Introduction

Cold environments encompass many of the Earth’s biomes, including polar regions and alpine environments. Along with frequent and often long-lasting sub-zero temperatures, these environments are often characterized by frequent freeze–thaw cycles, high salt concentrations, low moisture and nutrient availability, and extreme ultraviolet (UV) and solar radiation. Despite the harshness, they are inhabited by a high diversity of biota, composed predominantly of microorganisms such as bacteria, protists, and fungi [13, 20].

Cold-adapted (psychrotolerant) fungi provide a large portion of low-temperature biodiversity and are essential for maintaining ecosystem processes in cold environments [20, 37]. However, while they grow at sub-zero temperatures, most can cope with wide temperature ranges and have a growth optimum above 15°C [20, 28, 36]. Fungi are also highly abundant and widespread in temperate zones and are even found in artificial habitats, such as refrigerated environments [20, 37]. For the genus Pseudogymnoascus, a multilocus phylogenetic analyses and morphological characterizations have determined four new species in Antarctica: Pseudogymnoascus antarcticus sp. nov., Pseudogymnoascus australis sp. nov., Pseudogymnoascus griseus sp. nov., and Pseudogymnoascus lanuginosus sp. nov. [35]. Moreover, new secondary metabolites have been described for these fungi isolated from Antarctica, demonstrating the recent interest in this group of organisms [19, 31].

Studies have demonstrated that the great efficiency of cold-adapted fungi and their ability to cope with extreme environmental conditions depends on various molecular and physiological adaptations, including the production of antifreeze proteins, compatible solutes (i.e., glycerol, trehalose, polyols) and cold-active enzymes [20, 34]. These findings are crucial from both evolutionary and biotechnological points of view [5, 17, 23]. Various cosmopolitan model organisms such as Saccharomyces cerevisiae Hansen and Aspergillus nidulans (Eidam) G. Winter are important model organisms of fungal stress tolerance. Nonetheless, psychrophilic and psychrotolerant fungi have also been studied to provide specific details and information on their cold-adapted properties. Improving understanding of the cold stress responses of psychrotolerant fungi is nevertheless an important research field. However, it should also be noted that the natural micro-environments of many fungi are often extremely variable [18]. Therefore, relatively stable experimentally-applied cold stress and non-stress conditions do not replicate the natural environmental variation [3]. Thus, caution must be applied when interpreting data from experimental laboratory studies. From a proteomic perspective, cold stress responses are expected to involve a balanced production of protein networks within cells to eliminate the damaging effects of low-temperature stress while sustaining normal cell processes. Various mechanisms are proposed to underlie the overall complexity of fungal cold stress responses [15, 20]. Many of the proposed mechanisms involve a range of cold-adapted metabolic pathways [16, 22] and translation-related processes [10, 14]. For instance, the cold stress responses of Aspergillus flavus Link and Exophiala dermatitidis (Kano) de Hoog showed increased activity of metabolic pathways involved in amino acid and carbohydrate metabolism [4, 33]. In Flammulina velutipes (Curtis) Singer and Umbelopsis isabellina (Oudem.) Gams, the upregulation of energy metabolism pathways, and ATP production were reported [21, 27]. Various lipid metabolic pathways are also involved in the cold stress response of fungi, including the metabolism of sphingolipids, phospholipids, and unsaturated fatty acids [15, 32]. Lipid modulation is significantly related to the stability of fungal membrane structures and their integrity, allowing survival after freezing [26, 29]. The cold stress response of fungi also includes various translation-related processes, such as the upregulation of SRP-dependent co-translational protein targeting membrane pathway, different cold-adapted ribosomal protein biosynthesis, and translation elongation pathways [4, 14, 32].

Progressive advances in proteomic technologies have enhanced our understanding and biotechnological application of psychrotolerant fungi [1, 27]. For instance, some fungal cold stress response mechanisms are applicable in producing antibiotics, antifungal molecules, secondary metabolites, and methane metabolism [25, 38]. However, their potential biotechnological application value is still underexplored and unrecognized. Considering these limitations, we aimed to investigate the cold stress response mechanisms of Pseudogymnoascus spp. using a proteomic approach. To elucidate potential broad-scale differences in cold stress response mechanisms, isolates from three different and geographically distant regions were selected, including polar regions (i.e., Arctic and Antarctic) and Europe as a temperate region. Our findings provide important baseline data on cold stress responses of soil microfungi that are needed to enhance further research on their biotechnological applications.

