Issatchenkia orientalis, a non-Saccharomyces yeast that can tolerate a variety of stressful environments, is potentially useful in winemaking and bioethanol production. However, it is less tolerant to ethanol than S. cerevisiae (Archana et al. 2015), and can grow and ferment only when ethanol concentrations are under 10%. In S. cerevisiae, a cluster of environmental stress response (ESR) family genes have coordinated expression under a variety of stress conditions (Gasch et al. 2001), and 73 genes in the ESR family are up-regulated during ethanol stress (Alexandre et al. 2001). In contrast, little is known about gene and protein expression in I. orientalis under environmental stress. In this study, RNA-Seq was used to conduct a genome-wide transcriptional survey of I. orientalis during a short period of ethanol stress (4 h). 502 genes were identified as differentially expressed under these conditions. Among these, 451 and 51 genes were up-regulated and down-regulated, respectively, with fold change > 3 (P < 0.05) and FDR < 0.01.
KEGG enrichment analysis identified the steroid biosynthesis pathway (ko00100) as highly enriched (Fig. 5) including many DEGs associated with steroid biosynthesis (especially ergosterol biosynthesis). In S. cerevisiae, ergosterol protects cell membrane integrity and enhances membrane fluidity in response to stress (Chi and Arneborg 2000; Ren et al. 2014), but genes associated with ergosterol biosynthesis are transcriptionally down-regulated (Alexandre et al. 2001).
We found that ergosterol accumulates after ethanol stress (Fig. 1). Transcripts for the ergosterol biosynthesis genes ERG2, ERG3, and ERG27 are significantly more abundant in ethanol-stressed cells, in contrast to results reported for these genes in S. cerevisiae. ECM22, which encodes a sterol element-binding transcription factor that regulates sterol uptake and sterol biosynthesis (Woods and Höfken 2016), is also more abundant. ERG25 is an exception, and is less abundant under ethanol stress. The results confirm the role of ergosterol in I. orientalis as an important cytoplasmic membrane protectant in response to ethanol stress.
Analyses (Table 1, Fig. 4) show that genes involved in trehalose and glycogen metabolism are up-regulated during ethanol stress. The intracellular carbohydrates trehalose and glycogen are compatible solutes that resist osmotic pressure across the cytoplasmic membrane. Trehalose is involved in ethanol tolerance in S. cerevisiae (Mahmud et al. 2009; Wang et al. 2013; Yi et al. 2016). The up-regulation of trehalose and glycogen synthesis genes, and the accumulation of trehalose (Fig. 1), are consistent with this role. Stress tolerance in yeast may rely on trehalose-6p synthase (TPS1), the first enzyme in trehalose biosynthetic pathway, rather than on trehalose itself (Petitjean et al. 2015). In fact, we found that several genes in trehalose biosynthetic pathway, including TPS1, are up-regulated during ethanol stress. We conclude that the regulation of the trehalose pathway plays an important role in protecting cells against ethanol stress in I. orientalis.
Response to stress and stimulus
Genes involved in the response to biotic and abiotic stimulus, including heat and pH, were also enriched (Table 1, Fig. 6). Up-regulation of heat stress response genes, such as LRE1, WSC1, SGT2, and a variety of heat shock proteins, was observed in all samples in response to ethanol. In stress-tolerant S. cerevisiae strains, intracellular trehalose accumulates and heat shock protein genes are continuously induced in response to stresses that damage proteins, including heat, ethanol, osmotic, and oxidative stress (Kitichantaropas et al. 2016).
Expression of RIM101, a pH-response transcription factor, was up-regulated in response to ethanol. The homologous gene in S. cerevisiae regulates response and resistance to low pH and acidic conditions (Mira et al. 2009). In S. cerevisiae, high concentrations of ethanol affect the integrity of the cell membrane, changing proton permeability and causing intracellular acidification (Rosa and Sá-Correia 1996; Teixeira et al. 2009). Vacuolar acidification is a potential mechanism to recover cytosolic homeostasis after ethanol-induced intracellular acidification in S. cerevisiae (Martínez-Muñoz and Kane 2008). Similar mechanisms in I. orientalis may help I. orientalis maintain pH stability in the presence of ethanol.
HSP90, HSP70, and ubiquitin
Genes associated with protein folding and refolding (Fig. 6) are up-regulated under ethanol stress, such as HSP42, HSP78, and HSP104 (Fig. 4). PPI analysis suggests an important role for HSP82 (homolog of yeast HSP90) and HSA1 (HSP70 1) in protein folding and refolding (Additional file 2: Figure S1). Based on our RNA-Seq results, other genes encoding HSP binding proteins and co-chaperones such as STI1, AHA1, SSE1, MAS5, FES1, and SIS1 are also up-regulated.
