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Article

Characterization and Expression Analysis of the Sterol C-5 Desaturase Gene PcERG3 in the Mycobiont of the Lichen Peltigera canina Under Abiotic Stresses

by
Moatasem A. Swid
1,2,
Milana V. Koulintchenko
1,
Alfred O. Onele
1,
Ilya Y. Leksin
1,
Daniya F. Rakhmatullina
1,
Ekaterina I. Galeeva
1,
Julia N. Valitova
1,
Farida V. Minibayeva
1 and
Richard P. Beckett
1,*
1
Kazan Institute of Biochemistry and Biophysics, FRC Kazan Scientific Center, RAS, P.O. Box 261, 420111 Kazan, Russia
2
Institute of Fundamental Medicine and Biology, Kazan Federal University, Kremlyovskaya 18, 420008 Kazan, Russia
*
Author to whom correspondence should be addressed.
Microbiol. Res. 2025, 16(7), 139; https://doi.org/10.3390/microbiolres16070139
Submission received: 20 May 2025 / Revised: 23 June 2025 / Accepted: 24 June 2025 / Published: 1 July 2025
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Abstract

Lichens, symbiotic organisms with a high tolerance to harsh environments, possess a greater diversity of sterols than other organisms. Sterols are involved in maintaining membrane integrity, hormone biosynthesis, and signal transduction. (1) Background: A characteristic feature of lichen sterols is a high degree of unsaturation, which influences membrane properties. Desaturases play an important role in the synthesis of unsaturated sterols, in particular, sterol C-5 desaturase (ERG3), which controls the conversion of episterol to ergosterol. Earlier, we demonstrated that the treatment of the lichen Peltigera canina with low and elevated temperatures results in changes in the levels of episterol and ergosterol. (2) Methods: Here, for the first time, we identified ERG3 in P. canina and, using an in silico analysis, we showed that PcERG3 belongs to the superfamily of fatty acid hydrolyases. A phylogenetic analysis was conducted to determine the evolutionary relationships of PcERG3. (3) Results: A phylogenetic analysis showed that PcERG3 clusters with ERG3 from other Peltigeralian and non-Peltigeralian lichens and also with ERG3 from free-living fungi. This suggests that PcERG3 has an ancient evolutionary origin and is related to fungi with lichenized ancestors, e.g., Penicillium. The differential expression of PcERG3 in response to temperature stress, a dehydration/rehydration cycle, and heavy metal exposure suggests that it plays a crucial role in maintaining the balance between more and less saturated sterols and, more generally, in membrane functioning. The multifaceted response of P. canina to abiotic stresses was documented by simultaneously measuring changes in the expression of PcERG3, as well as the genes encoding the heat shock proteins, PcHSP20 and PcHSP98, and PcSOD1, which encodes the antioxidant enzyme superoxide dismutase. (4) Conclusions: These findings suggest that PcERG3 is similar to the sterol C-5 desaturases from related and free-living fungi and plays important roles in the molecular mechanisms underlying the tolerance of lichens to environmental stress.

