Exogenous NO affected the energy metabolism of mycelia and reduced the production and accumulation of ROS
NO, an important signaling molecule, plays an important regulatory role in the growth and development of organisms and their responses to stress. Our previous study showed that NO plays an important role in the response of P. ostreatus to HS. In this study, the addition of exogenous NO promoted the growth recovery of P. ostreatus mycelia after HS, as shown in Fig. 1A (the red arrow indicates regenerated mycelia after HS). H2O2 and superoxide anion (O2-) are important components of ROS, and as shown in Fig. 1B, the content of H2O2 in mycelia increased significantly after HS. Exogenous sodium nitroprusside (100 µM SNP) could significantly reduce the accumulation of H2O2 in mycelia after HS, but the level was still significantly higher than that in the control group. As shown in Figs. 1C and 1D, HS increased the O2- content in mycelia by 1.64-fold and the production rate of O2- by 60.74 %. In addition, compared with the levels found in the HS group, exogenous SNP decreased the O2- content and production rate by 12.85 % and 9.34 % respectively, whereas, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl3-oxidec (cPTIO (250 µM) significantly increased the content and production rate of O2-. The results showed that exogenous NO could affect not only the content of H2O2, but also the content and production rate of O2-.
Nicotinamide adenine dinucleotide (NAD) is a coenzyme that exists in all cell and is found in two forms: oxidized (NAD+) and reduced (NADH). NADH is produced during the TCA cycle, participates in material and energy metabolism in cells and serves as a control marker in the energy production chain of mitochondria. As shown in Fig. 1E, HS significantly increased the content of NADH in mycelia compared with the level found in control group, and this increase could be partially offset by treatment with SNP. HS also significantly affected the NAD+/NADH ratio, and the addition of exogenous SNP alleviated the imbalance in NAD caused by HS. In contrast, the addition of cPTIO significantly decreased the NAD+/NADH ratio compared with that observed in the HS group (Fig. 1F). Because NAD+ is reduced to NADH by the TCA cycle in mitochondria and NADH serves as a substrate for the generation of ROS in the respiratory chain [26], it can be assumed that the TCA cycle is accelerated by HS and that NO can affect the accumulation of NADH by regulating the TCA cycle. ATP is commonly considered as an intracellular energy currency molecule, and the mitochondrial respiratory chain is the site of ATP production. As shown in Fig. 1G, the total respiration rate of mycelia decreased after HS, and this decrease might be caused by mitochondrial damage induced by HS. The addition of exogenous NO further inhibited the total respiratory rate. It has been suggested that NO can regulate the respiration rate of mycelium under stress and that NO might reduce ROS production by inhibiting respiration rate. Interestingly, the ATP content increased by 39.27 % after HS treatment compared with the level found in control group, and this increase is synchronous with the outbreak of ROS. The increase in the ATP content might be due to the observed increase in the NADH level (Fig. 1E). Exogenous NO significantly inhibited the increase in ATP induced by HS, whereas the addition of exogenous cPTIO significantly increased the ATP content under HS (Fig. 1H). The results further proved that NO can regulate the respiratory chain.
In conclusion, NO might alleviate ROS outbreaks by regulating the mitochondrial respiratory chain and thereby reducing mycelial damage induced by HS.
RNA-Seq analysis of the regulatory mechanism through which NO alleviates mycelial damage induced by HS
To fully understand the effect of NO on the transcriptome of P. ostreatus under HS, 12 RNA-Seq cDNA libraries were prepared. As shown in Additional file 1: Table S2 , after removing adapters, low-quality regions and all possible contamination, each treatment group contained an average of 47.29 M clean reads with Q30 > 94.93 % and a GC percentage between 53.03 % and 53.64 %. The ratio of reads mapping to the P. ostreatus genome was high, ranging from 77.26 % to 82.93 %. This result indicated that the accuracy of the sequencing results was high and could be used for subsequent analysis.
The RNA-Seq analysis showed that HS caused significant changes in gene expression in P. ostreatus. To further explore the relationships among differentially expressed genes (DEGs) identified from the four different comparisons, Venn diagrams were constructed using all the identified DEGs (a total of 2400). Among the HS-responsive genes, 742 DEGs were significantly upregulated, and 1079 DEGs were significantly downregulated. A total of 817 DEGs were regulated by the addition of SNP or cPTIO. As shown in Fig. 2, 63 upregulated and 38 downregulated DEGs were found in CK_HS and SNP_cPTIO. Moreover, 626 specifics upregulated DEGs and 957 specifics downregulated DEGs were found in the CK_HS group, whereas 429 and 150 DEGs were significantly upregulated and significantly downregulated, respectively, in the SNP_cPTIO group. These results indicated that the molecular responses to HS between the group treated with SNP and cPTIO were strikingly different. The specific upregulated and downregulated DEGs identified from the SNP_cPTIO comparison were selected for further functional characterization to explore the possible molecular mechanism through which NO alleviates the oxidative damage to mycelia caused by HS.