Methodology

Fungal Cultivation and Cold Stress Experimental Design

Four isolates of Pseudogymnoascus spp. from the polar regions, including the Arctic (HND16 R4-1 sp.1 and HND16 R2-1 sp.2) and Antarctic (AK07KGI1202 R1-1 sp.3 and AK07KGI1202 R1-1 sp.4) were obtained from the culture collection of the National Antarctic Research Centre (NARC), Universiti Malaya, Malaysia. Isolation, identification, and phylogenetic analysis of these isolates are described in a prior publication from our group [39]. Phylogenetic analysis clustered all isolates within an undescribed group of Pseudogymnoascus sequences; thus, they were described as Pseudogymnoascus sp. [39]. All the isolates were kept in pure culture, and their sequences were deposited into the GenBank database. Two isolates of Pseudogymnoascus pannorum (Link) Minnis & D.L. Lindner (CBS 106.13 and CBS 107.65) that originated from the temperate region (Switzerland and Germany) were purchased from the Westerdijk Fungal Biodiversity Institute which was previously known as Centraalbureau voor Schimmelcultures (CBS-KNAW) Fungal Biodiversity Centre (Utrecht, The Netherlands). The list of the investigated isolates with information on their origin and identification codes is given in Table 1. Fungal colony plugs (ca. 5 mm in diameter) were inoculated onto 100 mm Petri dishes of Czapek-Dox agar (CDA, Oxoid) and incubated in cold stress (CS) conditions (5 °C) and at optimal temperature (15 °C) for control (C) for 5 days. In this work, 5°C and an incubation period of 5 days were chosen to represent cold stress conditions for all isolates. Our previous work showed a clear indication of stress-related changes in colony morphology at 5°C, and no growth was observed below 5°C for all isolates. [2]. The incubation period was fixed at 5 days to ensure that all fungi cells were maintained in log phase growth since no significant difference in growth rates between day 5 and day 7 for all six isolates was observed [2].

Table 1 Pseudogymnoascus spp. isolates used in this study with information on their origin and identification codes
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Preparation of Protein Extracts

Mycelia of Pseudogymnoascus spp. (from 10-day cultures) were carefully scraped using a sterile spatula. An average of 1 g of fungi mycelia (initial wet mass) was inoculated into 300 mL of Czapek-Dox liquid cultures in three replicates and grown for 5 days at the selected experimental temperatures (i.e., 5 °C and 15 °C). After 5 days, fungal biomass was harvested using a 0.45-µm filter paper and transferred to sterile tubes for weighing. Then, the harvested biomass was immediately flash-frozen and ground into fine powder in liquid nitrogen. Further steps of protein extract preparation were carried out following Tesei et al. [33] with modifications. Briefly, 1 g of ground mycelia was incubated in lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS, 30 mM tris HCl, pH 8.5) for 1 h. The mixture was bath-sonicated for 15 min at 20 °C. Then 5 mL of tris-buffered phenol solution pH 8.0 (Sigma Aldrich) was added to the cell lysate, and the phenolic phase was collected after centrifugation (3300 × g for 20 min). Proteins were precipitated overnight at -20 °C by adding 5 volumes of 20% (w/v) ice-cold TCA/acetone (with the addition of 0.2% DTT, w/v). After centrifugation at 10,000 × g for 30 min, the precipitate was washed twice with ice-cold acetone (80%, v/v). The resulting pellet was air-dried and resuspended in 100 µl of modified lysis buffer (2 M urea, 30 mM tris HCl, pH 8.5). Total protein content was determined using a standard Bradford protein assay [8].

In-solution Protein Digestion

In-solution protein digestion was carried out following Lau and Othman [24]. The extracted proteins (50 µg) were suspended in 100 µL of 50 mM ammonium bicarbonate and 1 M urea. The proteins were reduced and alkylated using 100 mM tris(2 carboxyethyl)phosphine and 200 mM iodoacetamide. Sodium deoxycholate in 5 mM ammonium bicarbonate [1% (w/v)] was added to the reduced and alkylated proteins to enhance the tryptic digestion at 37 °C for 10 min. Tryptic digestion using 1 µg of sequencing grade trypsin (Promega, Madison, WI, USA) per 50 µg protein was performed at 37 °C for 17 h. The resulting peptide mixture was then acidified with 0.5% formic acid to precipitate sodium deoxycholate through centrifugation at 14,000 × g (Eppendorf, Thermo Scientific) at ambient room temperature for 15 min. The remaining solvents and acids were removed using a centrifugal evaporator (CentriVap Concentrator, Labconco, MO, USA). The desiccated peptides were suspended in 100 µL of 0.1% formic acid and gently mixed before peptide purification. An Empore™ solid phase extraction disk (3 M Purification, Inc., MN, USA), conditioned with acetonitrile and methanol, was added into the peptide solution and incubated at the ambient temperature for 3 h to bind the peptides. Elution of the peptides from the disk was done twice using 50% ACN in 0.1% FA for 30 min each.

