-Seq from three control samples (T1- KEGG (http://www.genome.jp/kegg/) is a database T3; NCBI Accessions: SRX3277329, SRX3277330 and resource for understanding functions and utilities of SRX3277331), and three 10% ethanol-stressed samples the biological system from molecular-level informa- (TE1-TE3; NCBI Accessions: SRX3277326, SRX3277327 tion, especially large-scale molecular datasets generated and SRX3277328). After setting aside reads with adaptor by genome sequencing. KEGG is often used to tenta- contamination, ambiguous base calls, insufficient length, tively assign functions and other properties to genes. We or unacceptable numbers of low quality base scores, used KOBAS (Mao et  al. 2005) to determine if any dif- 27.15  Gb of high-quality data were obtained with aver- ferentially expressed genes were significantly enriched age quality values ≥ 30 for more than 85% of the reads. in KEGG pathways. To determine which Gene Ontology 76.01% of reads from the control libraries, and 77.17% (GO) categories were statistically overrepresented among of reads from the ethanol-stressed libraries, mapped the DEGs, topGO and Cytoscape version 3.4.0 with to the I. orientalis genome, indicating successful library BiNGO plugin version 3.0.3 (Maere et al. 2005) were used construction. to identify significantly enriched biological networks and to output the results as graphs. Identification of differentially expressed genes Gene expression levels were calculated using FPKM val- Quantitative PCR for selected DEGs ues. As shown in Fig.  3, DEGs detected between con- Real time quantitative PCR (qPCR) primers for selected trol and ethanol stressed transcriptomes were required DEGs were designed using Primer 5.0 (Additional file  1: to meet criteria for fold change > 3 (P < 0.05) as well as Miao et al. AMB Expr (2018) 8:39 Page 4 of 13 Fig. 1 Intracellular concentrations of trehalose and ergosterol in ethanol-stressed I. orientalis. The data were obtained at the indicated times (h) after ethanol was introduced into the culture. Values are represented as mean ± S.D. Three biological replicates were used. a Intracellular trehalose. b Intracellular ergosterol Fig. 2 SEM images of control and ethanol-stressed I. orientalis cells. SEM images were captured after ethanol stress for 24 h. A Control cells at a magnification of 10,000×. B Ethanol-stressed cells at a magnification of 10,000×. C Control cells at a magnification of 20,000×. D Ethanol-stressed cells at a magnification of 20,000× Miao et al. AMB Expr (2018) 8:39 Page 5 of 13 Fig. 3 Distribution of DEGs in ethanol-stressed and control I. orientalis. a Volcano plot showing all DEGs. Dashed lines indicate inclusion criteria for false discovery rate (FDR < 0.01; = line at 2 on the y-axis) and Fold Change (FC > 3; line at ~ 1.6 on the x-axis). Red, more abundant in ethanol-stressed cells; green, more abundant in control cells; black, does not meet inclusion criteria and assumed to be unchanged. b MA plot showing FC vs. FPKM for all DEGs. Colors are as in (a). c Heatmap showing all DEGs. Colors indicate expression levels of DEGs FDR < 0.01. Of 502 transcripts with threefold or greater KEGG pathway analysis change, 451 were more abundant and 51 less abundant DEGs were annotated using KEGG to identify ortholo- in ethanol-stressed cells. With four exceptions, all suc- gous genes, and KOBAS was used to test for statistically cessfully matched with entries in the nr (498), Swiss-Prot significant enrichment of DEGs in KEGG pathways. (379), KEGG (205), or GO (224) databases, yielding a Q-values (Storey 2003) were generated by KOBAS, and total of 498 unique and annotated DEGs. are analogous to P-values in the context of our analysis. Transcript levels for a subset of DEGs in several func- As shown in Fig.  5, DEGs were significantly enriched tional groups were determined by real time quantitative in pathways used for protein processing in endoplas- PCR. The results are shown in Fig. 4. mic reticulum (ko04141, q < 0.01) and meiosis (ko04113 Miao et al. AMB Expr (2018) 8:39 Page 6 of 13 Fig. 4 Normalized transcript levels for selected I. orientalis DEGs determined by RT-qPCR. Transcript levels (fold changes) for DEGs are shown relative to levels in I. orientalis before ethanol stress. Bars represent mean values ± S.D. for