Based on the above-described analysis, the functions of 579 DEGs identified from the SNP_cPTIO comparison were examined to elucidate the possible mechanism through which NO alleviates the oxidative damage to mycelia induced by HS. In the gene ontology (GO) analysis, the DEGs identified from the SNP_cPTIO comparison were classified into three categories: ‘biological process’, ‘cellular component’ and ‘molecular function’ (Fig. 3). Among the biological process category, genes corresponding to metabolic process, cellular process and single organism process were the most abundant. Membrane and membrane parts were the most abundant of the cellular components, and in the molecular function category, catalytic activity and binding were the most abundant. These results indicated that NO might participate in the response of mycelia to HS by regulating cell metabolism, affecting cell membrane components and structure, and affecting the catalytic activity of proteins.
To understand the functions of the DEGs identified from the SNP_cPTIO comparison, a pathway enrichment analysis was performed. The results showed that the DEGs were mainly concentrated in the following pathways: oxidoreductase activity, oxidation-reduction process, cofactor binding, protein kinase activity, phosphotransferase activity, alcohol group as acceptor, and protein phosphorylation (Fig. 4A). Previous studies have shown that HS can lead to the production and accumulation of ROS in mycelia, and ROS can further cause oxidative damage. As shown in Fig. 4A, exogenous NO can affect the oxidation-reduction process and oxidoreductase activity. In addition, considering the close relationship between the antioxidant system and ROS clearance, the expression pattern of the genes after the addition of a NO donor or scavenger was further analyzed with a heatmap. As shown in Fig. 4B, 69 DEGs identified after the addition of SNP or cPTIO were enriched in the oxidation-reduction process pathway and oxidoreductase activity, and these included 62 significantly upregulated DEGs and seven downregulated DEGs. It can thus be hypothesized that NO can activate the activity of oxidoreductase and accelerate redox reactions under HS.
NO affected the expression of key genes in the respiratory chain under HS
The respiratory chain is closely related to ROS. Based on previous studies, to explore whether NO can regulate the respiratory chain to alleviate mycelial damage under HS, six DEGs related to the respiratory chain were identified from 579 DEGs via functional enrichment (Table 1). Two of these DEGs are not annotated, and the remaining four DEGs are g3097, g11376, g12148 and g12952, which encode the succinate dehydrogenase iron-sulfur subunit, AOX, hypothetical protein and mitochondrial chaperone bcs1, respectively. As illustrated in the heatmap shown in Fig. 5, an exogenous NO donor (SNP) inhibited the expression of five DEGs and upregulated the expression of g11376 (AOX). Mitochondrial chaperone bcs1 is a transmembrane chaperone found in the mitochondrial inner membrane and is required for the assembly of mitochondrial respiratory chain complex III (http://www.ebi.ac.uk/interpro/entry/InterPro/IPR027243/) [27]. The succinate dehydrogenase iron-sulfur subunit is involved in the synthesis and assembly of mitochondrial respiratory chain complex II (https://www.uniprot.org/uniprot/A1AZJ0). These results indicate that exogenous NO can inhibit the cytochrome pathway and activate the alternative oxidation pathway.
NO induced aox gene expression under HS
ROS are mainly produced by a high-oxygen environment and the respiratory chain at a high reduction state during the transition of mitochondria from complex III to complex IV, which results in the leakage of a large number of electrons and the reduction of oxygen molecules. AOX prevents excessive reduction of downstream complexes (cytochrome pathway) by introducing a branch into the ETC at the ubiquinone pool. When AOX bypasses complexes III and IV of the cytochrome pathway, it significantly reduces ATP production and single electron leakage, which results in the reduction of ROS production [18]. Moreover, as determined through RNA-Seq analysis, aox can be regulated by NO, participates in the oxidation-reduction process pathway and has oxidoreductase activity.