Liquid Chromatography Tandem Mass Spectrometry Analysis (LC-MS/MS)

Peptides were reconstituted in 30 µL of 0.1% FA and 5% ACN. Then, 2 µL of the digest was loaded onto an Acclaim PepMap 100 C18 column (3 µm, 0.075 × 150 mm) (Thermo Scientific, MA, USA). The reverse phase column was equilibrated with 0.1% FA (mobile phase A) and 80% ACN in 0.1% FA (mobile phase B). A gradient of 5–35% mobile phase B over 70 min, at a flow rate of 300 nL min−1, was applied to elute the peptides. The peptides were separated using the EASY-nano liquid chromatography (EASY-nLC) 1200 System (Thermo Scientific, MA, USA). An online Q Exactive Plus Hybrid Quadrupole-Orbitrap mass spectrometer system (Thermo Scientific, MA, USA) generated the peptide ions with a spray voltage of 1800 V in positive mode. The precursor ion scan was conducted with a resolution of 70,000 and a mass range of m/z 310–1800. Precursors containing charge states from 2 + to 8 + were fragmented further. The fragmentation was done via collision-induced and high-energy collision-induced at a normalized energy of 28%. The resolution, isolation window, and ion injection time were set at 17,500, 0.7 Da, and 60 ms, respectively. The scanned precursor mass range was set at m/z 110–1800.

Protein Identification and Bioinformatic Analysis

Mass spectra of the peptides were acquired using Xcalibur (Ver. 4.1.31.9) (Thermo Scientific, MA, USA) and deconvoluted with Proteome Discoverer (Ver. 2.4) (Thermo Scientific, MA, USA) to create the peptide mass list. SEQUEST HT search engine, incorporated in the Proteome Discoverer, was used to match the generated mass list against Pseudogymnoascus destructans (Blehert & Gargas) Minnis & D.L. Lindner (Taxonomy ID is 655981, 82,900 sequences). Mass tolerance for the proteins and their fragments was fixed at 10 ppm and 0.02 Da, respectively. Trypsin was indicated as the digestion enzyme, with up to two missed cleavages allowed during the search. Carbamidomethylation modification on cysteine residues was set as a static modification, whereas variable amino acid modifications included deamidation (asparagine and glutamine residues) and oxidation (methionine residues). The mass list was also searched against a decoy database generated from randomized protein sequences of the taxonomy mentioned earlier. Only proteins having at least the Rank 1 peptide and a false discovery rate of 1% were accepted. Spectra that matched the sequences were further validated using the Percolator algorithm (Ver. 2.04) with q-value at 1% false discovery rate. Venn diagrams were generated using the web-based Venny v2.1 software available at https://bioinfogp.cnb.csic.es/tools/venny/index.html (Oliveros 2007–2015).

Peptide Quantification and Bioinformatics Analysis

The protein function was determined by inputting protein identifiers (NCBI accession number) into the UniProtKB database (http://www.uniprot.org/blast/) and assigning the respective Gene Ontology (GO) terms and annotations. Protein abundance values were used to calculate each isolate’s log2 ratios of CS:C for each isolate. A microarray (MA) plot was constructed using log2 CS:C ratios against -log10 local false discovery rate (FDR) values. A cut-off value of 1% FDR was applied to all data obtained from LC–MS/MS and quantification before performing MA plot analysis. Relative abundances (RA) were identified from the protein abundance data with a minimum of ± 0.1-fold change. The proteins that were significantly increased and decreased in relative abundance were determined with a 1.5-fold change as the cut-off value. Venn diagrams were also constructed to compare RAs of isolates within regions.

Differences in protein abundances between cold stress (CS) and control (C) conditions were analyzed by label-free relative quantitation method with the Proteome Discoverer v2.4 software. Briefly, the following parameters were used: precursor quantitation was based on intensity; normalization mode and scaling mode were set as “total peptide amount” and “on all average.” Protein abundances and ratios were calculated using the summed abundances and pairwise ratios. In this method, protein ratios were calculated as the median of all possible pairwise peptide ratios between the replicates of all related peptides. These values were normalized by the sum of their abundances for each channel over all peptides identified.

Gene Ontology Enrichment Analysis

KOBAS v2.0 (http://kobas.cbi.pku.edu.cn) was used to search for gene enrichment. This software uses gene-level statistics called overrepresentation analysis [40]. The analysis is based on the hypergeometric distribution/Fisher’s exact test with the addition of Benjamini and Hochberg [7] false discovery rate (FDR) correction. FASTA sequences were used to identify enriched pathways in the KEGG, BioCyc, and Reactome databases based on changes in protein abundances between CS and C conditions. GO terms with p ≤ 0.05 were considered significantly enriched. Saccharomyces cerevisiae was selected as the reference Ascomycota species.