1. Introduction

Lichens are fascinating symbiotic organisms, comprising fungal (the mycobiont) and algal or cyanobacterial (the photobiont) partners. Lichens are highly tolerant to a range of abiotic stresses, enabling them to grow in extreme habitats, which would be lethal for many other organisms [1]. For example, lichens are desiccation-tolerant, meaning that they can lose most of their water and then recover normal function when water becomes available again. Furthermore, when dry, they can tolerate very high temperatures. However, when hydrated, they have a similar thermotolerance to other photosynthetic organisms. Even in lichens, stress increases the production of reactive oxygen species (ROS) [2]. While low concentrations of ROS are thought to serve as cellular-signaling molecules, oxidative damage happens when the antioxidant defense system is overwhelmed [3]. Interestingly, despite their high tolerance to many abiotic stresses, they are sensitive to pollution and climate change, which threatens the diversity of life on Earth [4]. For example, lichens only adapt very poorly to the increases in temperature that accompany global warming [5]. Furthermore, their lack of a cuticle makes them susceptible to airborne pollutants, including heavy metals. Nevertheless, the high tolerance of lichens to abiotic stresses makes them an attractive model in studying how “extremophiles” can survive in harsh environments.
Studies on lichen sterols suggest that their high resistance to abiotic stress may result, in part, from their unique sterol composition, which differs from that of free-living fungi and algae [6]. For example, we have shown that the lichen Peltigera canina contains dehydroergosterol, lichesterol, ergosterol, fungesterol, and episterol [7]. However, in common with other fungi, the main sterol in lichens is ergosterol [6,8]. Ergosterol plays important roles, including maintaining membrane structural stability and fluidity; controlling selective permeability; helping to organize lipid rafts; and generally protecting fungi from environmental stresses, such as heat and chilling [9,10]. Furthermore, ergosterol also acts as an antioxidant, helping to neutralize reactive oxygen species (ROS), thus enhancing cellular resilience under oxidative stress conditions [8]. Ergosterol displays high reactivity due to the presence of conjugated double bonds within its ring [8]. In lichens, ergosterol is a metabolic marker, as levels of ergosterol are positively correlated with respiration and oxidative phosphorylation in mitochondria [11]. The antioxidant function of ergosterol may be particularly relevant in lichens, which often experience desiccation and high light intensity, which can promote ROS generation [1,2]. Numerous enzymes are involved in ergosterol biosynthesis; sterol desaturases, in particular, play an important role by increasing sterol unsaturation. Specifically, the enzyme sterol C-5 desaturase (ERG3) introduces an ∆5-bond into episterol, a precursor of ergosterol biosynthesis [12]. ERG3 is cyanide-sensitive, requires iron and molecular oxygen for activity, and is coupled to the NAD(P)H-cytochrome b/cytochrome b5 reductase microsomal electron transport system [13]. ERG3 has been conserved throughout evolution, as demonstrated by the presence of ∆5 sterols in all organisms from the protist, fungal, plant, and animal kingdoms [14]. For example, ERG3 in plants produces campesterol by catalyzing the conversion of episterol to 5-dehydroepisterol [15]. Campesterol is a precursor to the plant hormones brassinosteroids, which are crucial for growth and development. Furthermore, ERG3 catalyzes the transformation of avenosterol into 5-dehydroavenosterol, which is then transformed into β-sitosterol, a crucial membrane component [16]. In the model plant Arabidopsis, the mutation of ERG3 results in the accumulation of ∆7 sterols and delayed seed germination [17]. Therefore, ERG3 plays many essential roles in fungal and higher plant metabolism.
Despite the importance of ergosterol in lichen biology, the structure and function of ERG3 has not been characterized from any mycobiont. Therefore, the first aim of the present study was to identify the gene sequence of ERG3, using the lichen P. canina as a model species. An in silico analysis of the structure and phylogeny of the corresponding protein, PcERG3, was then carried out. The second aim of this study was to investigate the role of PcERG3 in stress tolerance. We used quantitative real-time PCR to analyze changes in the expression of PcERG3 in response to abiotic stress treatments, specifically temperature extremes, desiccation, and the heavy metal copper. To confirm that the lichens were indeed stressed, we analyzed the expression of other classical stress-upregulated genes, including those encoding heat-shock proteins (HSPs) and the antioxidative enzyme superoxide dismutase (SOD). The results showed the following: first, the PcERG3 gene is similar to the sterol C-5 desaturases from free-living fungi and, second, the expression analyses suggested that, together with other stress-upregulated enzymes, it is likely to play an important role in the tolerance of lichens to environmental stress.

2. Materials and Methods

2.1. Lichen Material

Thalli of P. canina (L.) Willd. were collected in the territory of the Aishinsky nature reserve of the Republic of Tatarstan, Russia (55°53 21.3 N 48°38 14.3 E) (Figure 1). The thalli were placed between sheets of filter paper, left to air-dry slowly at room temperature for 2 d, and then stored at −20 °C; until they were used.

2.2. Cloning of PcERG3 Gene

Total P. canina RNA was extracted using the RNeasy Plant Mini Kit (Qiagen, Venlo, The Netherlands). First-strand and double-strand cDNA were synthesized using the Evrogen Mint 2 synthesis kit, according to the manufacturer’s protocols (Evrogen, Moscow, Russia). cDNA was obtained with standard PCR, with Taq polymerase (Evrogen) and specific primers.
The primers for the ERG3 were designed based on metagenomic data derived from the sequencing of the lichen P. canina (SRA accession SRR15568737; BioProject PRJNA756680). Protein-coding genes were detected using the Augustus program, based on metagenomic assembly (GenBank GCA_020063925.1). To search for PcERG3, a screening of ERG3 homologues was performed using BLAST (v. 2.2.18) among the CDSs of the predicted genes. The best results were compared with ERG3 from other Lecanoromycetes, including Peltigera leucophlebia, allowing us to predict the CDS of the ERG3 of P. canina. The detected ERG3 sequence was used for the primer construction. The size of the PCR products was confirmed by agarose gel electrophoresis, then the cDNA of the predicted size was eluted from the gel and cloned into the pAL2-T vector (Evrogen).

2.3. Sequence Analysis

Plasmid DNA containing the cDNA insert (200 ng) of PcERG3 was used for sequencing. The Sanger reaction was performed using the Big-Dye Terminator v3.1 Cycle Sequencing kit (Thermo Scientific, Waltham, MA, USA). The reaction was carried out using SP6-Forward and T7-Reverse primers. BLASTN software (v. 2.2.18), available online at BLAST: Basic Local Alignment Search Tool (nih.gov), was used to perform a homology search to compare the sequenced gene with other genes in the database. Files in the Fasta format were downloaded from the NCBI database after a BLAST search and then subjected to sequence alignments.

