Transcriptome Analysis to Elucidate the Response of Sanghuangporus baumii (Pilát) L.W. Zhou & Y.C. Dai to Sodium Treatment

Abstract

To investigate the effects and underlying mechanisms of sodium (Na⁺) on the growth characteristics of Sanghuangporus baumii mycelia, a single-factor Na⁺ addition experiment was performed. We found that treatment with 10 mmol/L Na⁺ (Na10) significantly increased the growth rate (0.41 ± 0.01 cm/d) and biomass (4.27 ± 0.05 g/L) of S. baumii mycelia, surpassing the control (Ck) group by 3.14% and 4.06%, respectively. In contrast, treatment with 100 mmol/L Na⁺ (Na100) resulted in a significant reduction in growth rate (0.34 ± 0.01 cm/d) and biomass (3.25 ± 0.02 g/L) of S. baumii mycelia compared to the Ck group. Transcriptome analysis further revealed that low Na⁺ concentrations (10 mmol/L) promoted the accumulation of soluble sugars (7.63 ± 0.54 mg/g) and upregulated the expression of pertinent genes, thereby accelerating mycelial growth. On the other hand, high Na⁺ concentrations (100 mmol/L) led to H₂O₂ accumulation (12.18 ± 0.24 μmol/g), causing toxicity in S. baumii mycelia. High Na⁺ concentrations also significantly boosted the production of valuable metabolites, such as triterpenoids (19.65 ± 0.22 mg/g), although the exact mechanisms remain to be elucidated. Overall, we suggest an effective approach for accelerating mycelial growth cycles and enhancing the production of high-value bioactive compounds from S. baumii.

1. Introduction

Sanghuangporus baumii (Pilát) L.W. Zhou & Y.C. Dai, a medicinal macrofungus growing on Syringa reticulata [1], has been used in traditional Chinese medicine for over 2000 years, with references in The Divine Farmer’s Materia Medica [2]. S. baumii produces several bioactive compounds, such as triterpenoids, ergosterol, and polysaccharides, which exhibit anti-tumor [3], anti-inflammatory [4], and immune-enhancing properties [5], highlighting its promising commercial potential. Although bioactive compounds can be extracted from both the fruiting bodies and mycelia of S. baumii [2], mycelia are preferred for large-scale production due to ease of cultivation and comparable compound content [6–8]. Consequently, producing bioactive compounds from S. baumii mycelia has become the primary approach to meeting the increasing demand for such compounds sustainably and efficiently [9,10].

The growth cycle of S. baumii mycelia is longer than that of other fungi, such as those in the Pleurotus genus [9,11], significantly influencing biomass accumulation and large-scale production of bioactive compounds. Studies suggest that metal ions stimulate fungal growth and enhance bioactive compound accumulation [12]. For example, Bystrzejewska-Piotrowska et al. reported that Na⁺ is absorbed by P. eryngii and that Na⁺ regulates its growth [13]. Further, Hou et al. reported that Na⁺ promotes substrate degradation by fungal mycelia, increasing yields by 89.16% [14]. In addition to growth promotion, metal ions enhance active compound accumulation. In Ganoderma lucidum, 100 mmol/L sodium chloride (NaCl) was found to increase triterpenoid content by 2.8-fold [15]. Metal ions like Mg²⁺, Zn²⁺, and Fe²⁺ have also been shown to enhance the bioactivity of polysaccharides [16]. Thus, the exogenous addition of metal ions has emerged as a promising strategy to improve fungal growth and bioactive compound production.

At present, there are no studies on the use of metal ion induction to enhance growth and bioactive compound production in S. baumii mycelia. Given concerns over the safety of exogenous additives, NaCl has been identified as the most suitable exogenous inducer [15]. Herein we aimed to investigate the effects of Na⁺ treatment on S. baumii growth rate and biomass accumulation. We performed transcriptome analysis to assess potential changes in metabolic pathways induced by Na⁺ treatment. We simultaneously measured soluble sugar content and hydrogen peroxide (H₂O₂) levels, along with the expression of key genes involved in these pathways. Finally, we quantified total triterpenoid content and analyzed the expression of pertinent genes. Our findings are significant for optimizing high-quality S. baumii mycelial production.

2. Materials and methods

2.1. Strain preservation and activation

Sanghuangporus baumii strain DL101 was preserved in the protection laboratory at Northeast Forestry University. ITS identification results have been submitted to NCBI GenBank (no. KP974834). S. baumii mycelia were transferred to Petri dishes containing potato dextrose (PD) agar and cultured at 25 °C in the dark. The mycelia of the parent strain were then inoculated in PD broth and cultured with shaking for seed liquid preparation.