three biological replicates. DEGs are grouped by function. a DEGs associated with the ergosterol pathway. b DEGs associated with the trehalose pathway. c DEGs associated with responses to stress and stimulus. d DEGs associated with heat shock proteins (HSPs). e DEGs associated with the ubiquitin–proteasome proteolytic pathway. f DEGs associated with meiosis, sporulation, and ascospore cell wall assembly q < 0.05). Pathways involving lipoic acid metabolism GO annotation and analyses (ko00785) and steroid biosynthesis (ko00100) also had DEGs were annotated and classified by GO category, high enrichment scores, but with q values > 0.05. which are divided into three ontologies: molecular func- tion, cellular component, and biological process. TopGO Miao et al. AMB Expr (2018) 8:39 Page 7 of 13 Fig. 5 KEGG pathways enrichment analysis. The diameter of the circle is proportional the number of DEGs enriched in each pathway. The color of circle represents the q-value for enrichment analysis revealed that several biological process catego- A protein–protein interaction (PPI) network was gen- ries (Table  1) were enriched for DEGs, including car- erated to identify key proteins involved in the response bohydrate metabolism, transmembrane transport, ion made by I. orientalis to ethanol stress (Additional file  2: homeostasis, nuclear or cell division, and process in Figure S1). Five proteins in this network (DSK2, HSP82, response to stress or stimuli. HSA1, BiP, and SMK1) are significantly and differentially Figure  6 shows molecular interaction graphs for expressed. These may play important roles in the stress the three GO ontology classifiers, generated using response. Cytoscape-BiNGO. In the biological process ontology (Fig.  6a), statistically overrepresented GO categories Discussion can be divided into five groups (process in response to Issatchenkia orientalis, a non-Saccharomyces yeast stimuli, protein folding and refolding, sugar transport, that can tolerate a variety of stressful environments, is DNA repair and flocculation). In the molecular function potentially useful in winemaking and bioethanol pro- ontology (Fig. 6b), overrepresented GO categories can be duction. However, it is less tolerant to ethanol than S. divided into four clusters. The largest group consists of cerevisiae (Archana et  al. 2015), and can grow and fer- binding functions, specifically nucleotide binding, pro - ment only when ethanol concentrations are under 10%. tein binding, and sugar binding. Other groups involved In S. cerevisiae, a cluster of environmental stress response ATP hydrolase activities, ubiquitin protein ligase activi- (ESR) family genes have coordinated expression under ties, and sugar transmembrane transport activities. In a variety of stress conditions (Gasch et  al. 2001), and 73 the cellular component ontology (Fig. 6c), the overrepre- genes in the ESR family are up-regulated during ethanol sented GO categories included cell wall, ER, and plasmid stress (Alexandre et al. 2001). In contrast, little is known membrane. about gene and protein expression in I. orientalis under Miao et al. AMB Expr (2018) 8:39 Page 8 of 13 Table 1 Enriched biological process terms of the DEGs Table 1 continued after ethanol stress (KS < 0.05) GO:ID Term Annotated DEGs KS GO:ID Term Annotated DEGs KS GO:0031349 Positive regulation of 26 6 0.047 defense response GO:0005991 Trehalose metabolic process 19 7 0.0018 GO:0052510 Positive regulation by organ- 26 6 0.047 GO:0048284 Organelle fusion 13 2 0.0025 ism of defense response of GO:0015833 Peptide transport 14 7 0.0039 other organism involved in symbiotic interaction GO:0005978 Glycogen biosynthetic 7 3 0.0048 process GO:2000241 Regulation of reproductive 13 1 0.0476 process GO:0042981 Regulation of apoptotic 8 2 0.0101 process GO:0055085 Transmembrane transport 129 20 0.0143 environmental stress. In this study, RNA-Seq was used to GO:0005992 Trehalose biosynthetic 7 4 0.0144 process conduct a genome-wide transcriptional survey of I. ori- GO:0075136 Response to host 46 6 0.0175 entalis during a short period of ethanol stress (4  h). 