Results

Response of Proteomic Profiles to Cold Stress

The intracellular protein extracts from Pseudogymnoascus spp. isolates were analyzed using tandem liquid chromatography-mass spectrometry (LCMS/MS). A total of 2541 proteins were identified with high confidence (p < 0.01) from all six isolates in cold stress (CS) and control (C) conditions (Supplementary 1). The shift in the distribution of protein abundances under CS was demonstrated on a microarray analysis (MA) plot (Fig. 1). The fold change ratios of increased or decreased relative protein abundance (RA) with a minimum of ± 0.1-fold were plotted against -log10 local false discovery rate (FDR). The MA analysis identified 720 RA in all six isolates, with relatively similar proportions being increased and decreased in RA; 383 (53.2%) and 337 (46.8%) proteins, respectively. The majority of identified proteins (i.e., 71.7%) were clustered close to 0 and had relatively high confidence values (− log10 FDR > 800). All isolates showed a similar distribution pattern of RA under CS with no indication of differences related to geographical origin.

Fig. 1
figure 1

The microarray analysis (MA) plot showing the distribution of 2,541 proteomic profiles of proteins identified in all six isolates of Pseudogymnoascus spp. under cold stress (CS). Proteins that pass a threshold of 1.5-fold change were determined as significantly up- or downregulated (red- and blue-shaded area, respectively). Colors and shapes represent different isolates; Arctic: sp1 – grey circle, sp2 – grey diamond, Antarctic: sp3 – yellow circle, sp4 – yellow diamond, and temperate region: C106 – green circle, C107 – green diamond

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A stacked bar graph was constructed to better visualize the distribution patterns of RA for each isolate (Fig. 2). The total number of RA differed noticeably among isolates from different geographical regions, with the highest numbers for the Arctic (168–271), intermediate for the Antarctic (97–102), and the lowest for the temperate region (38–44). This pattern seems to be consistent for both increased and decreased RA. However, due to the high variation, no clear pattern was visible in the proportion of increased and decreased RA. For instance, one of the Arctic isolates (i.e., sp2) exhibited a considerably higher number of increased than decreased RA proteins: 161 (59.4%) vs. 110 (40.6%), respectively. The other Arctic isolate (i.e., sp1) provided the opposite proportion, with only 70 (41.7%) vs. 98 (58.3%) increased and decreased RA proteins, respectively. The numbers of decreased RA proteins in both Arctic isolates were relatively similar, so these reverse proportions were mostly due to the very high difference in number of increased RA proteins. On the other hand, both Antarctic isolates (i.e., sp3 and sp4) produced very similar numbers and proportions of increased and decreased RA proteins, i.e., 59 (69.8%) vs. 38 (39.2%) for sp3 and 56 (54.9%) vs. 46 (45.1%) for sp4, respectively. Similarly, the temperate isolates (i.e., C106 and C107) did not differ significantly in the number and proportion of increased and decreased RA proteins. Although, some proteins demonstrated the opposite pattern compared to the Antarctic isolates, with 20 (45.5%) vs. 24 (54.5%) for C106 and 17 (44.7%) vs. 21 (55.3%) for C106 increased and decreased RA proteins, respectively.

Fig. 2
figure 2

Bar graph showing the number of differentially expressed proteins (DEPs) in response to cold stress (CS) in isolates of Pseudogymnoascus spp. from different geographical regions. Values by the bars represent the number of differentially expressed proteins (DEPs); + values, upregulated proteins; – values, downregulated proteins. The Arctic isolates: sp1, sp2; Antarctic isolates: sp3, sp4; temperate isolates: C106, C107