2.4. Alignment and Phylogenetic Analysis

The EMBOSS web service (EMBOSS Transeq < Sequence Translation Sites < EMBL-EBI) was used to translate the nucleic acid sequences of PcERG3 cDNA to their corresponding peptide sequences. BLASTP software (v. 2.2.18), available online, was used to perform a homology search in the database of predicted protein sequences, with proteins from other species. Homologous sequences of the PcERG3 protein were obtained after using BLASTX and other known homologous ERG3 proteins from the NCBI database. These protein sequences were aligned with the MUSCLE algorithm [18], with standard parameters, and were trimmed by the trimAl v.1.2 program [19], using the «automated1» mode. The phylogenetic tree was constructed using the IQ-TREE program (v. 3.0.1) [20], using an ultrafast bootstrap [21]. The phylogenetic trees were constructed using the iTOL tool (v. 7.2) [22] and edited in the Inkscape (v.1.4) program.

2.5. Structural Analysis of Predicted PcERG3 from P. canina

The Expasy ProtParam tool was used to estimate the physicochemical characteristics of PcERG3, such as its molecular weight, theoretical pI, amino acid composition, atomic composition, extinction coefficient, estimated half-life, instability index, aliphatic index, and its grand average of hydropathy (GRAVY). Secondary structure properties, such as the α-helix, β-sheet, and β-turns of PcERG3, were predicted using the PSI-blast-based secondary structure PREDiction (PSIPRED) and the self-optimized prediction method with alignment (SOPMA). Structural analyses of the protein, including its classification into a family and a prediction of conserved domains, were performed using the software InterProScan (v. 5.52-86.0) and PROSITE—Expasy.

2.6. Stress Treatments

Prior to the stress treatments, dry thalli were pre-hydrated at +10 °C in a semi-transparent container on a damp cloth, in dim laboratory lighting, for 48 h. The thalli were then gently blotted with filter paper before the experiment. On the day of the experiment, the containers with the hydrated thalli of P. canina were kept at room temperature under a lamp at c. 30 µmol m−2 s−1 for 2–3 h for acclimation. For temperature treatment, the lichens were exposed to −20 °C or +40 °C for 3 h in a temperature-controlled chamber (TSO-1/80 SPU thermostat, Moscow, Russia) under lighting 16–20 µmol m−2 s−1, and then were immediately sampled. The hydrated thalli were kept at room temperature and were used as controls. For the heavy metal treatment, the thalli (0.3 g fresh mass) were incubated in 5 mL of 250 µM CuSO4 for 10 h in a Petri dish with gentle shaking under laboratory lighting, and were then sampled. To induce dehydration/rehydration stress, the lichens were dehydrated to a relative water content (RWC) of 12% by placing them in a desiccator above saturated CaCl2 solution for 8 h at room temperature, giving a relative humidity of c. 30%. The thalli were rehydrated by spraying them with distilled water every 20 min for 1 h and then sampled.

2.7. RNA Extraction, cDNA Synthesis, and RT-qPCR

The samples exposed to stresses were immersed in liquid nitrogen, then each sample was ground into a fine powder. For RT-qPCR, 0.1 g of material from each replicate was frozen in liquid nitrogen and stored at −80 °C until use. The extraction of total RNA was performed using the RNeasy Plant Mini kit (Qiagen, Hilden, Germany), according to the manufacturer’s protocol. The RNA concentration and purity were measured with the NanoDrop® ND-1000 spectrophotometer (Thermo Scientific, Waltham, MA, USA) and the integrity was further evaluated by gel electrophoresis in a 1% (w/v) agarose gel. First-strand cDNA was synthesized using protocols from the Evrogen Mint 2 synthesis kit (Evrogen, Moscow, Russia).
For RT-qPCR, PcGAPDH (glyceraldehyde-3-phosphate dehydrogenase), PcKG (α-ketoglutarate dehydrogenase complex subunit E1) were used as reference genes. The vector NTI Suite 9 software was used to design the RT-qPCR primers, with the following parameters: an amplicon length from 60 to 300 bp and a Tm range of 55 to 65 °C. The primers used in this study are shown in Table S1. The RT-qPCR conditions used in our study followed the protocol described in [23].

2.8. Data Analysis

Three biological replicates, each with two analytical replicates, were used to run all reactions. Gene expression differences were assessed using normalized expression (Cq) in the Bio-Rad CFX Maestro Software version 2.3. The statistical analysis was conducted using one-way ANOVA and Tukey’s post hoc test, and differences where p < 0.05 were considered significant. The standard errors of the mean (SE) were shown as vertical bars (n = 3).