502 GO:0012501 Programmed cell death 11 2 0.0182 genes were identified as differentially expressed under GO:0006875 Cellular metal ion homeo- 21 4 0.0187 these conditions. Among these, 451 and 51 genes were stasis up-regulated and down-regulated, respectively, with fold GO:0042173 Regulation of sporulation 17 3 0.022 change > 3 (P < 0.05) and FDR < 0.01. resulting in formation of a cellular spore Ergosterol biosynthesis GO:0006915 Apoptotic process 9 2 0.0251 KEGG enrichment analysis identified the steroid bio - GO:0008643 Carbohydrate transport 16 6 0.0262 synthesis pathway (ko00100) as highly enriched (Fig.  5) GO:0044003 Modification by symbiont 38 7 0.0265 of host morphology or including many DEGs associated with steroid biosyn- physiology thesis (especially ergosterol biosynthesis). In S. cer- GO:0006879 Cellular iron ion homeostasis 10 4 0.0281 evisiae, ergosterol protects cell membrane integrity and GO:0006566 Threonine metabolic process 16 2 0.0326 enhances membrane fluidity in response to stress (Chi GO:0006139 Nucleobase-containing 823 58 0.0334 and Arneborg 2000; Ren et al. 2014), but genes associated compound metabolic with ergosterol biosynthesis are transcriptionally down- process regulated (Alexandre et al. 2001). GO:0005993 Trehalose catabolic process 13 3 0.0357 We found that ergosterol accumulates after ethanol GO:0043940 Regulation of sexual sporula- 9 1 0.0358 tion resulting in formation stress (Fig.  1). Transcripts for the ergosterol biosynthe- of a cellular spore sis genes ERG2, ERG3, and ERG27 are significantly more GO:0040020 Regulation of meiosis 9 1 0.0358 abundant in ethanol-stressed cells, in contrast to results GO:0055082 cellular chemical homeo- 27 4 0.0397 reported for these genes in S. cerevisiae. ECM22, which stasis encodes a sterol element-binding transcription factor that GO:0006540 glutamate decarboxylation 7 1 0.0398 regulates sterol uptake and sterol biosynthesis (Woods to succinate and Höfken 2016), is also more abundant. ERG25 is an GO:0009068 Aspartate family amino acid 11 2 0.0429 catabolic process exception, and is less abundant under ethanol stress. The GO:0046187 Acetaldehyde catabolic 11 2 0.0429 results confirm the role of ergosterol in I. orientalis as an process important cytoplasmic membrane protectant in response GO:0006567 Threonine catabolic process 11 2 0.0429 to ethanol stress. GO:0006117 Acetaldehyde metabolic 11 2 0.0429 process Trehalose metabolism GO:0090304 Nucleic acid metabolic 621 50 0.0429 Analyses (Table 1, Fig. 4) show that genes involved in tre- process halose and glycogen metabolism are up-regulated during GO:0043650 Dicarboxylic acid biosyn- 6 1 0.0435 ethanol stress. The intracellular carbohydrates trehalose thetic process and glycogen are compatible solutes that resist osmotic GO:0006457 protein folding 38 10 0.0439 pressure across the cytoplasmic membrane. Trehalose GO:0030003 Cellular cation homeostasis 25 4 0.0453 is involved in ethanol tolerance in S. cerevisiae (Mah- GO:0051701 Interaction with host 88 11 0.0454 mud et  al. 2009; Wang et  al. 2013; Yi et  al. 2016). The GO:0052173 Response to defenses of 56 6 0.0468 other organism involved in up-regulation of trehalose and glycogen synthesis genes, symbiotic interaction and the accumulation of trehalose (Fig. 1), are consistent Miao et al. AMB Expr (2018) 8:39 Page 9 of 13 Fig. 6 Gene Ontology enrichment analysis using Cytoscape-BiNGO. The number of enriched DEGs in each GO category is proportional to node diameter. Darker nodes are associated with lower P-values. a Biological process. b Cellular component. c Molecular function Miao et al. AMB Expr (2018) 8:39 Page 10 of 13 with this role. Stress tolerance in yeast may rely on tre- and assembly of client proteins, and also work in concert halose-6p synthase (TPS1), the first enzyme in treha - with the ubiquitin–proteasome system  (UPS), direct- lose biosynthetic pathway, rather than on trehalose itself ing misfolded proteins for degradation (Li et  al. 2012). (Petitjean et al. 2015). In fact, we found that several genes HSP42, HSP78, and HSP104, which were mentioned in trehalose biosynthetic pathway, including TPS1, are above, also help process aggregations of unfolded or mis- up-regulated during ethanol stress. We conclude that folded proteins (Glover and Lindquist 1998). the regulation of the trehalose pathway plays an impor- Cytoscape-BiNGO analysis suggests that proteins tant role in protecting cells against ethanol stress in I. with