2.2. Treatment of S. baumii mycelia with Na⁺

NaCl was dissolved in deionized water, sterilized by filtration through a 0.45 μm Millipore filter, and added to PD agar medium. Final Na⁺ concentrations of 1 mmol/L (Na1), 10 mmol/L (Na10), and 100 mmol/L (Na100) were prepared, as previously reported [15], with sterile water as a control (Ck group). Fungal cakes of S. baumii (1 cm diameter) were placed in Petri dishes, incubated at 25 °C in the dark, and their growth rate was calculated by averaging the vertical and horizontal colony diameters.

The seed liquid was homogenized into 250 mL PD media at a 4% inoculum volume (v/v) and incubated at 25 °C with shaking at 180 rpm for 8 days. Afterward, the same concentration of Na⁺ was then added to the media, followed by cultivation for another 48 h before harvesting S. baumii mycelia. one part of each sample was oven-dried at 45 °C until a constant weight was achieved and weighed. The other part of the sample used for further analysis.

2.3. Transcriptome analysis and quantitative reverse transcription PCR (qRT-PCR) validation

RNA from S. baumii samples with significant growth differences was extracted using an RNAiso Plus Kit (Takara, Dalian, China). For transcriptome sequencing, high-quality RNA was sequenced on an Illumina HiSeq 2500 platform by Suzhou PANOMIX Biomedical Tech Co., LTD (China). Raw data were filtered to remove reads containing adapters, reads < 50 bp, and those with an average sequence quality score of < Q20. High-quality sequences were spliced from scratch to obtain transcripts, and the longest transcript was selected as a unigene. Unigenes were subsequently annotated through Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and open reading frame (ORF) prediction. Based on these results, differential expression and enrichment analyses were performed. Gene expression levels were calculated as FPKM; genes with |log₂FoldChange| of >1 and P-value of <0.05 were considered differentially expressed genes (DEGs). DEGs were validated by qRT-PCR, with primers designed using Primer Premier 5.0 (Table S1). Relative expression levels were calculated using the 2^−ΔΔCt method, with the α-tubulin gene serving as an internal control [17].

2.4. Quantification of soluble sugar content, H₂O₂ levels, and total triterpenoid content

For metabolite analysis, 0.1 g sample was homogenized with 1 mL distilled water, followed by incubation in a 95 °C water bath for 10 min. The homogenate was then centrifuged at 8,000 ×g for 10 min at 25 °C. A mixture of 0.1 mL supernatant and 0.9 mL distilled water was prepared and thoroughly mixed. Soluble sugar content was measured using a commercial KT-2-Y kit (Suzhou Comin, Suzhou, China), as per manufacturer instructions. Similarly, 0.1 g sample was homogenized with 1 mL acetone on ice, followed by centrifugation at 8,000×g for 10 min at 4 °C. The supernatant thus obtained was collected, and H₂O₂ content was determined using the Suzhou Comin H₂O₂-2-Y test kit according to manufacturer instructions. Total triterpenoid content was quantified as previously described [18].

2.5. Evaluation of the antioxidant system

To assess the tolerance of S. baumii to Na⁺-induced stress, the functionality of its antioxidant system was evaluated. A 0.1 g sample was homogenized with 1 mL extraction solution on ice to prevent enzymatic degradation. This homogenate was centrifuged at 8,000 ×g for 10 min at 4 °C, and the supernatant was collected for analyses. The activities of superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) were determined using commercial kits (Suzhou Comin).

2.6. Statistical analysis

Experimental data, including mycelial growth rate, biomass, gene expression, metabolite content, and enzyme activity, were derived from three biological replicates. Statistical significance between samples was determined using Duncan’s test. Data were analyzed with SPSS 17.0. Differences were considered significant at p < 0.05, and values represent mean ± standard deviation.

3. Results

3.1. Tolerance analysis of S. baumii mycelia treated with Na⁺

Different Na⁺ concentrations caused phenotypic differences in S. baumii mycelia, significantly affecting mycelial growth (Figure 1A). Mycelial growth rate gradually increased with an increase in Na⁺ concentration from 1 mmol/L to 10 mmol/L, reaching 0.41 ± 0.01 cm/d at 10 mmol/L Na⁺. However, at 100 mmol/L Na⁺, mycelial growth rate significantly decreased to 0.34 ± 0.01 cm/d (Figure 1B). Mycelial biomass exhibited a similar trend, with maximum biomass at 10 mmol/L Na⁺ (4.27 ± 0.05 g/L; Figure 1C). These findings suggest that moderate Na⁺ levels promote the growth of S. baumii mycelia, whereas excessive levels lead to growth inhibition.

Figure 1.