3. Results

3.1. In Silico Analysis of Predicted PcERG3 Protein

One ORF corresponding to the known PcERG3 gene was detected using the cDNA library. Using total RNA from P. canina, cDNA was obtained, cloned, and sequenced. The size of the obtained cDNA product corresponded to the size of the transcripts predicted in silico. The BLAST analysis of the cloned PcERG3 cDNA from P. canina did not reveal sequence homology with the known transcripts of lichens or fungi. However, when the predicted peptide sequence (355 aa) was applied for analysis in BLASTp alignment, the highest homology was found between P. canina and sequences from other lichens (Table 1), suggesting that ERG3 from P. canina is conserved within lichens.
To investigate the evolutionary relationships between ERG3 in P. canina and other organisms, a neighbor-joining phylogenetic tree was constructed using homologous peptide sequences. A phylogenetic analysis of the obtained homologous peptide sequences revealed that the sequence of PcERG3 is quite conservative, with a high similarity to sterol C-5 desaturase from other lichens (almost 75%), as well as from numerous Candida spp. and Saccharomyces cerevisiae (close to 55%). It is worth noting that PcERG3 shares more homology with the animal proteins involved in cholesterol biosynthesis (approximately 50%) than those in plant species such as Arabidopsis (32%) (Figure 2, Table 1). PcERG3 clustered most tightly with sterol C-5 desaturase from P. leucophlebia within the Order Peltigerales (89%), suggesting a common ancestry among these proteins.
The physicochemical properties of PcERG3 were predicted using the ExPASy ProtParam tool. The molecular weight of PcERG3 was estimated to be 41.45 kDa, with a theoretical pI of 6.97 and an aliphatic index of 85.64. The instability index was 32.10 and GRAVY was −0.073. The secondary structure of the protein chain was analyzed by SOPMA, which predicted the presence of α-helix, β-sheet, and β-turns (Figure S1A). In the predicted secondary structure of PcERG3, α-helix comprised 43.63%, random coils comprised 34.56%, and extended strands comprised 18.13%. The predominance of α-helix and coiling in the structure and the presence of four transmembrane domains (Figure S1A) suggested that PcERG3 is a transmembrane protein with extensive intramolecular interactions, making it highly stable and resistant to denaturation.
An analysis using InterPro showed that PcERG3 belongs to the superfamily of fatty acid hydroxylases and to the sterol desaturase family. The results from InterPro also showed that PcERG3 contains three non-cytoplasmic domains and two cytoplasmic domains (Figure S1B).

3.2. Expression of ERG3 Gene of P. canina Under Abiotic Stresses

When the lichen thalli were exposed to freezing temperatures, PcERG3 expression slightly decreased. However, after heat stress, PcERG3 expression significantly increased in comparison to the control (Figure 3A). Treatment with CuSO4 caused a slight (non-significant) decrease in PcERG3 expression (Figure 3B). Dehydration for 8 h greatly reduced PcERG3 expression, while rehydration rapidly increased PcERG3 expression back to initial levels (Figure 3B).

3.3. Expression of General Stress Responsive Genes of P. canina

Subjecting the lichen thalli to −20 °C for 3 h significantly decreased the expression of the HSPs PcHSP20 and PcHSP98, while subjecting them to +40 °C significantly increased expression (Figure 4). For PcSOD1, −20 °C significantly increased expression, whereas heat stress had no significant effect (Figure 5A). There was a small (non-significant) increase in PcSOD1 expression after 10 h of exposure to CuSO4 (Figure 5A). Dehydration had no effect on PcSOD1 expression, whereas expression greatly increased during rehydration (Figure 5B).

4. Discussion

Ergosterol is an important sterol in lichenized and free-living fungi, and is generally considered to have important roles in protecting fungi from environmental stresses [9]. Ergosterol biosynthesis is a multi-step process that requires several specific enzymes, including desaturases. Here, for the first time, we identified a gene encoding the sterol C-5 desaturase in the lichen P. canina (PcERG3). The differential expression of PcERG3 and of stress-responsive genes such as PcHSP20, PcHSP98, and PcSOD1 following abiotic stresses suggest that ergosterol, together with HSPs and ROS-scavenging antioxidative enzymes, play significant roles in the survival of the lichen P. canina in harsh environments.

4.1. Phylogenetic Analysis of PcERG3

As expected, the phylogenetic analysis showed that the ERG3 sequence from P. canina clusters with that of P. leucophlebia (Figure 2, Table 1). Notably, PcERG3 also groups with ERG3 homologs from other lichens and from some free-living fungi, such as Penicillium that are believed to have evolved from lichenized ancestors [24]. The presence of highly conserved ERG3 homologs across both lichen-forming and free-living fungi highlights the essential and evolutionarily conserved role of sterol C-5 desaturase activity. In fungi, the primary role of this enzyme is to contribute to ergosterol biosynthesis.