ubiquitin-protein ligase activity are up-regulated, orientalis. including genes encoding ubiquitin-associated proteins (UBP16, BUL2, TOM1, HUL4, BRE1, and CUE2). The Response to stress and stimulus UPS degrades proteins that have exceeded their func- Genes involved in the response to biotic and abiotic stim- tional lifetime and destroys most unfolded and misfolded ulus, including heat and pH, were also enriched (Table 1, proteins (Amm et  al. 2014). Proteins with ubiquitin- Fig. 6). Up-regulation of heat stress response genes, such protein ligase activity, mainly E3 ligases, often work with as LRE1, WSC1, SGT2, and a variety of heat shock pro- HSP90/HSP70 chaperone systems and recognize mis- teins, was observed in all samples in response to etha- folded proteins (Berndsen and Wolberger 2014; Petrucelli nol. In stress-tolerant S. cerevisiae strains, intracellular et  al. 2004). The gene encoding ubiquitin domain-con - trehalose accumulates and heat shock protein genes are taining protein DSK2, which involved in the ubiquitin– continuously induced in response to stresses that damage proteasome proteolytic pathway and in spindle pole body proteins, including heat, ethanol, osmotic, and oxidative duplication, was identified by PPI analysis as a key factor stress (Kitichantaropas et al. 2016). in the response to ethanol stress (Additional file  2: Figure Expression of RIM101, a pH-response transcription S1). factor, was up-regulated in response to ethanol. The The up-regulation of genes encoding HSP proteins homologous gene in S. cerevisiae regulates response and and E3 ubiquitin ligases suggests that protein misfold- resistance to low pH and acidic conditions (Mira et  al. ing occurs under ethanol stress, possibly affecting pro - 2009). In S. cerevisiae, high concentrations of ethanol teins that help maintain plasma membrane integrity and affect the integrity of the cell membrane, changing pro - function. Since the accumulation of improperly folded ton permeability and causing intracellular acidification proteins is toxic, the HSP90/HSP70 based chaperone (Rosa and Sá-Correia 1996; Teixeira et  al. 2009). Vacu- machinery and the ubiquitin–proteasome proteolytic olar acidification is a potential mechanism to recover pathway may be essential in the response to ethanol cytosolic homeostasis after ethanol-induced intracellular stress. acidification in S. cerevisiae (Martínez-Muñoz and Kane 2008). Similar mechanisms in I. orientalis may help I. ori- Starvation effect and transport entalis maintain pH stability in the presence of ethanol. Genes associated with meiosis, reproduction, sporula- tion, ascospore cell wall assembly, and membrane bio- HSP90, HSP70, and ubiquitin genesis were up-regulated (Fig.  6, Table  1). For example, Genes associated with protein folding and refolding RRT12 encodes a spore wall-localized subtilisin-family (Fig.  6) are up-regulated under ethanol stress, such as protease required for spore wall assembly (Suda et  al. HSP42, HSP78, and HSP104 (Fig. 4). PPI analysis suggests 2009). GAS4 encodes a 1,3-beta-glucanosyltransferase an important role for HSP82 (homolog of yeast HSP90) that elongates 1,3-beta-glucan chains during spore wall and HSA1 (HSP70 1) in protein folding and refolding assembly (Ragni et  al. 2007). FLO1 encodes a cell wall (Additional file  2: Figure S1). Based on our RNA-Seq protein that participates directly in adhesive cell–cell results, other genes encoding HSP binding proteins and interactions during yeast flocculation (Fichtner et  al. co-chaperones such as STI1, AHA1, SSE1, MAS5, FES1, 2007). IFF6 encodes a GPI-anchored cell wall protein and SIS1 are also up-regulated. involved in cell wall organization and hyphal growth. In eukaryotes, HSP90 proteins are conserved, abundant Finally, CZF1 is a transcription factor involved in the molecular chaperones involved in many essential cellular regulation of filamentous growth in yeasts that responds processes (Li et al. 2012). Two cytosolic HSP90 isoforms to temperature and carbon source (Brown et  al. 1999; exist in yeast: an inducible form HSP82, and a constitu- Vinces et  al. 2006). It is possible that CZF1 is involved tive form HSC82. The association of HSP90 with HSP70 in the flocculation of I. orientalis cells that we observed and a variety of