Growth characteristics of Sanghuangporus baumii mycelia under Na⁺ treatment (the mean difference being significant at the 0.05 level, p < 0.05). (A) Colony morphology. (B) Growth rate. (C) Biomass.

3.2. Transcriptome analysis of differential pathways and genes in S. baumii mycelia under Na⁺ treatment

To elucidate the mechanisms underlying the effects of Na⁺ on the growth of S. baumii mycelia, high-quality RNA was extracted from the Ck, Na10, and Na100 groups, which exhibited significant growth differences, for transcriptome analysis (Figure 2A). Transcriptome data confirmed sequencing quality (Figure S1), with clean reads ratio > 98.54%, Q20 > 98.61%, and Q30 > 95.96% (Table S2). The Ck, Na10, and Na100 groups produced approximately 43450915, 42389260, and 40773272 clean reads, respectively. Consistent clustering among biological replicates (Figure 2B) confirmed RNA-seq data suitability for further analyses.

Figure 2.

Transcriptome analysis of growth differences in Sanghuangporus baumii mycelia under Na⁺ treatment (the mean difference being significant at the 0.05 level, p < 0.05). (A) RNA gel electrophoresis. (B) PCA score plot of transcript profiles (Ck, Na10, and Na100 groups). (C) Volcano plot of up- and downregulated DEGs from pairwise comparisons. (D) qRT-RCR validation of gene expression. (E) KEGG pathway enrichment analysis.

DEG analysis led to the identification of 1,013 DEGs (614 up- and 399 downregulated) between the Ck and Na10 groups, 760 DEGs (459 up- and 301 downregulated) between the Ck and Na100 groups, and 468 DEGs (208 up- and 260 downregulated) between the Na10 and Na100 groups (Figure 2C). qRT-PCR data that DEG expression profiles were consistent with transcriptome-derived FPKM values (Figure 2D). KEGG pathway enrichment analysis indicated that DEGs between the Ck and Na10 groups were primarily enriched in starch and sucrose metabolism and glycolysis/gluconeogenesis. In contrast, DEGs between the Ck and Na100 groups were enriched mainly in the starch and sucrose metabolism pathway, peroxisome pathway, and terpenoid backbone biosynthesis pathway (Figure 2E). These differences potentially explain growth variations caused by Na⁺ treatment.

3.3. Changes in soluble sugar content in S. baumii mycelia treated with Na⁺

The starch and sucrose metabolism pathway was significantly enriched under Na⁺ treatment. At 10 mmol/L Na⁺, the growth rate of S. baumii mycelia was significantly higher, with increased expression of soluble sugar synthesis genes, including EG2, MGAM, and PYG (Figure 3A; Table S3), resulting in higher soluble sugar content (7.63 ± 0.54 mg/g; Figure 3B). While 100 mmol/L Na⁺ inhibited S. baumii mycelial growth, the expression of soluble sugar synthesis genes (Figure 3A) and soluble sugar content continued to increase (Figure 3B), indicating a role for starch and sucrose metabolism in regulating the growth of S. baumii mycelia under Na⁺ treatment.

Figure 3.

Soluble sugar content and gene expression levels in Sanghuangporus baumii under Na⁺ treatment (the mean difference being significant at the 0.05 level, p < 0.05). (A) Expression levels of soluble sugar-related genes. (B) Soluble sugar content.

3.4. Antioxidant system alterations in S. baumii mycelia treated with Na⁺

Abiotic stress often triggers physiological responses in organisms [19]. The peroxisome pathway was found to be enriched under Na⁺ treatment, with an increase in SOD gene expression (Figure 4A; Table S3) and enzymatic activity (Figure 4B) at 10 mmol/L Na⁺. Elevated CAT and POD gene expression and enzymatic activities facilitated timely clearance of H₂O₂ (Figure 4C-D), significantly reducing its content (0.33 ± 0.11 μmol/g; Figure 4E). However, at 100 mmol/L Na⁺, SOD activity and gene expression continued to increase (Figure 4A–B), leading to H₂O₂ accumulation beyond CAT and POD clearance capacity (12.18 ± 0.24 μmol/g; Figure 4E), potentially slowing down the growth rate of S. baumii mycelia. The results indicate that excessive accumulation of H₂O₂ is the main reason for the inhibition of S. baumii mycelia under Na⁺ treatment.

Figure 4.

Antioxidant system alterations in Sanghuangporus baumii under Na⁺ treatment (the mean difference being significant at the 0.05 level, p < 0.05). (A) Gene expression levels. (B) SOD, (C) CAT, and (D) POD activities. (E) H₂O₂ content.

