Analysis of the growth state of Pleurotus pulmonarius after heat stress
Under normal culture conditions, mycelium grows well (Fig. 1A, B). Mycelia was transferred to 26℃ for recovery after heat stress (heat stress group, HS), and it’s growth was inhibited compared with mycelia without heat stress (controlled group, CK) (Fig. 1C, D).
Identification of DEGs of Pleurotus pulmonarius after heat stress
In order to explore the molecular mechanism of Pleurotus pulmonarius response to heat stress, samples from CK and HS were collected for RNA-Seq. A total of 5906 differentially expressed genes (DEGs) were identified, of which 1086 genes were up-regulated and 4820 genes were down-regulated after heat stress (Fig. 2A, B). A volcano plot was used to directly show the degree of genetic difference between two groups (Fig. 2B), and the closer the genes are to the two ends of the graph, the greater the degree of difference.
GO enrichment and KEGG pathway analysis of DEGs
Based on GO functional classification, all DEGs were classified into three different categories: biological process, cellular component and molecular function (Fig. 2C). The significant enrichment analysis of KEGG pathway is shown in Fig. 2D. Among the top 20 pathways with significant enrichment of DEGs, the most enriched pathway is related to aminoacyl tRNA synthesis, pyrimidine metabolism, arginine and proline metabolism, fructose and mannose metabolism, and glutathione metabolism.
Analysis of glutathione metabolic pathway
Transcriptomic results showed that heat stress treatment could affect glutathione metabolism of Pleurotus pulmonarius (Fig. 3A). Three genes involved in glutathione metabolism were obtained, with ggt (γ-glutamyl transferase) involved in glutathione degradation, and gst2 and gst3 involved in glutathione catabolism. KEGG pathway analysis showed that the expression level of ggt (γ-glutamyl transferase) was downregulated while the expression level of gst2 and gst3 were upregulated (Fig. 3B). These suggest that the glutathione metabolic pathway may be involved in the process of adaptation to heat stress.
PPI network analysis of DEGs
In order to systematically analyze the function of DEGs involved in glutathione metabolism after heat stress, these DEGs were mapped to obtain the PPI network (Fig. 4). The DEGs associated with glutathione metabolism formed a highly complex network, indicating the complexity of glutathione metabolism in Pleurotus pulmonarius. Glutathione is an important regulatory protein in plant cells and plays a key role in a variety of cellular activities, including maintaining the integrity of cell membranes, scavenging free radicals in body, participating in the absorption and transport of amino acids, chelating endogenous and exogenous toxins, resisting heavy metal stress, and participating in cold and heat stress in plants. These results suggest that Pleurotus pulmonarius may be resistant to heat stress through regulation of these genes and pathways.
Validation of transcriptomics data by qRT-PCR
In order to verify the results of transcriptome sequencing and further analyze the expression patterns of genes related to glutathione metabolism under heat stress, four representative genes involved in glutathione metabolism were selected for qRT-PCR analysis: ggt, gst1, gst2 and gst3. As shown in Fig. 5, ggt in Pleurotus pulmonarius was significantly downregulated after heat stress, gst2 and gst3 were significantly upregulated, but the expression of gst1 did not change much. The result indicates that qRT-PCR expression patterns and RNA-Seq results have similar trends, further confirming the reliability of the transcriptome data.
Determination of reduced glutathione content of Pleurotus pulmonarius after heat stress
As shown in Fig. 6, reduced glutathione content in Pleurotus pulmonarius mycelia after heat stress was significantly higher than that in the controll group, which was consistent with the results of gene expression. It indicated that Pleurotus pulmonarius may change the expression of glutathione metabolism related genes and thereby regulate the endogenous reduced glutathione content to adapt to heat stress.