co-chaperones generates large dynamic under ethanol stress (Fig. 2). multi-chaperone complexes known as HSP90/HSP70 Genes with transporter activities were also up-reg- machinery. These play critical roles in the recruitment ulated. These include genes involved in amino acid and Miao et al. AMB Expr (2018) 8:39 Page 11 of 13 peptide transport (transporter specific for methio - Additional files nine, cysteine and oligopeptide), carbohydrate trans- port (transporter specific for hexose such as mannose, Additional file 1: Table S1. RT-qPCR Primers used in this study. fructose and glucose) and transmembrane transport. In Additional file 2: Figure S1. Protein–Protein Interaction network. addition, genes involved in protein transport, coenzyme transport, lipid transport, a-factor pheromone transport, and genes in the major transporter facilitator superfamily Abbreviations DEGs: differentially expressed genes; FC: fold change; FDR: false discovery rate; (MFS) were up-regulated. GO: gene ontology; NGS: next generation sequencing; PDB: protein data bank; Nitrogen starvation in S. cerevisiae induces meiosis, PIR: protein information resource; PPI: protein–protein interaction; PRF: Protein pseudohyphal growth, and sporulation. The presence Research Foundation; RNA-seq: RNA-sequencing; FPKM: fragments per kilo- bases per million mapped reads; RT-qPCR: reverse transcription quantitative of ethanol may affect the transmembrane transport of PCR; SEM: scanning electron microscopy. nutrients, leading to a pseudo-starvation state that elicits a nitrogen starvation response by the cell (Chandler et al. Authors’ contributions Corresponding author ZW conceived and designed the study, and was the 2004; Kasavi et  al. 2016; Stanley et  al. 2010). Consistent guarantor of integrity for the entire project. YM and GX contributed equally with this hypothesis, up-regulation of meiosis, sporula- to the work. YM contributed to experimental design, data analysis/interpreta- tion, and transportation-associated genes suggests that tion, manuscript preparation, and manuscript editing. GX conducted literature research, experimental studies, data acquisition, and statistical analysis. RL I. orientalis responds to ethanol stress as if it were expe- worked primarily on experimental studies and data acquisition. XZ and PW riencing nitrogen starvation. In effect, I. orientalis cells reviewed and edited the manuscript. All authors read and approved the mistakenly perceive that they are growing in a nutrient- manuscript. deficient environment, rather than in a nutrient-complete culture medium. The up-regulation of transmembrane Competing interests transport genes is thus an attempt by the cell to cope with The authors declare that they have no competing interests. the pseudo-starvation state caused by ethanol stress. Availability of data and materials The pseudo-starvation state may be due to the lack RNA-Seq raw data for the six libraries (NCBI Accession: PRJNA413795), repre- of coenzymes such as NAD + and coenzyme A (CoA). senting reads from the control samples T1-T3 (NCBI Accessions: SRX3277329, SRX3277330 and SRX3277331) and the 10% ethanol-stressed samples TE1-TE3 NAD + is an important cofactor for the glycolysis enzyme (NCBI Accessions: SRX3277326, SRX3277327 and SRX3277328) are available glyceraldehyde 3-phosphate dehydrogenase (GAPDH), online. while CoA is required for fatty acid metabolism and the Consent for publication oxidation of pyruvate in the citric acid cycle. We found Not applicable. that several genes encoding NAD(P) + -dependent enzymes were up-regulated, which implies that demand Ethics approval and consent to participate Not applicable. for NAD(P) + had increased. This is consistent with the transcriptional activation of Liz1 (Stolz et  al. 2004), Funding which encodes a plasma membrane-localized transport This study was funded by National Natural Science Foundation, China (NNSF No. 31471709) and the K.C. Wong Magna Fund at Ningbo University. protein for the uptake of pantothenate, the precursor of coenzyme A (CoA). A lack of pantothenate would result Publisher’s Note in slow growth, delayed septation, and mitotic defects. Springer Nature remains neutral with regard to jurisdictional claims in pub- In conclusion, our data provide a global view of tran- lished