4.2. Theoretical Prediction of the Physicochemical Characteristics of PcERG3

Expasy ProtParam predicted that PcERG3 has a molecular weight of 41,449.69 Da and a theoretical isoelectric point (pI) of 6.97, indicating its stability in environments with a near-neutral pH. The high aliphatic index of this protein (85.64) indicates that it possesses a significant proportion of hydrophobic residues, which may contribute to its thermal stability. With an instability index of 32.10, PcERG3 is expected to be stable under physiological conditions. The negative GRAVY value of −0.073 suggests that it is hydrophilic and likely to be soluble in aqueous media. The secondary structure of PcERG3 consists mainly of α-helices (44%), followed by random coils (35%) and extended strands (18%) (Figure S1A). Taken together, these physicochemical properties indicate that PcERG3 is a stable, soluble, and structurally flexible protein, which can be involved in the regulation of ergosterol levels in stressful environments.

4.3. Protein Domain Architecture of PcERG3

The in silico structural analysis of PcERG3 (Figure S1B) and the prediction of the presence of a conserved FA hydroxylase domain (Table S2) suggested that PcERG3 belongs to the fatty acid hydroxylase superfamily, which comprises fatty acid and carotene hydroxylases and sterol desaturases [25]. Members of the sterol desaturase family are involved, not only in ergosterol biosynthesis in fungi [26], but also in cholesterol synthesis in mammals [27] and in the synthesis of sterols, brassinosteroids, and cuticular wax in plants [28]. This family contains sterol C-5 desaturase (ERG3) and C-4 sterol methyl oxidase, which both contain two copies of an HXHH motif that coordinates two Fe3+ ions at the catalytic center. Being ER-integral membrane proteins, they share a mushroom-shaped fold, consisting of four transmembrane (TM1–TM4) helices with a cytoplasmic domain that contains a histidine-coordinating catalytic center [28]. PcERG3 also contains two cytoplasmic domains (Figure S1B), which are involved not only in catalytic activity, but also in the stabilization of protein structure [29].

4.4. Changes in the Expression of PcERG3 Genes During Stress

The final stages of sterol biosynthesis, particularly those catalyzed by desaturase enzymes, are known to be highly sensitive to abiotic stresses [30]. In our previous work, we showed that both freezing and elevated temperatures significantly alter the sterol profile in P. canina, with a decline in the proportion of ergosterol in total sterols and an increase in the proportion of episterol, its more saturated precursor [7]. In this study, we found that these environmental stresses modulate the expression of PcERG3, the gene-encoding sterol C-5 desaturase. Notably, the elevated temperatures upregulated the expression of PcERG3, while the expression was downregulated by freezing (Figure 3A).
The downregulation of PcERG3 during freezing likely reflects a general suppression of metabolic activity under cold conditions rather than an adaptive mechanism aimed at increasing membrane saturation. In general, decreased rather than increased sterol saturation would be expected to increase membrane fluidity and, thereby, contribute to improved cold tolerance. In contrast, the marked upregulation of PcERG3 under heat stress likely represents an active adaptive response. Ergosterol plays a critical role in maintaining membrane integrity under thermal stress by stabilizing lipid bilayers and preventing excessive fluidization [10]. Increased sterol desaturation may help to fine-tune membrane properties, ensuring an optimal balance between fluidity and rigidity during heat exposure.
The importance of sterol desaturation in stress tolerance in fungi is supported by several other studies in the literature. For example, the overexpression of a C-5 sterol desaturase from the edible mushroom Schizosaccharomyces pombe confers enhanced tolerance to both heat and ethanol, directly linking sterol desaturase activity with stress resilience [31]. Similarly, a comparable increased tolerance to heat and ethanol in a sterol auxotroph of Saccharomyces cerevisiae was correlated with higher ergosterol levels, and was independent of classical heat shock responses such as HSP accumulation or trehalose synthesis [32]. These findings suggest that ergosterol plays a specific role in protecting cells under thermal stress.
In addition to conferring heat tolerance, other studies have shown that ergosterol stabilizes membranes during stress, influences lipid raft formation, and reduces ROS damage by acting as an antioxidant [10]. These functions are likely to be particularly important in lichens, which are frequently exposed to fluctuating and extreme environmental conditions. Therefore, the strong upregulation of PcERG3 in response to heat in P. canina may represent a conserved thermoprotective strategy, which ensures adequate ergosterol production for membrane homeostasis, antioxidant defense, and stress adaptation.
In this study, the expression of PcERG3 was slightly reduced following the exposure of the thalli to 250 µM CuSO4 (Figure 3B), suggesting that copper toxicity can disrupt sterol synthesis. Apart from directly binding to enzymes, copper ions can induce oxidative stress, reducing the activity of enzymes, such as ERG3, that are involved in sterol biosynthesis [6]. Similarly, dehydration significantly decreases the expression of PcERG3, likely reflecting a general reduction in metabolic processes in the dry state of lichen. However, upon rehydration, the expression of PcERG3 rapidly returned to initial levels, indicating that normal sterol biosynthesis can quickly resume (Figure 3B). Taken together, the variable patterns of PcERG3 expression indicate that this gene is important in the maintenance of cellular homeostasis in P. canina under abiotic stresses, contributing to lichen stress tolerance.