To further verify the regulatory effect of NO on aox under HS, the effects of exogenous NO donors and scavengers on aox gene expression were detected. As shown in Fig. 6A, the relative expression of the aox gene in P. ostreatus mycelia changed steadily with increases in the time of exposure to HS. During the first 24 h of exposure to HS, aox gene expression first increased and then decreased within a small range. An increase in the exposure time to 48 h significantly increased the relative expression of the aox gene, and the level detected after 48 h was approximately 8-fold higher than that at 0 h. As shown in Fig. 6B, the relative expression of the aox gene was significantly increased after HS, whereas exogenous SNP treatment almost completely enhanced this effect, and cPTIO blocked the effect of SNP on aox gene expression. The results showed that NO can promote the expression of the aox gene in mycelia after HS. In conclusion, NO might participate in the response of P. ostreatus to HS by regulating the expression of the aox gene.
The OE of aox promoted the recovery of mycelial growth after HS
Using previously reported methods, we successfully constructed an RNAi-aox plasmid (Additional file 1: Fig. S1) and established aox-transformed strains via Agrobacterium mediated transformation. The hyg gene fragment was amplified for preliminary selection, and the relative expression of this target gene was amplified by (quantitative PCR) qPCR to screen the RNAi-aox strains. The results are shown in Additional file 1: Fig. S2. To further explore the function of the aox gene in the response of P. ostreatus to HS, the plates containing the various strains were cultured at 28 °C for 5 d, transferred to 40 °C for 48 h, and then incubated at 28 °C to allow growth recovery. After 3 d of growth recovery after HS, mycelial germination was observed in the wild type (WT), OE-aox and RNAi-aox strains, as shown in Fig. 7A. After 5 d of growth recovery, compared with the WT strain, the OE-aox strains exhibited a faster recovery rate and a complete colony edge, whereas RNAi-aox strains presented a slower mycelial recovery rate and showed defects on the edge of the colony. In conclusion, the aox gene plays an active role in the recovery of P. ostreatus mycelia after HS. Figs. 7B, 7C and 7D show the changes in the H2O2 content and the O2- content and production rate of the aox-transformed strains under HS. Under HS, the accumulation of H2O2 in the OE-aox 47 and OE-aox 71 strains was significantly lower (by 9.05 % and 12.28 % respectively) than that in the WT strain. In addition, the H2O2 content in the OE-aox 34 strain was 4.29 % lower than that in the WT strain. In contrast, the H2O2 contents in the RNAi-aox 12, RNAi-aox 29 and RNAi-aox 7 strains were 4.59 %, 17.71 % and 21.11 % higher, respectively, than that in the WT strain. As shown in Fig. 7C-D, the O2- production rate and content of the OE-aox strains under HS were significantly lower than those of the WT strain, whereas that of the RNAi -aox strains increased significantly. These results indicate that the aox gene can regulate the production and accumulation of ROS. ROS are mainly caused by electron leakage in respiratory chain. As shown in Figs. 7E, 7F and 7G, compared with the WT strain, the average NADH and ATP contents in the OE-aox strains were decreased by 26.47 % and 9.82 %, respectively, and the average NAD+/NADH ratio in these strains was 1.55-fold higher. In contrast, the average NADH and ATP contents of the RNAi-aox strains were 66.86 % and 29.79 %, respectively, higher than those in the WT strain, and the NAD+/NADH ratio was decreased by 26.52 %. Therefore, it can be speculated that the aox gene plays an important role in regulating the mitochondrial ETC, energy metabolism and maintaining mitochondrial homeostasis.
In conclusion, the aox gene can affect the production and accumulation of ROS by regulating mitochondrial respiration, and participates in the response of mycelia to HS.