maps and institutional affiliations. scriptional changes in I. orientalis under ethanol stress. Received: 4 November 2017 Accepted: 8 March 2018 The changes are likely to reflect adaptation to stressful conditions at multiple levels. We observed modifications in the trehalose and ergosterol biosynthetic pathways, and also activation of various genes related to stress. Examples include heat shock proteins and their co-chap- References Alexandre H, Ansanaygaleote V, Dequin S, Blondin B (2001) Global gene erones, which refold aggregated and misfolded proteins, expression during short-term ethanol stress in Saccharomyces cerevisiae. and the ubiquitin–proteasome system, which targets FEBS Lett 498(1):98–103. https://doi.org/10.1016/S0014-5793(01)02503-0 misfolded proteins for degradation. Finally, ethanol stress Amm I, Sommer T, Wolf DH (2014) Protein quality control and elimination of protein waste: the role of the ubiquitin–proteasome system. BBA Mol Cell appears to induce a nutrition starvation effect, which is Res 1843(1):182–196. https://doi.org/10.1016/j.bbamcr.2013.06.031 associated with changes in cellular uptake, pseudohyphal Archana KM, Ravi R, Anu-Appaiah KA (2015) Correlation between etha- growth, and sporulation. These results provide a basis nol stress and cellular fatty acid composition of alcohol producing non-Saccharomyces in comparison with Saccharomyces cerevisiae by for future investigations of the mechanisms that regulate multivariate techniques. J Food Sci Technol 52(10):6770–6776. https://doi. ethanol stress in I. orientalis. org/10.1007/s13197-015-1762-y Miao et al. AMB Expr (2018) 8:39 Page 12 of 13 Arthington-Skaggs BA, Jradi H, Desai T, Morrison CJ (1999) Quantitation of nov., Lindnera gen. nov. and Wickerhamomyces gen. nov. FEMS Yeast Res ergosterol content: novel method for determination of fluconazole sus- 8(6):939–954. https://doi.org/10.1111/j.1567-1364.2008.00419.x ceptibility of Candida albicans. J Clin Microbiol 37(10):3332–3337 Kwon Y-J, Wang F, Liu C-Z (2011) Deep-bed solid state fermentation of sweet Berndsen CE, Wolberger C (2014) New insights into ubiquitin E3 ligase sorghum stalk to ethanol by thermotolerant Issatchenkia orientalis IPE mechanism. Nat Struct Mol Biol 21(4):301–307. https://doi.org/10.1038/ 100. Bioresour Technol 102(24):11262–11265. https://doi.org/10.1016/j. nsmb.2780 biortech.2011.09.103 Brown DH Jr, Giusani AD, Chen X, Kumamoto CA (1999) Filamentous growth Lackner DH, Schmidt MW, Wu S, Wolf DA, Bähler J (2012) Regulation of of Candida albicans in response to physical environmental cues and transcriptome, translation, and proteome in response to environmental its regulation by the unique CZF1 gene. Mol Microbiol 34(4):651–662. stress in fission yeast. Genome Biol 13(4):R25. https://doi.org/10.1186/ https://doi.org/10.1046/j.1365-2958.1999.01619.x gb-2012-13-4-r25 Chandler M, Stanley G, Rogers P, Chambers P (2004) A genomic approach to Lewis JA, Broman AT, Will J, Gasch AP (2014) Genetic architecture of ethanol- defining the ethanol stress response in the yeast Saccharomyces cerevi- responsive transcriptome variation in Saccharomyces cerevisiae strains. siae. Ann Microbiol 54(4):427–454 Genetics 198(1):369–382. https://doi.org/10.1534/genetics.114.167429 Chi Z, Arneborg N (2000) Saccharomyces cerevisiae strains with different Li J, Soroka J, Buchner J (2012) The Hsp90 chaperone machinery: conforma- degrees of ethanol tolerance exhibit different adaptive responses to tional dynamics and regulation by co-chaperones. Biochim et Biophys produced ethanol. J Ind Microbiol Biotechnol 24(1):75–78. https://doi. Acta (BBA) Mol Cell Res 1823(3):624–635. https://doi.org/10.1016/j. org/10.1038/sj.jim.2900769 bbamcr.2011.09.003 Ciani M, Comitini F, Mannazzu I, Domizio P (2010) Controlled mixed culture Maere S, Heymans K, Kuiper M (2005) BiNGO: a Cytoscape plugin to assess fermentation: a new perspective on the use of non-Saccharomyces overrepresentation of gene ontology categories in biological networks. yeasts in winemaking. FEMS Yeast Res 10(2):123–133. https://doi. Bioinformatics 21(16):3448–3449. https://doi.org/10.1093/bioinformatics/ org/10.1111/j.1567-1364.2009.00579.x bti551 Fichtner L, Schulze F, Braus GH (2007) Differential Flo8p-dependent regula- Mahmud SA, Nagahisa K, Hirasawa