Differences in the growth of two S. rugosoannulata strains under low-temperature stress
To test the performance of the DQ-1 and DQ-3 strains under low-temperature stress, plates and liquid mycelia of the two strains were placed at 10 °C for 3 days and mycelia grown at 25 °C were used as controls. As shown in Fig. 1, we found that the growth rate of both S. rugosoannulata strains decreased under low-temperature stress (Fig. 1A). The growth rate of the DQ-1 strain dropped from 0.293±0.014 to 0.187±0.012 mm/d, while that of DQ-3 dropped from 0.181±0.013 to 0.150±0.017 mm/d (Fig. 1B). In addition, the dry weight of the mycelia of the DQ-1 strain decreased by 60.63% and that of the DQ-3 strain decreased by 42.21% after low-temperature stress. However, the difference in the dry weight of mycelia between DQ-1 and DQ-3 was not obvious under low-temperature stress (Fig. 1C). Therefore, the growth rate of DQ-1 mycelia and the decrease in biomass were significantly higher than those of DQ-3 under low-temperature stress. Moreover, these findings indicate that the DQ-1 strain is high-sensitive to low temperature while the DQ-3 strain is low-sensitive.
Transcriptome sequencing, gene mapping and differential expression analysis
RNA-Seq was performed using 12 S. rugosoannulata cDNA libraries. A total of 592.79 million raw reads were generated by Illumina sequencing. Then, after applying cleaning and quality control, 525.63 million clean reads were obtained, and the Q30 value of the base ratio was higher than 92.26%. The proportion of reads mapping to the S. rugosoannulata genome was 95.56–96.89% (Additional file 1: Table S2). Up to 9646 genes and 11496 transcripts were identified. The expression analysis of SR1 vs. SR6, SR3 vs. SR8, SR1 vs. SR3 and SR6 vs. SR8 generated 9499 DEGs. The partitioning of this value is shown in Fig. 2A. Compared with the DQ-1 strain, DQ-3 had more DEGs after low-temperature stress and showed a higher number of upregulated and downregulated genes. More DEGs were produced between the two strains after low-temperature stress (Fig. 2B). In addition, compared with the DQ-3 strain, the DQ-1 strain had more downregulated genes.
A Venn diagram comparison among DEGs is shown in Fig. 2B, and it revealed that 284 DEGs overlap among the experimental groups. After 10 °C low-temperature treatment, 774 DEGs overlapped between SR1 vs. SR6 and SR3 vs. SR8. After the two varieties were treated with varying temperatures, 1025 DEGs overlapped between SR1 vs. SR3 and SR6 vs. SR8. Moreover, SR3 vs. SR8 had the most unique DEGs. In addition, the DQ-1 and DQ-3 strains had the same overall change trend of differentially expressed genes after low-temperature stress. However, differences in gene expression were still observed between them (Fig. 2C). This result suggests that these differentially expressed genes may be caused by the different responses of the two strains to low temperature.
GO and KEGG enrichment analyses of DEGs between DQ-1 and DQ-3
Gene Ontology and KEGG pathway enrichment analyses of DEGs were performed to investigate the effect of low-temperature stress on the up- and downregulation of genes. The number of DEGs upregulated by the two strains increased significantly after low-temperature stress. Based on the GO enrichment analysis, the sample DEGs were assigned to three categories (biological process, cellular component, and molecular function). After the same variety was treated at 10 °C, DQ-1 corresponded to more gene processes, including “catalytic activity”, “hydrolase activity”, “oxidoreductase activity”, “response to stimulus”, “response to chemical”, “oxidation-reduction process” and “extracellular region” (Fig. 3A). However, DQ-3 mainly corresponded to “structural constituent of ribosome”, “unfolded protein binding”, “cytosolic part”, “cytosolic ribosome”, “ribonucleoprotein complex assembly” and “cytoplasmic translation” (Fig. 3B). After treating the different strains at 25 °C, more genes were involved in “transporter activity”, “transmembrane transporter activity”, “active transmembrane transporter activity”, “response to chemical” and “anion transport” (Fig. 3C), whereas after treatment at 10 °C, these genes were mostly involved in “oxidoreductase activity”, “anion transmembrane transport” and “nucleobase-containing compound transport” (Fig. 3D). However, none of them involved cellular components, and a more detailed GO enrichment process is shown in Additional file 1: Table S3.