4.5. Changes in Expression of HSP and SOD Genes During Stress

In addition to modifying their lipid composition, lichens respond to abiotic stress in a variety of other ways, including the upregulation of HSPs and antioxidative enzymes. The upregulation of the genes encoding HSPs is a common stress response in many organisms. This appears to be primarily because HSPs can unfold and refold proteins that become misfolded during stress, in particular heat stress [33]. HSPs are classified into the following families based on their molecular masses: HSP100, HSP90, HSP70, HSP60, HSP40, and low-molecular-weight HSPs. The higher molecular mass proteins from the HSP100 family, including HSP98, play important roles in reactivating aggregated proteins by resolubilizing non-functional protein aggregates, which facilitates the degradation of irreversibly damaged polypeptides [34]. Thermal stress has been shown to upregulate genes encoding HSP98 in the free-living ascomycete Aspergillus fumigatus [35]. In contrast, small HSPs are ATP-independent chaperones that delay the onset of protein misfolding and the initiation of aggregation. Furthermore, small HSPs are important in maintaining membrane functioning during stress [36], playing similar roles to those of the membrane sterol component. In the free-living fungus A. nidulans, small HSPs are upregulated following exposure to temperature, osmotic, and oxidative stresses [37]. Surprisingly, there have been few studies on the roles of HSPs in stress tolerance in lichens. However, heat stress increases the expression of HSPs in the lichens Peltigera brittanica, P. membranacea, and Lobaria pulmonaria, as demonstrated in [38], [39], and [34], respectively. In the present study, we analyzed the expression of genes encoding representative low- and high-molecular-mass HSPs, specifically PcHSP20 and PcHSP98. The results showed that low temperatures reduce the expression of PcHSP20 and PcHSP98, while an increase in temperature upregulates the expression of these genes (Figure 4). The downregulation observed following freezing conditions probably reflected the general reduction in metabolic activity that occurs at low temperatures. Conversely, the upregulation of HSPs during heat stress is probably a more active response, which helps to prevent protein aggregation and facilitates refolding [40]. Taken together, the results presented here show that the treatments used did indeed cause stress, and that HSPs probably work together with ERG3 to enable lichens to tolerate stress.
Temperature stress increases the production of potentially harmful ROS in free-living fungi [41]. Therefore, not surprisingly, it also upregulates ROS-scavenging enzymes [42]. In the present study, freezing temperatures increased the expression of PcSOD1 (Figure 5A), presumably to scavenge ROS-induced O2− radicals [43]. Interestingly, PcSOD1 expression did not change following heat stress. Although heat stress has been shown to induce oxidative stress, presumably in P. canina, other antioxidant mechanisms, such as ergosterol or HSPs, are involved in ROS detoxification. Heavy metals can also cause oxidative stress. It has been observed in other organisms that enhanced SOD activity can occur, which may reduce oxidative damage and preserve cellular redox balance [44,45]. In the present study, PcSOD1 expression increased in response to CuSO4 exposure. This was probably in response to increases in the levels of ROS in the lichens, which was caused by heavy metals (Figure 5B) [46].
Lichens are poikilohydric organisms that can survive severe desiccation due to numerous protective mechanisms, including the activity of antioxidants [2,47,48]. However, the expression of PcSOD1 did not change significantly during dehydration (Figure 5B), which was consistent with the observation that SOD activity is typically very low during the early stages of the rehydration of desiccated lichens [49]. Presumably, other enzymatic or non–enzymatic ROS-scavenging systems protect lichens during desiccation and early imbibitional stress. However, the large increase in PcSOD1 expression 1 h after rehydration (Figure 5B) suggested that this enzyme plays a role in restoring redox balance and promoting the recovery of lichens following desiccation stress.

5. Conclusions

In free-living fungi, ergosterol, an important component of fungal membranes, is not only essential for fungal growth and development, but also very important for adaptation to stress [10]. Here, for the first time in P. canina, we identified ERG3, the gene encoding sterol C-5 desaturase (ERG3). ERG3 is a key enzyme responsible for converting episterol to ergosterol. Bioinformatic analyses showed that ERG3 from P. canina belongs to the fatty acid hydrolyase superfamily. A phylogenetic analysis showed that PcERG3 clusters with ERG3 from other peltigeralian and non-peltigeralian lichens, but also with ERG3 from free-living fungi, including lichenized ancestors, e.g., Penicillium. This suggests that the gene has an ancient evolutionary origin. Among the various stress conditions tested, heat stress caused a particularly strong upregulation of PcERG3. The differential expression of PcERG3 in response to chilling and heat stresses, a dehydration/rehydration cycle, and heavy metal exposure suggests that regulating the balance between more- and less-saturated sterols is key to maintaining membrane functioning during stress. Further studies are needed to explore how ergosterol levels correlate with stress tolerance in lichens. This could include the experimental manipulation of ergosterol contents to assess the physiological impacts under stress, as well as broader transcriptional or proteomic analyses of the entire ergosterol biosynthetic pathway to identify other stress-responsive regulatory nodes. Such investigations will help to clarify the role of sterols in the remarkable resilience of lichens to extreme environmental conditions.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/microbiolres16070139/s1. Figure S1: In silico analysis of PcERG3; Table S1: Primers of RT-qPCR.; Table S2: Prediction of the presence of a conserved FA hydroxylase domain in sterol C-5 desaturase.