T, Yoshikawa K, Ashitani K, Shimizu H (2009) tion of FLO1 and FLO11 for cell–cell and cell–substrate adherence Eec ff t of trehalose accumulation on response to saline stress in Saccharo - of S. cerevisiae S288c. Mol Microbiol 66(5):1276–1289. https://doi. myces cerevisiae. Yeast 26(1):17–30. https://doi.org/10.1002/yea.1646 org/10.1111/j.1365-2958.2007.06014.x Mao X, Cai T, Olyarchuk JG, Wei L (2005) Automated genome annotation and Fleet GH (2003) Yeast interactions and wine flavour. Int J Food Microbiol pathway identification using the KEGG Orthology (KO) as a controlled 86(1–2):11–22. https://doi.org/10.1016/S0168-1605(03)00245-9 vocabulary. Bioinformatics 21(19):3787–3793. https://doi.org/10.1093/ Gasch AP, Spellman PT, Kao CM, Carmelharel O, Eisen MB, Storz G, Botstein D, bioinformatics/bti430 Brown PO (2001) Genomic expression programs in the response of yeast Martínez-Muñoz GA, Kane P (2008) Vacuolar and plasma membrane proton cells to environmental changes. Mol Biol Cell 11(12):4241–4257. https:// pumps collaborate to achieve cytosolic pH homeostasis in yeast. J Biol doi.org/10.1091/mbc.11.12.4241 Chem 283(29):20309–20319. https://doi.org/10.1074/jbc.M710470200 Giovani G, Rosi I, Bertuccioli M (2012) Quantification and characterization of Masneufpomarede I, Bely M, Marullo P, Albertin W (2015) The genetics of cell wall polysaccharides released by non-Saccharomyces yeast strains non-conventional wine yeasts: current knowledge and future challenges. during alcoholic fermentation. Int J Food Microbiol 160(2):113. https:// Front Microbiol. 6:1563. https://doi.org/10.3389/fmicb.2015.01563 doi.org/10.1016/j.ijfoodmicro.2012.10.007 Mira NP, Lourenço AB, Fernandes AR, Becker JD, Sa-Correia I (2009) The RIM101 Glover JR, Lindquist S (1998) Hsp104, Hsp70, and Hsp40: a novel chaperone pathway has a role in Saccharomyces cerevisiae adaptive response and system that rescues previously aggregated proteins. Cell 94(1):73–82. resistance to propionic acid and other weak acids. FEMS Yeast Res https://doi.org/10.1016/S0092-8674(00)81223-4 9(2):202–216. https://doi.org/10.1111/j.1567-1364.2008.00473.x Götz S, García-Gómez JM, Terol J, Williams TD, Nagaraj SH, Nueda MJ, Robles Pedersen LL, Owusukwarteng J, Thorsen L, Jespersen L (2012) Biodiversity and M, Talón M, Dopazo J, Conesa A (2008) High-throughput functional probiotic potential of yeasts isolated from Fura, a West African spontane- annotation and data mining with the Blast2GO suite. Nucleic Acids Res ously fermented cereal. Int J Food Microbiol 159(2):144–151. https://doi. 36(10):3420–3435. https://doi.org/10.1093/nar/gkn176 org/10.1016/j.ijfoodmicro.2012.08.016 Greppi A, Rantisou K, Padonou W, Hounhouigan J, Jespersen L, Jakobsen Petitjean M, Teste M-A, François JM, Parrou J-L (2015) Yeast tolerance to vari- M, Cocolin L (2013) Yeast dynamics during spontaneous fermenta- ous stresses relies on the trehalose-6P synthase ( Tps1) protein, not on tion of mawè and tchoukoutou, two traditional products from trehalose. J Biol Chem 290(26):16177–16190. https://doi.org/10.1074/jbc. Benin. Int J Food Microbiol 165(2):200–207. https://doi.org/10.1016/j. M115.653899 ijfoodmicro.2013.05.004 Petrucelli L, Dickson D, Kehoe K, Taylor J, Snyder H, Grover A, De Lucia M, Isono N, Hayakawa H, Usami A, Mishima T, Hisamatsu M (2012) A compara- McGowan E, Lewis J, Prihar G (2004) CHIP and Hsp70 regulate tau ubiq- tive study of ethanol production by Issatchenkia orientalis strains under uitination, degradation and aggregation. Hum Mol Genet 13(7):703–714. stress conditions. J Biosci Bioeng 113(1):76–78. https://doi.org/10.1016/j. https://doi.org/10.1093/hmg/ddh083 jbiosc.2011.09.004 Ragni E, Coluccio A, Rolli E, Rodriguez-Peña JM, Colasante G, Arroyo J, Neiman Kasavi C, Eraslan S, Oner ET, Kirdar B (2016) An integrative analysis of tran- AM, Popolo L (2007) GAS2 and GAS4, a pair of developmentally regulated scriptomic response of ethanol tolerant strains to ethanol in Saccha- genes required for spore wall assembly in Saccharomyces cerevisiae. romyces cerevisiae. Mol BioSyst 12(2):464–476. https://doi.org/10.1039/ Eukaryot Cell 6(2):302–316. https://doi.org/10.1128/ec.00321-06 c5mb00622h Ren B, Dai H-Q, Pei G, Tong Y-J, Zhuo Y, Yang N, Su M-Y, Huang P, Yang Y-Z, Kitagawa T, Tokuhiro K, Sugiyama H, Kohda K, Isono