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Simple Venn diagrams were used to illustrate the numbers of shared and unique proteins found in isolates from the same geographical region (Fig. 3). This analysis is crucial to show the degree of similarity or differences among isolates within regions to understand the relationship of RA proteins between isolates and to identify common proteins that potentially play a major role during cold stress in Pseudogymnoascus spp. The two Arctic isolates shared only two increased RA proteins (Fig. 3a), representing hypothetical proteins with molecular weights (MW) below 30 kDa. On the other hand, the Arctic isolates shared as much as 10 decreased RA proteins (Fig. 3d). These proteins were a mixture of enzymes (pyruvate dehydrogenase complex dihydrolipoamide acetyltransferase, ATP synthase F1, and isocitrate dehydrogenase), small subunit ribosomal proteins (S3e, S13, S20, and S22), structural protein (tubulin alpha-β chain), transporter protein (protein transporter sec-23) and degradation component protein (proteasome core particle subunit alpha 2). The Antarctic isolates shared 16 RA proteins, including eight increased and eight decreased RA proteins (Fig. 3b, e). Three of the shared RA proteins among increased RA proteins belonged to hypothetical proteins with MW over 30 kDa, and only one (GI number 1040529802) hypothetical protein VE03_04039 with a MW equal to 21.9 kDa. The other four of the eight shared RA proteins among increased RA proteins included translation initiation factor eIF4, guanine nucleotide-binding protein subunit beta-like protein, isocitrate dehydrogenase, and 60S ribosomal protein L20. The decreased RA proteins shared by the Antarctic isolates included NADP-specific glutamate dehydrogenase, mitochondrial heat shock protein 60, glucose-regulated protein, and 60S ribosomal protein L11. Surprisingly, the temperate isolates did not share any increased RA protein and had in common only one decreased RA protein, i.e., the plasma membrane ATPase (Fig. 3c, f).

Fig. 3
figure 3

Venn diagrams showing the relationship between common and unique proteins in isolates from the same geographical region. a–c Upregulated proteins, d–f downregulated proteins. a and d the Arctic isolates, b and e the Antarctic isolates, c and f the temperate isolates

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Gene Ontology Enrichment Analysis of Proteins Significantly Increased in Abundance (Fold Change of ≥ 1.5)

The significantly increased RA of Pseudogymnoascus spp. was further analyzed to identify significantly increased RA proteins with a fold change of ≥ 1.5 (Table 2). A total of 176 proteins were significantly increased in abundance across all isolates, with 47.7% (84) of them identified as hypothetical proteins. Among all the analyzed isolates, Arctic sp2 had the highest numbers of significantly increased RA proteins, 91. Whereas temperate C106 had the lowest numbers of significantly increased RA proteins, 5. In general, all Pseudogymnoascus spp. isolates significantly increased the abundance of various species of large ribosomal subunit proteins (i.e., L4e, L10a, L12, L16, L20, L21e, L26e, L35, and P0) and enzymes (e.g., catalase, pyruvate carboxylase, malate dehydrogenase, fatty acid synthase, transketolase, aldehyde dehydrogenase, enolase). Furthermore, 45 (25.6%) proteins were highly abundant with a fold change of ≥ 3.0. Surprisingly, only the Arctic sp2 and Antarctic sp4 isolates showed a significant increase in abundance of heat shock proteins and hsp-like protein species (i.e., heat shock protein SSB1 and hsp70-like proteins).

Table 2 List of significantly upregulated proteins under cold stress (fold change, log2 ratios of ≥ 1.5) as recorded in Supplementary 1
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GO enrichment analysis was carried out for all 176 significantly increased RA proteins in response to cold stress using KOBAS v2.0 to search for over-represented categories of molecular pathways in the databases Kyoto Encyclopedia of Genes and Genomes (KEGG), Panther, BioCyc, and Reactome. A complete list of enriched pathways with p values ≤ 0.05 for each isolate is given in Supplementary 2. The top 10 pathways and their respective p values for each isolate are presented in Fig. 4. Surprisingly, the increased RA proteins represented a high variety of pathways. Still, no common pathways were shared between pairs of isolates from the same geographical region. For instance, in the Arctic sp1 and temperate P. pannorum C107 isolates, the majority of enriched pathways were related to various translation processes, including the SRP-dependent co-translational protein targeting to membrane, cap-dependent translation initiation, eukaryotic translation initiation, various nonsense-mediated decay (NMD) processes, and ribosomal-related pathways (i.e., the formation of a pool of free 40S subunits, GTP hydrolysis and joining of the 60S ribosomal subunits) (Fig. 4a, f). On the other hand, in the Arctic sp2 and temperate P. pannorum C106 isolates, metabolic-related pathways were enriched, including tryptophan, carbon, glyoxylate, and dicarboxylate metabolism pathways, and biosynthesis of secondary metabolites (Fig. 4b, e). In addition, the Arctic sp2 isolate showed enrichment of cellular responses to stress, biosynthesis of antibiotics, and activation of the innate immune system (Fig. 4b). In contrast, the temperate P. pannorum C106 isolate also demonstrated enrichment of some additional pathways, such as methane metabolism, peroxisomal protein import, longevity regulating pathway and detoxification of reactive oxygen species (ROS) (Fig. 4e). A more distinct profile was observed in the Antarctic sp3 isolate with the majority of enriched pathways related to energy production, such as the glycolysis, gluconeogenesis, and respiratory electron transport (ETC), and flavin/riboflavin metabolism pathways (Fig. 4c). However, the Arctic sp3 isolate showed the same enriched methane metabolism pathway, as the temperate P. pannorum C106 isolate. On the other hand, in the Antarctic sp4 isolate, the enriched pathways showed similarities with both the Arctic and temperate isolates (Fig. 4d). The increased RA proteins in that isolate showed enrichment of various metabolic pathways, mainly the biosynthesis of secondary metabolites, antibiotics, and amino acids. Moreover, the Arctic sp4 isolate also showed enrichment of protein and carbon metabolism pathways and various translation processes such as ribosomal scanning and start codon recognition, cap-dependent, and eukaryotic translation initiation pathways.