3.5. Changes in triterpenoid levels in S. baumii mycelia treated with Na⁺

Triterpenoids, key components contributing to the medicinal properties of S. baumii, and most of them have important medicinal value. For example, Ganoderol A, Ganoderic acid F, Ganoderic acid H show the ability to inhibit tumor growth, anti-inflammatory and anti-aging pharmaceutical activities [7,20], and their accumulation can be enhanced by exogenous inducers [15]. While low Na⁺ concentrations did not stimulate triterpenoid accumulation (13.59 ± 0.27 mg/g), high Na⁺ concentrations enriched the terpenoid backbone biosynthesis pathway (Figure 2E), increasing the expression of triterpenoid biosynthesis-related genes (AACT, HMGS, MVK, and GPS) (Figure 5A; Table S3) and total triterpenoid content (19.65 ± 0.22 mg/g; Figure 5B). These results indicate that high Na⁺ concentrations induce triterpenoid biosynthesis in S. baumii.

Figure 5.

Total triterpenoid content and gene expression levels in Sanghuangporus baumii under Na⁺ treatment (the mean difference being significant at the 0.05 level, p < 0.05). (A) Triterpenoid-related gene expression levels. (B) Total triterpenoid content.

4. Discussion

Metal ions are essential for organismal growth and development as well as for the synthesis of bioactive compounds [13–15,21]. Herein we found that low Na⁺ concentrations activated the starch and sucrose metabolism pathway in S. baumii, leading to a significant increase in both soluble sugar content and the expression of related genes. After being metabolized through glycolysis/gluconeogenesis (Figure 2E), these soluble sugars supply energy that promotes S. baumii mycelial growth, contributing to faster growth rates and increased biomass [22]. This is of significance for the study of shortening the growth cycle of S. baumii mycelia. However, high Na⁺ concentrations also led to increased soluble sugar content and related gene expression. This suggests that soluble sugars are involved in maintaining cellular osmotic stability, an effective strategy for organisms to cope with stress, similar to previous findings indicating that soluble sugars can enhance salt tolerance [23]. This finding indicates the feasibility for the production of soluble sugars from S. baumii mycelia treated with Na⁺.

In addition, we found that low Na⁺ concentrations did not activate the peroxisome pathway and were associated with reduced H₂O₂ levels. Under high Na⁺ concentrations (100 mmol/L), however, the peroxisome pathway was activated, leading to the accumulation of H₂O₂ and toxicity in S. baumii mycelia (Figure 1A). This explains why reactive oxygen species (ROS) are often regarded as “toxic cellular waste” [24]. However, recent studies suggest that ROS play beneficial roles in cellular function regulation. For instance, H₂O₂ can reversibly oxidize critical, redox-sensitive cysteine residues on target proteins [25]. Moreover, H₂O₂ can activate AP1-like transcription factors and regulate genes involved in triterpenoid biosynthesis, thereby enhancing the production of beneficial metabolites [26]. We also observed a significant increase in H₂O₂ under high Na⁺ concentrations, suggesting that high Na⁺ concentrations activate the peroxisome pathway, resulting in H₂O₂ accumulation. While H₂O₂ is partially degraded by CAT and SOD (Figure 4C–D), excess H₂O₂ may trigger the activation of the expression of triterpenoid biosynthesis-related genes, thus increasing triterpenoid content in S. baumii. Although previous studies have shown that H₂O₂ can elevate triterpenoid levels via signal transduction, the precise mechanisms underlying H₂O₂-mediated triterpenoid production remain unclear and warrant further investigation [26].

5. Conclusions

Our findings confirmed that low Na⁺ concentrations positively affect the growth rate and biomass of S. baumii mycelia, primarily by activating the starch and sucrose metabolism pathway, which stimulates soluble sugar accumulation and related gene expression. This results in the generation of sufficient energy for optimal growth. In contrast, high Na⁺ concentrations inhibit the growth rate and biomass of S. baumii mycelia due to H₂O₂ toxicity associated with peroxisome pathway activation. Moreover, high Na⁺ concentrations promote the accumulation of valuable metabolites, including soluble sugars and triterpenoids. These findings offer new insights into optimizing the growth cycle of S. baumii mycelia and producing high-value bioactive compounds, thus ensuring that higher yields can be achieved in a short period of time.

Funding Statement

This research was supported by the Fundamental Research Funds for the China Postdoctoral Science Foundation [grant number 2023M740560], and National Natural Science Foundation of China [grant number 32301597].

Author contributions

Writing—original draft preparation, Zengcai Liu; Data curation, Ying Yu, Jingwei Hu. and Ge Lv; Writing – review and editing, Li Zou. All authors have read and agreed to the published version of the manuscript.

Disclosure statement

The authors declare that they have no competing interests.