N, Hisamatsu M, Takahashi Zhang L-X (2014) ABC transporters coupled with the elevated ergos- H, Imaeda T (2010) Construction of a β-glucosidase expression system terol contents contribute to the azole resistance and amphotericin B using the multistress-tolerant yeast Issatchenkia orientalis. Appl Microbiol susceptibility. Appl Microbiol Biotechnol 98(6):2609–2616. https://doi. Biotechnol 87(5):1841–1853. https://doi.org/10.1007/s00253-010-2629-9 org/10.1007/s00253-013-5425-5 Kitichantaropas Y, Boonchird C, Sugiyama M, Kaneko Y, Harashima S, Auesu- Rosa MF, Sá-Correia I (1996) Intracellular acidification does not account karee C (2016) Cellular mechanisms contributing to multiple stress for inhibition of Saccharomyces cerevisiae growth in the presence tolerance in Saccharomyces cerevisiae strains with potential use in high- of ethanol. FEMS Microbiol Lett 135(2–3):271–274. https://doi. temperature ethanol fermentation. AMB Express 6(1):107. https://doi. org/10.1016/0378-1097(95)00465-3 org/10.1186/s13568-016-0285-x Sadoudi M, Tourdotmaréchal R, Rousseaux S, Steyer D, Gallardochacón JJ, Bal- Koutinas M, Patsalou M, Stavrinou S, Vyrides I (2016) High temperature alco- lester J, Vichi S, Guérinschneider R, Caixach J, Alexandre H (2012) Yeast– holic fermentation of orange peel by the newly isolated thermotolerant yeast interactions revealed by aromatic profile analysis of Sauvignon Pichia kudriavzevii KVMP10. Lett Appl Microbiol 62(1):75–83. https://doi. Blanc wine fermented by single or co-culture of non-Saccharomyces org/10.1111/lam.12514 and Saccharomyces yeasts. Food Microbiol 32(2):243–253. https://doi. Kurtzman CP, Robnett CJ, Basehoar-Powers E (2008) Phylogenetic relationships org/10.1016/j.fm.2012.06.006 among species of Pichia, Issatchenkia and Williopsis determined from multigene sequence analysis, and the proposal of Barnettozyma gen. Miao et al. AMB Expr (2018) 8:39 Page 13 of 13 Stanley D, Bandara A, Fraser S, Chambers PJ, Stanley GA (2010) The ethanol Efg1p and Czf1p. Eukaryot Cell 5(5):825–835. https://doi.org/10.1128/ stress response and ethanol tolerance of Saccharomyces cerevisiae. J Appl EC.5.5.825-835.2006 Microbiol 109(1):13–24. https://doi.org/10.1111/j.1365-2672.2009.04657.x Wang PM, Zheng DQ, Chi XQ, Li O, Qian CD, Liu TZ, Zhang XY, Du FG, Sun PY, Stolz J, Caspari T, Carr AM, Sauer N (2004) Cell division defects of Schizosac- Qu AM (2013) Relationship of trehalose accumulation with ethanol fer- charomyces pombe liz1 − mutants are caused by defects in pantoth- mentation in industrial Saccharomyces cerevisiae yeast strains. Bioresour enate uptake. Eukaryot Cell 3(2):406–412. https://doi.org/10.1128/ Technol 152C(1):371–376. https://doi.org/10.1016/j.biortech.2013.11.033 ec.3.2.406-412.2004 Woods K, Höfken T (2016) The zinc cluster proteins Upc2 and Ecm22 promote Storey JD (2003) The positive false discovery rate: a Bayesian interpretation filamentation in Saccharomyces cerevisiae by sterol biosynthesis- and the q-value. Ann Stat 31(6):2013–2035. https://doi.org/10.1214/ dependent and-independent pathways. Mol Microbiol 99(3):512–527. aos/1074290335 https://doi.org/10.1111/mmi.13244 Suda Y, Rodriguez RK, Coluccio AE, Neiman AM (2009) A screen for spore wall Xiao H, Shao Z, Jiang Y, Dole S, Zhao H (2014) Exploiting Issatchenkia orientalis permeability mutants identifies a secreted protease required for proper SD108 for succinic acid production. Microb Cell Fact 13:121. https://doi. spore wall assembly. PLoS ONE 4(9):e7184. https://doi.org/10.1371/jour- org/10.1186/s12934-014-0121-4 nal.pone.0007184 Yi C, Wang F, Dong S, Li H (2016) Changes of trehalose content and expression Teixeira MC, Raposo LR, Mira NP, Lourenço AB, Sá-Correia I (2009) Genome- of relative genes during the bioethanol fermentation by Saccharo- wide identification of Saccharomyces cerevisiae genes required for myces cerevisiae. Can J Microbiol 1:827–835. https://doi.org/10.1139/ maximal tolerance to ethanol. Appl Environ Microbiol 75(18):5761–5772. cjm-2015-0832 https://doi.org/10.1128/AEM.00845-09 Vinces MD, Haas C, Kumamoto CA (2006) Expression of the Candida albicans morphogenesis regulator gene CZF1 and its regulation by

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Published: Mar 13, 2018

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