The DEGs from SR1 vs. SR6, SR3 vs. SR8, SR1 vs. SR3 and SR6 vs. SR8 revealed 10, 6, 11 and 8 significantly enriched pathways (P < 0.01), respectively (Additional file 1: Table S4). After the same variety was treated at 10 °C, the pathways enriched by the DQ-1 strain included “microbial metabolism in diverse environments”, “aminobenzoate degradation”, “fatty acid degradation” and “pentose and glucuronate interconversions”, while those enriched by the DQ-3 strain included “proteasome”, “ribosome”, “ribosome biogenesis in eukaryotes” and “steroid biosynthesis”. At the same time, the “steroid biosynthesis” pathway was shared by the two strains after low-temperature stress (Additional file 1: Fig. S1). After the different strains were treated at 25 °C, the DEG enrichment pathways included “microbial metabolism in diverse environments”, “glycine, serine and threonine metabolism”, “degradation of aromatic compounds” and “carbon metabolism”. However, low-temperature treatment at 10 °C induced some specific enriched significant enrichment pathways (steroid biosynthesis and unsaturated fatty acid biosynthesis) (Additional file 1: Table S4).
The results of the GO and KEGG functional classification and enrichment analyses indicated that there were certain differences between the two strains of S. rugosoannulata after low-temperature stress. However, they were more involved in certain processes, such as xenobiotic biodegradation and metabolism, carbohydrate metabolism, lipid metabolism and oxidoreductase activity. Thus, these processes may play an important role in resisting low-temperature stress.
Differential expression of CAZyme genes and analysis of cellulase activity in S. rugosoannulata under low-temperature stress
Carbohydrate enzymes are the key enzymes used by fungi for nutrient transformation and utilization and mainly include six families of enzymes (auxiliary activities (AAs), glycosyl hydrolases (GHs), carbohydrate binding modules (CBMs), carbohydrate esterases (CEs), glycosyl transferases (GTs), and polysaccharide lyases (PLs)) (Hao et al., 2019). By comparing the whole transcriptome of S. rugosoannulata with the CAZy database, we identified 181 DEGs as CAZyme genes. After the two strains were subjected to low-temperature stress, we found that their carbohydrate enzyme expression patterns had more downregulated genes, with the DQ-3 strain showing more DEGs (Fig. 4A, B). At the same time, the top ten gene cluster analyses for six carbohydrate enzymes with significant differences were selected, as shown in Fig. 4. We found that the DQ-1 strain showed greater downregulation of AA, GH, CE, and GT family genes, while the DQ-3 strain showed greater upregulation of the GH, CE, and GT family genes (Fig. 4E, F). In addition, when treated at 25 ℃ and 10 ℃, the GH family genes of the DQ-3 strain were downregulated considerably, while other enzyme families did not change much (Fig. 4C, D). In addition, after culturing the two strains at 25 °C, the AA, GH, CE and GT genes of the DQ-3 strain were significantly downregulated (Fig. 4G). However, certain carbohydrate-enzyme genes were upregulated under 10 °C low-temperature stress (Fig. 4H).
To further illustrate the effect of low-temperature stress on the carbohydrate metabolism of S. rugosoannulata, we measured the activities of four important carbohydrate enzymes (cellulase, exo/endo-β-1,4-glucanase and β-glucosidase) and found that the four enzymes showed a downward trend after low-temperature stress (Fig. 5). In addition, there was no significant change in endo-β-1,4-glucanase activity of the two strains, while the enzymes showed significantly lower activity in the DQ-3 strain than the DQ-1 strain under the 25℃ treatment. After low-temperature stress, the DQ-1 strain showed reductions in cellulase and endo-β-1,4-glucanase activity by 48.24–48.71%, and that of the DQ-3 strain decreased by 42.42–43.50% (Fig. 5A, C). Exo-β-1,4-glucanase activity also decreased by 25.34% and 33.70% in the DQ-1 and DQ-3 strains, respectively (Fig. 5B). In addition, the activity of β-glucosidase decreased most significantly, with the DQ-1 strain showing a decrease of 80.99 % and the DQ-3 strain showing a decrease of 67.2% (Fig. 5D).