Author Contributions

Authors’ contributions to the manuscript: Conceptualization, M.A.S., J.N.V. and F.V.M.; data curation, J.N.V.; Formal analysis, M.A.S., M.V.K., A.O.O. and I.Y.L.; funding acquisition, J.N.V.; investigation, M.A.S., M.V.K., A.O.O. and I.Y.L.; methodology, D.F.R. and E.I.G.; project administration, J.N.V. and F.V.M.; supervision, J.N.V. and F.V.M.; validation, R.P.B.; writing—original draft, M.A.S. and J.N.V.; and writing—review & editing, M.A.S., J.N.V., F.V.M. and R.P.B. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Russian Science Foundation (grant № 22-14-00362, gene cloning and sequencing, gene expression).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in the study are included in the article/Supplementary Materials section. Further inquiries can be directed to the corresponding authors.

Acknowledgments

The research was carried out within the framework of the state assignment of the FRC Kazan Scientific Center, the Russian Academy of Sciences (phylogenetic analysis).

Conflicts of Interest

The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Abbreviations

The following abbreviations are used in this manuscript:
HSPHeat shock protein
ROSReactive oxygen species
SODSuperoxide dismutase
FAFatty Acid

References

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Microbiolres 16 00139 g001
Figure 1. Gross morphology of the lichen Peltigera canina (taken from https://en.wikipedia.org/wiki/Peltigera_canina, accessed on 23 June 2025).
Figure 1. Gross morphology of the lichen Peltigera canina (taken from https://en.wikipedia.org/wiki/Peltigera_canina, accessed on 23 June 2025).
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Figure 2. A phylogenetic tree showing the relationships between retrieved homologous peptide sequences for PcERG3, constructed in IQ-TREE using the maximum likelihood (ML) method, with 2500 replicates of ultrafast bootstraps. Branch support is given using numbers. ERG3 from P. canina is indicated in red. Colors indicate general evolutionary relationships. The scale represents 1.0 amino acid sequence substitutions per site.
Figure 2. A phylogenetic tree showing the relationships between retrieved homologous peptide sequences for PcERG3, constructed in IQ-TREE using the maximum likelihood (ML) method, with 2500 replicates of ultrafast bootstraps. Branch support is given using numbers. ERG3 from P. canina is indicated in red. Colors indicate general evolutionary relationships. The scale represents 1.0 amino acid sequence substitutions per site.
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Microbiolres 16 00139 g003
Figure 3. The effect of abiotic stresses on the relative expression of PcERG3. Thalli were exposed to (A) −20 °C and +40 °C for 3 h or (B) 250 µM CuSO4 for 10 h, and dehydration for 8 h followed by rehydration for 1 h (B). Hydrated thalli were kept at room temperature and used as controls. Statistical analysis was conducted using one-way ANOVA and Tukey’s post hoc test. Bars with different letters on top significantly differ (p < 0.05). The standard errors of the mean are shown as vertical bars (n = 3).
Figure 3. The effect of abiotic stresses on the relative expression of PcERG3. Thalli were exposed to (A) −20 °C and +40 °C for 3 h or (B) 250 µM CuSO4 for 10 h, and dehydration for 8 h followed by rehydration for 1 h (B). Hydrated thalli were kept at room temperature and used as controls. Statistical analysis was conducted using one-way ANOVA and Tukey’s post hoc test. Bars with different letters on top significantly differ (p < 0.05). The standard errors of the mean are shown as vertical bars (n = 3).
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Microbiolres 16 00139 g004
Figure 4. Relative expression of PcHSP20 and PcHSP98 of P. canina exposed to −20 °C and +40 °C for 3 h. Statistical analysis was conducted using one-way ANOVA and Tukey’s post hoc test. Bars with different letters on top significantly differ (p < 0.05) The standard errors of the mean are shown as vertical bars (n = 3).
Figure 4. Relative expression of PcHSP20 and PcHSP98 of P. canina exposed to −20 °C and +40 °C for 3 h. Statistical analysis was conducted using one-way ANOVA and Tukey’s post hoc test. Bars with different letters on top significantly differ (p < 0.05) The standard errors of the mean are shown as vertical bars (n = 3).