Fig. 4
figure 4

GO enrichment analysis of significantly upregulated proteins of Pseudogymnoascus spp. in response to cold stress (top 10 pathways). The Arctic isolates: a sp1 and b sp2; Antarctic isolates c sp3 and d sp4; and temperate isolates: e C106 and f C107

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Gene Ontology Enrichment Analysis of the Proteins Significantly Decreased in Abundance (with a Fold Change of ≥ −1.5)

Significantly decreased RA proteins (with a fold change of ≥  − 1.5) recorded in isolates of Pseudogymnoascus spp. in response to cold stress are listed in Table 3. A total of 148 proteins were significantly decreased in abundance across all six isolates, with 77 (52.0%) identified as hypothetical proteins. Forty-six (31.1%) of the proteins were decreased in abundance with a fold change of at least -3.0-fold. Arctic isolates (i.e., sp1 and sp2) showed many significantly decreased RA proteins (54 and 55, respectively). In contrast, one of the Antarctic isolates (i.e., sp3) had the lowest number of significantly decreased RA proteins (5), all were identified as hypothetical proteins. In general, the significantly decreased RA proteins included various enzymes in energy production processes such the TCA cycle, glycolysis and gluconeogenesis, glyceraldehyde 3-phosphate-dehydrogenase, ATP-citrate synthase subunit 1, phosphoglycerate kinase, succinyl-CoA ligase subunit beta, isocitrate dehydrogenase, acetyl Co-A hydrolase, fatty acid synthase subunit beta, and pyruvate kinase. Heat shock proteins or hsp-like proteins were only significantly decreased in abundance in the Arctic sp1 isolate (heat shock protein SSB1 and hsp70-like protein).

Table 3 List of significantly downregulated proteins under cold stress (fold change, log2 ratios of ≥ -1.5) recorded in Supplementary 1
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GO enrichment analysis of all 148 significantly decreased RA proteins showed a variety of metabolic and biosynthesis pathways enriched in all isolates (Fig. 5). Metabolic pathways related to protein homeostasis, such as protein metabolism and the biosynthesis of amino acids, were enriched in the Arctic sp1, Antarctic sp4 and both temperate isolates (C106 and C107) (Fig. 5a, d–f). The biosynthesis of secondary metabolites was also enriched in the Arctic sp1 isolate and temperate isolates (C106 and C107) (Fig. 5a, e–f). Carbon metabolism and biosynthesis of antibiotics were enriched in the Arctic sp1 and temperate C106 isolates (Fig. 5a, e), and glycogen degradation II in the Antarctic sp4 and temperate C107 isolate (Fig. 5d, f). The decreased protein pathways in the Antarctic sp4 isolate also involved the wingless-related integration site (wnt) signaling pathway, neutrophil degranulation, respiratory electron transport (ETC), urea cycle, and activation of antigen pathway (Fig. 5d). In the Arctic sp2 isolate, the majority of decreased protein pathways were related to translation processes such as the eukaryotic and cap-dependent translation initiation, nonsense-mediated decay (NMD) processes, and the formation of a pool of free 40S subunits (Fig. 5b). Pyruvate metabolism was also decreased in the Arctic sp2 isolate. In the Antarctic sp3 isolate, most decreased protein pathways were related to phospholipid metabolism involving pathways such as the phospho-PLA2, hydrolysis of lysophosphatidylcholine (LPC), acyl chain remodeling of cardiolipin (CL), phosphatidylcholine (PC), and phosphatidylinositol (PI). This isolate also showed downregulation of starch and sucrose metabolism and COPI-independent Golgi-to-ER retrograde traffic and signal amplification pathways (Fig. 5c).