To confirm the reliability of the carbohydrate enzyme gene expression pattern, the six selected enzyme family genes were validated using qRT–PCR. These genes included 2 AA genes [AA2 (DQGG003998) and AA5 (DQGG004011)], 3 GH genes [GH3 (DQGG000752), GH5 (DQGG009707) and GH9 (DQGG002370)], 1 CE4 (DQGG002082) gene, 1 GT2 (DQGG005286) gene, 1 CBM50 (DQGG000758) gene and 1 PL14 (DQGG010936) gene. As shown in Fig. 5E-F, gene expression profiling of carbohydrate enzymes using qRT–PCR revealed variation trends similar to those observed in the RNA-Seq results. Low-temperature stress has a certain effect on carbohydrate enzymes of S. rugosoannulata, and it reduces the process of carbohydrate metabolism by downregulating the expression of AA, GH, CE and GT family genes.
Differential expression of antioxidant enzyme genes and enzyme activity analysis of S. rugosoannulata under low-temperature stress
Studies have shown that low-temperature stress can cause oxidative stress in fungi, which can resist oxidative damage through antioxidant enzymes (Hu et al., 2009). In the GO enrichment analysis, we also found that the process of “oxidoreductase activity” can be significantly enriched after low-temperature stress; therefore, the differential expression of oxidoreductase may play an important role in resisting low-temperature stress. Therefore, we analyzed the expression of genes with antioxidant enzymes and found that the SOD1 (DQGG001858), SOD2 (DQGG002408) and SOD3 (DQGG002593) genes of the two strains were all downregulated under low-temperature stress. Among them, more significant downregulation was observed for the DQ-1 strain. However, the CAT1 (DQGG003981), CAT2 (DQGG007488), glutathione reductase (GR, DQGG007487) and peroxidase (POD, DQGG003554) genes of the two strains were upregulated after low-temperature stress, and the upregulated expression of the DQ-1 strain was more significant. It is worth noting that the expression level of the glutathione peroxidase (GPX, DQGG003145) gene of the DQ-1 strain was downregulated after low-temperature stress, while the expression level of the DQ-3 strain was upregulated (Fig. 6A).
We further determined the SOD and CAT activities of the two strains after low-temperature stress. The SOD enzyme activity of the DQ-1 strain was 3.09 times that of DQ-3 under mycelial culture conditions at 25 °C, whereas the difference in CAT enzyme activity was not obvious. After 10 °C low-temperature stress, the SOD enzyme activity of the DQ-1 and DQ-3 strains decreased by 85.75% and 77.08%, respectively (Fig. 6B, C). However, the CAT enzyme activity of the DQ-1 and DQ-3 strains increased by 3.45 times and 3.06 times, respectively (Fig. 6B, C). At the same time, we also verified the expression of all antioxidant enzyme genes through qRT–PCR, which is consistent with the RNA-Seq and enzyme activity results (Fig. 6D, E). This result indicates that S. rugosoannulata might resist low-temperature stress by activating CAT, GR and POD and the DQ-3 strain may also upregulate GPX gene expression to increase its tolerance to low-temperature stress.
Heat shock proteins of S. rugosoannulata in response to low-temperature stress
Heat shock proteins (HSPs) are proteins synthesized by biological cells to resist high-temperature stress. However, studies have also shown that heat shock proteins can maintain the normal structure of functional proteins, prevent protein degeneration, enhance cell membrane fluidity and maintain the normal physiological functions of cells under low-temperature stress (Renaut et al., 2006). After 10 °C low-temperature stress, the DQ-1 strain had 3 downregulated HSP expression levels and 9 upregulated HSP gene expression levels. In addition, the DQ-3 strain had 6 downregulated HSP expression levels and 13 upregulated HSP gene expression levels (Fig. 7A, B). It is worth noting that the expression of 6 small HSPs was significantly upregulated in both strains. In addition, we selected 1 HSP (DQGG006217), 3 sHSPs [sHSP1 (DQGG002510), sHSP2 (DQGG002656) and sHSP3 (DQGG004546)] and 1 HSP78 (DQGG008487) for the qRT–PCR verification experiments, which is consistent with the RNA-Seq results (Fig. 7A and B). This result indicates that HSPs may play an important role in resisting low-temperature stress in S. rugosoannulata. In addition, the expression of more HSP genes in the DQ-3 strain was upregulated, which may be related to low-temperature tolerance.