In eukaryotes, HSP90 proteins are conserved, abundant molecular chaperones involved in many essential cellular processes (Li et al. 2012). Two cytosolic HSP90 isoforms exist in yeast: an inducible form HSP82, and a constitutive form HSC82. The association of HSP90 with HSP70 and a variety of co-chaperones generates large dynamic multi-chaperone complexes known as HSP90/HSP70 machinery. These play critical roles in the recruitment and assembly of client proteins, and also work in concert with the ubiquitin–proteasome system (UPS), directing misfolded proteins for degradation (Li et al. 2012). HSP42, HSP78, and HSP104, which were mentioned above, also help process aggregations of unfolded or misfolded proteins (Glover and Lindquist 1998).
Cytoscape-BiNGO analysis suggests that proteins with ubiquitin-protein ligase activity are up-regulated, including genes encoding ubiquitin-associated proteins (UBP16, BUL2, TOM1, HUL4, BRE1, and CUE2). The UPS degrades proteins that have exceeded their functional lifetime and destroys most unfolded and misfolded proteins (Amm et al. 2014). Proteins with ubiquitin-protein ligase activity, mainly E3 ligases, often work with HSP90/HSP70 chaperone systems and recognize misfolded proteins (Berndsen and Wolberger 2014; Petrucelli et al. 2004). The gene encoding ubiquitin domain-containing protein DSK2, which involved in the ubiquitin–proteasome proteolytic pathway and in spindle pole body duplication, was identified by PPI analysis as a key factor in the response to ethanol stress (Additional file 2: Figure S1).
The up-regulation of genes encoding HSP proteins and E3 ubiquitin ligases suggests that protein misfolding occurs under ethanol stress, possibly affecting proteins that help maintain plasma membrane integrity and function. Since the accumulation of improperly folded proteins is toxic, the HSP90/HSP70 based chaperone machinery and the ubiquitin–proteasome proteolytic pathway may be essential in the response to ethanol stress.
Starvation effect and transport
Genes associated with meiosis, reproduction, sporulation, ascospore cell wall assembly, and membrane biogenesis were up-regulated (Fig. 6, Table 1). For example, RRT12 encodes a spore wall-localized subtilisin-family protease required for spore wall assembly (Suda et al. 2009). GAS4 encodes a 1,3-beta-glucanosyltransferase that elongates 1,3-beta-glucan chains during spore wall assembly (Ragni et al. 2007). FLO1 encodes a cell wall protein that participates directly in adhesive cell–cell interactions during yeast flocculation (Fichtner et al. 2007). IFF6 encodes a GPI-anchored cell wall protein involved in cell wall organization and hyphal growth. Finally, CZF1 is a transcription factor involved in the regulation of filamentous growth in yeasts that responds to temperature and carbon source (Brown et al. 1999; Vinces et al. 2006). It is possible that CZF1 is involved in the flocculation of I. orientalis cells that we observed under ethanol stress (Fig. 2).
Genes with transporter activities were also up-regulated. These include genes involved in amino acid and peptide transport (transporter specific for methionine, cysteine and oligopeptide), carbohydrate transport (transporter specific for hexose such as mannose, fructose and glucose) and transmembrane transport. In addition, genes involved in protein transport, coenzyme transport, lipid transport, a-factor pheromone transport, and genes in the major transporter facilitator superfamily (MFS) were up-regulated.
Nitrogen starvation in S. cerevisiae induces meiosis, pseudohyphal growth, and sporulation. The presence of ethanol may affect the transmembrane transport of nutrients, leading to a pseudo-starvation state that elicits a nitrogen starvation response by the cell (Chandler et al. 2004; Kasavi et al. 2016; Stanley et al. 2010). Consistent with this hypothesis, up-regulation of meiosis, sporulation, and transportation-associated genes suggests that I. orientalis responds to ethanol stress as if it were experiencing nitrogen starvation. In effect, I. orientalis cells mistakenly perceive that they are growing in a nutrient-deficient environment, rather than in a nutrient-complete culture medium. The up-regulation of transmembrane transport genes is thus an attempt by the cell to cope with the pseudo-starvation state caused by ethanol stress.
The pseudo-starvation state may be due to the lack of coenzymes such as NAD + and coenzyme A (CoA). NAD + is an important cofactor for the glycolysis enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH), while CoA is required for fatty acid metabolism and the oxidation of pyruvate in the citric acid cycle. We found that several genes encoding NAD(P) + -dependent enzymes were up-regulated, which implies that demand for NAD(P) + had increased. This is consistent with the transcriptional activation of Liz1 (Stolz et al. 2004), which encodes a plasma membrane-localized transport protein for the uptake of pantothenate, the precursor of coenzyme A (CoA). A lack of pantothenate would result in slow growth, delayed septation, and mitotic defects.
In conclusion, our data provide a global view of transcriptional changes in I. orientalis under ethanol stress. 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-chaperones, which refold aggregated and misfolded proteins, and the ubiquitin–proteasome system, which targets misfolded proteins for degradation. Finally, ethanol stress appears to induce a nutrition starvation effect, which is associated with changes in cellular uptake, pseudohyphal growth, and sporulation. These results provide a basis for future investigations of the mechanisms that regulate ethanol stress in I. orientalis.