Fig. 5
figure 5

GO enrichment analysis of significantly downregulated proteins of Pseudogymnoascus spp. isolates in response to cold stress (the top 10 pathways). The Arctic isolates: a sp1 and b sp2; Antarctic isolates: c sp3 and d sp4; and temperate isolates: e C106 and f C107

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Discussion

Variation in Proteomic Profiles of Pseudogymnoascus Spp. Isolates in Response to Cold Stress

All Pseudogymnoascus spp. isolates investigated in this work originated from environments that naturally experience cold temperatures, though they are exposed to wide variations in mean annual temperatures and distinct seasonal changes [6, 12]. Thus, all the investigated isolates from polar and temperate regions can be expected to share the same cold-adaptation mechanisms. However, it is worth noting that all six isolates are not of the same species, except for the temperate isolates (C106 and C107), that belong to the species P. pannorum (Table 1). Our previous work on these isolates, when exposed to heat stress, showed a diversity of protein profiling with protein homeostasis, energy production, and DNA repair pathways being enriched [2].

Similar patterns of relative protein abundances in cold stress (CS) compared to control (C) conditions (log2 ratios CS:C) did not demonstrate any apparent geographical differences in cold stress responses among the investigated isolates (Fig. 1). The individual plots of RA for each isolate (Fig. 2) showed similar findings to the distribution patterns of RA as observed in the overall MA plot (Fig. 1), again with no indication of any effect from different geographical origins. However, variation in the total number of significantly increased and decreased RA and visible shifts in proportion between increased and decreased RA (Fig. 2) suggested some likely geographical differences in cold stress responses among Pseudogymnoascus spp. isolates. For instance, the temperate isolates had the lowest number of RA, and decreased RA proteins were more abundant than increased RA proteins. The Antarctic isolates had over twice the number of RA than the temperate isolates, and increased RA proteins were dominant. Then, the Arctic isolates provided the highest number of RA, but shifts in the proportion of increased and decreased RA were inconsistent. However, a very small number of shared RA (Fig. 3) indicates the existence of very high variation, even among isolates originating from the same geographical region. From our previous work on temperature-dependent growth analysis of all six isolates, the four polar isolates (sp1, sp2, sp3, and sp4) had an optimal growth temperature at 15°C and 25°C with no significant difference between the two temperatures [2]. For the temperate isolates (C106 and C107), the optimal growth temperature was at 20°C. This suggests that the optimal growth temperature of isolates may contribute to the high number of increased and decreased RA proteins of all polar isolates compared to the temperate isolates exposed to cold stress. Therefore, analysis of a higher number of fungal isolates from all regions would be necessary to verify the consistency and significance of patterns apparent in our data.

The numbers of significantly increased and decreased RA proteins varied greatly between isolates, within a range of 5–91 proteins (Tables 2 and 3). It is noteworthy that 161 proteins were identified as hypothetical proteins from significantly increased and decreased RA proteins. These hypothetical proteins are important because they contribute to 49% of the overall significantly regulated proteins in all six isolates of Pseudogymnoascus spp. (161 from a total of 324 proteins). Our result suggests that Pseudogymnoascus spp. respond by altering only important proteins to preserve the lack of cumulative energy under cold stress. This is consistent with other studies, demonstrating a generally low number of differentially upregulated proteins. For instance, Flammulina velutipes (Curtis) Singer, a white-rot fungus that has a relatively low vegetative-growth temperature (20–24°C), under cold stress produced only 31 differentially upregulated proteins [27]. Likewise, a psychrophilic fungus Mrakia psychrophila M.X. Xin and P.J. Zhou showed increases in only 27 proteins when exposed to 4°C [32]. Similar findings were also reported for the mesophilic fungi Mortierella isabellina Oudem. M6-22 and Exophiala dermatitidis (Kano) de Hoog showed upregulation of only 29 and 33 proteins, respectively, when exposed to cold stress [21, 33]. However, while all previous studies investigated only single isolates, our work is the first to report the effects of cold stress on the proteome in several isolates of the same fungal genus from different geographical regions.

Gene Ontology Enrichment Analysis

The composition of increased RA proteins in Pseudogymnoascus spp. exposed to cold stress indicated enrichment of various metabolic and translation-related pathways. This included mostly pathways involved in the metabolism of carbon, glyoxylate, dicarboxylate, methane, and amino acids. In addition, an increment was also observed for translation-related pathways, such as forming a pool of free 40S subunits, nonsense-mediated decay (NMD), and eukaryotic and cap-dependant translation initiation pathways. Surprisingly, no increment of an identical pathway was identified, even for isolates from the same geographical region. Response mechanisms to cold stress have already been investigated in several cosmopolitan and common fungi, including Saccharomyces cerevisiae, Schizosaccharomyces pombe Lindner, and Aspergillus nidulans. Studies of these “model” fungal species have resulted in the discovery of numerous stress-related proteins [9, 30]. These include various cold-adapted enzymes and protective molecules that are produced in cold-stress conditions to increase fungal cell stability [37].