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Microbiolres 16 00139 g005
Figure 5. The effect of abiotic stresses on the relative expression of PcSOD1. Thalli were exposed to (A) −20 °C and +40 °C for 3 h and 250 µM CuSO4 for 10 h, and (B) dehydration for 8 h followed by rehydration for 1 h. Hydrated thalli were kept at room temperature and used as controls. Statistical analysis was conducted using one-way ANOVA and Tukey’s post hoc test. Bars with different letters on top significantly differ (p < 0.05). The standard errors of the mean are shown as vertical bars (n = 3).
Figure 5. The effect of abiotic stresses on the relative expression of PcSOD1. Thalli were exposed to (A) −20 °C and +40 °C for 3 h and 250 µM CuSO4 for 10 h, and (B) dehydration for 8 h followed by rehydration for 1 h. Hydrated thalli were kept at room temperature and used as controls. Statistical analysis was conducted using one-way ANOVA and Tukey’s post hoc test. Bars with different letters on top significantly differ (p < 0.05). The standard errors of the mean are shown as vertical bars (n = 3).
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Table 1. Homology of PcERG3, resulting from BLASTp alignment.
Table 1. Homology of PcERG3, resulting from BLASTp alignment.
SpeciesProteinHomologyID
Model Organisms
Danio rerioLathosterol oxidase53%NP_001004630.1
Homo sapiensSterol-C5-desaturase 47%BAA33729.1
Homo sapiensFungal sterol-C5-desaturase homolog 51%BAA18970.1
Mus musculusLathosterol oxidase 47%NP_766357.1
Saccharomyces cerevisiaeC-5 sterol desaturase47%NP_013157.1
Schizosaccharomyces pombeC-5 sterol desaturase Erg31 50%NP_593135.1
Schizosaccharomyces pombeC-5 sterol desaturase Erg32 45%NP_001018791.2
Arabidopsis thalianaFatty acid hydroxylase superfamily protein32%NP_186908.1
Lichens
Peltigera leucophlebiaC-5 sterol desaturase89%MCJ1342268.1
Crocodia aurataC-5 sterol desaturase79%MCJ1468455.1
Lobaria immixtaC-5 sterol desaturase79%MCJ1260240.1
Mycoblastus sanguinariusC-5 sterol desaturase74%MCJ1458375.1
Toensbergia leucococcaC-5 sterol desaturase74%MCJ1227742.1
Nostoc sp. ‘Lobaria pulmonaria (5183) cyanobiont’Sterol desaturase family protein32%WP_325034720.1
Fungi
Penicillium macrosclerotiorumDelta(7)-sterol 5(6)-desaturase 65%XP_056930843.1
Candida tropicalisDelta(7)-sterol 5(6)-desaturase ERG355%KAK6887551.1
Candida tropicalisC-5 sterol desaturase44%XP_002550182.1
Candida metapsilosisERG352%KAG5421701.1
Candida albicansC-5 (6) desaturase52%WCC72276.1
Candida albicansC-5 sterol desaturase 46%XP_713577.1
Candida pseudojiufengensisERG3 45%XP_051616781.1
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Swid, M.A.; V. Koulintchenko, M.; Onele, A.O.; Leksin, I.Y.; Rakhmatullina, D.F.; Galeeva, E.I.; Valitova, J.N.; Minibayeva, F.V.; Beckett, R.P. Characterization and Expression Analysis of the Sterol C-5 Desaturase Gene PcERG3 in the Mycobiont of the Lichen Peltigera canina Under Abiotic Stresses. Microbiol. Res. 2025, 16, 139. https://doi.org/10.3390/microbiolres16070139

AMA Style

Swid MA, V. Koulintchenko M, Onele AO, Leksin IY, Rakhmatullina DF, Galeeva EI, Valitova JN, Minibayeva FV, Beckett RP. Characterization and Expression Analysis of the Sterol C-5 Desaturase Gene PcERG3 in the Mycobiont of the Lichen Peltigera canina Under Abiotic Stresses. Microbiology Research. 2025; 16(7):139. https://doi.org/10.3390/microbiolres16070139

Chicago/Turabian Style

Swid, Moatasem A., Milana V. Koulintchenko, Alfred O. Onele, Ilya Y. Leksin, Daniya F. Rakhmatullina, Ekaterina I. Galeeva, Julia N. Valitova, Farida V. Minibayeva, and Richard P. Beckett. 2025. "Characterization and Expression Analysis of the Sterol C-5 Desaturase Gene PcERG3 in the Mycobiont of the Lichen Peltigera canina Under Abiotic Stresses" Microbiology Research 16, no. 7: 139. https://doi.org/10.3390/microbiolres16070139

APA Style

Swid, M. A., V. Koulintchenko, M., Onele, A. O., Leksin, I. Y., Rakhmatullina, D. F., Galeeva, E. I., Valitova, J. N., Minibayeva, F. V., & Beckett, R. P. (2025). Characterization and Expression Analysis of the Sterol C-5 Desaturase Gene PcERG3 in the Mycobiont of the Lichen Peltigera canina Under Abiotic Stresses. Microbiology Research, 16(7), 139. https://doi.org/10.3390/microbiolres16070139

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