However, only a limited number of studies reported on fungal cold stress response mechanisms with the application of proteomic profiling, and the majority of these focused on mesophilic fungi [21, 27, 33]. For instance, in Exophiala dermatitidis, cold stress-induced upregulation of the beta-oxidation of very long-chain fatty acids, glycolysis/gluconeogenesis, peroxisomal lipid metabolism, and cellular response to stress [33]. Flammulina velutipes also showed upregulation of amino acid biosynthesis, signaling pathways, and various energy metabolism pathways, such as the citrate cycle (TCA cycle), pentose phosphate pathway, glyoxylate, and dicarboxylate metabolism [27]. In comparison, the psychrophilic fungus Mrakia psychrophila demonstrated upregulation of energy metabolism and production of unsaturated fatty acids that regulate membrane fluidity [32]. In this work, we also showed a significant number of hypothetical proteins, demonstrating that functional studies on these proteins from polar fungi are still lacking. Hence, studying these functionally unknown sequences could provide additional insight into potential mechanisms governing cold adaptation of Pseudogymnoascus spp.

Previous studies have also demonstrated that low temperatures do not cause irreversible damage to fungal cells, and fungi respond to cold stress by modifying molecular content in their complex protein networks [27, 32]. Comparison across all six isolates of Pseudogymnoascus spp. studied here revealed no apparent geographical pattern in protein profiles or pathways involved. This was particularly the case for pathways of carbon metabolism, biosynthesis of amino acids, secondary metabolites and antibiotics, and translation-related pathways. Our findings suggest that Pseudogymnoascus spp. modulate various carbon and amino acid metabolism and translation-related pathways to minimize energy use for growth or cell division. We postulated that Pseudogymnoascus spp. adapt to cold stress by utilizing nutrient availability to support cell damage and repair and minimizing protein production for cell growth. Su et al. [32] reported that Mrakia psychrophila showed downregulation of TCA cycle, glycolysis, and ribosomal proteins. Similarly, Exophiala dermatitidis demonstrated downregulation of carbon and pyruvate metabolism and the pentose phosphate pathway [33]. In our study, only the Antarctic sp3 isolate showed a distinctive profile of cold stress response. The upregulated pathways of that isolate included mainly flavin/riboflavin biosynthesis, glycolysis/gluconeogenesis, respiratory electron transport (ETC), and methane metabolism. It is important to indicate that biogenic methane production is generally only associated with prokaryotic microorganisms such as methanogens and Archaea [11]. However, Lenhart et al. [25] have suggested that terrestrial vegetation and fungi can also be involved in the production of methane. There are a few decreased protein pathways that were identified only in sp3 and not in any others isolates, such as acyl chain remodeling of cardiolipin, phosphatidylcholine, phosphatidylinositol, and the hydrolysis of lysophosphatidylcholine. Phospholipids are key molecules involved in the maintenance of membrane fluidity and are also involved in signaling pathways. The metabolism of fungal phospholipids has been extensively studied in the model organism, S. cerevisiae [29].

Conclusions

When exposed to cold stress, our results showed a variation in increased and decreased protein abundances between different isolates of Pseudogymnoascus spp. Several metabolic enzymes and ribosomal proteins were significantly increased and decreased in abundance in all six isolates of Pseudogymnoascus spp. examined. Pathway enrichment analysis also showed diversity in the cold stress response pathways, with metabolic and translation-related processes being prominent in most isolates. However, the Antarctic isolate sp3 showed a distinctive cold stress response profile involving increased flavin/riboflavin biosynthesis and methane metabolism. The Antarctic sp3 isolate is also the only one that showed decreased phospholipid metabolism when exposed to cold stress. Our results suggest that Pseudogymnoascus spp. adapt to cold stress by utilizing nutrient availability to support cell damage and repair with minimal need for cell growth. The cold stress response of the Pseudogymnoascus spp. isolates examined, while showing wide variation in the pathways enriched, did not show any obvious association with the biogeographical regions of origins of the isolates. The data obtained in this study provides new information on how Pseudogymnoascus spp. respond to temperature variations in their environments. This work also improves our understanding of their responses and adaptions toward varying environmental temperatures that may affect their survival in soil ecosystems. We would like to emphasize the need for whole genome analysis of Pseudogymnoascus spp. for future works to support functional annotation of unknown sequences of various hypothetical proteins, thus providing additional insight into potential mechanisms governing cold adaptation of Pseudogymnoascus spp.