aox gene regulates the expression of key antioxidant enzyme genes in mycelia after HS
The signaling from organelles controlling nuclear gene expression is called retrograde signaling, and previous studies have shown that AOX serves as a marker gene for mitochondrial retrograde regulation [28]. AOX also acts as a facilitator for signaling molecules conveying the metabolic status of mitochondria to the nucleus and is thus able to influence nuclear gene expression [29]. Moreover, studies have shown that AOX can affect the production and accumulation of ROS. Antioxidant systems (such as antioxidant enzymes and nonenzymatic oxidants) play critical roles in the defense against oxidative stress [30]. We detected the changes in the expression of genes that encode four key antioxidant enzymes, namely catalase (CAT), superoxide dismutase (SOD), thioredoxin reductase (TrxR) and glutathione peroxidase (GSH-PX). In the genome of P. ostreatus, two genes (cat1 and cat2) encode CAT, four genes (SOD1, SOD2, SOD3 and SOD4) encode SOD, one gene encodes TrxR, and one gene encodes GSH-PX [31]. To study whether aox in P. ostreatus can alleviate ROS outbreaks by regulating the antioxidant enzyme system, we measured the expression of these key antioxidant enzyme genes in aox-transformed strains exposed to HS at 40 °C for 48 h. As shown in Fig. 8A, after 48 h of exposure to HS, the relative expression of cat1 in the OE-aox 47-, OE-aox 71- and OE-aox 34- transformed strains was significantly downregulated to 26.5 %, 25.96 % and 35.30 % of the level found in the WT strain, respectively. In RNAi-aox 12 and RNAi-aox 29 strains, the relative expression of cat1 was significantly increased by 1.57-fold and 7.28-fold compared with that found in WT strain, but no significant change in the expression of this gene was detected in the RNAi-aox 7 strain. As shown in Fig. 8B, compared with that in the WT strain, the relative expression of cat2 in the OE-aox and RNAi-aox strains was significantly downregulated and significantly increased, respectively. In conclusion, aox can negatively regulate cat gene expression under HS. As shown in Figs. 8C and 8D, the expression of the TrxR and GSH-PX genes was significantly downregulated in the OE-aox-transformed strains. In addition, the TrxR gene was significantly upregulated in all RNAi-aox strains, and the relative expression of the GSH-PX gene was significantly upregulated in RNAi-aox12 and RNAi-aox 29, and slightly upregulated in RNAi-aox 7. SOD is one of the major defense systems used to remove O2−. The relative expression level of the four SOD-encoding genes in the aox- transformed strains under HS are shown in Figs. 8E-8H. As shown in Fig. 8E, compared with the level in the WT strain, SOD1 gene expression was significantly increased in the OE-aox strains and decreased to 72.66 %, 67.71 % and 79.93 % in the RNAi-aox 12, RNAi-aox 29 and RNAi-aox 7, respectively. In addition, as shown in Figs. 8F-8H, the expression level of SOD2, SOD3 and SOD4 in the OE-aox strains was significantly downregulated, to 34.93 %, 60.33 % and 32.16 % of the level found in the WT strain, respectively. However, SOD2 and SOD3 gene expression was not significantly changed in the RNAi-aox strains, and the expression of SOD4 was slightly downregulated in these strains compared with the WT strain. Because the SOD1 gene encodes Gu-SOD, which mainly exists in the cytoplasm, it can be concluded that the OE of aox during HS can regulate the expression of SOD-encoding gene.
In conclusion, in addition to SOD1, the OE-aox strains exhibited downregulated expression of key antioxidant enzyme coding genes under HS (40 °C for 48 h), and the RNAi-aox strains exhibited upregulated CAT-, GSH-PX- and TrxR-encoding genes. It can thus be hypothesized that the significant upregulation of the aox gene after HS (40 °C for 48 h) can further affect the expression of key antioxidant enzyme-encoding genes.
Exogenous benzohydroxamate (BHAM) regulated the expression of antioxidant enzyme-encoding genes in mycelia under HS
AOX is sensitive to inhibitors of the cytochrome pathway such as cyanide, antimycin A, or miyxothiazol, but is inhibited by primary hydroxamic acids, such as BHAM [32, 33]. To further prove whether aox interference can regulate the expression of key antioxidant enzyme-encoding genes, an experiment in which different concentrations of an AOX inhibitor (BHAM) were added was then performed. The results showed that the growth rate of P. ostreatus mycelia at 28 °C was affected by exogenous BHAM. As shown in Additional file 1: Figs. S3A and S3B, a low concentration of BHAM (50-100 μM) had no significant effect on the colony and mycelial growth rate of P. ostreatus, but the addition of exogenous BHAM at a concentration higher than 200 μM affected mycelial growth of mycelia and significantly reduced the mycelial growth rate. Furthermore, whether exogenous BHAM could regulate the expression of the aox gene was assessed in the experiment. As shown in Additional file 1: Fig. S3C, the addition of BHAM at a concentration of 50-200 μM significantly downregulated the expression of the aox gene compared with the CK level, which indicated that low concentrations of BHAM can inhibit the expression of the aox gene. However, a BHAM concentration of 400 μM upregulated the expression of the aox gene, and because this concentration also significantly inhibited the mycelial growth rate, it can be speculated that 400 μM BHAM might affect the growth environment of mycelia and induce abiotic stress, which eventually leads to the induction of aox gene expression. In addition, because the mycelial growth of the RNAi-aox strains did not significantly differ from the normal growth of P. ostreatus mycelia, 50-200 μM was used as the BHAM concentration in the subsequent experiments.
As shown in Fig. 9A, the addition of exogenous BHAM at different concentrations could slow down the recovery of mycelial growth after HS compared with that obtained with the WT strain, and this result was consistent with the results obtained with the RNAi-aox strains. The effects of exogenous BHAM on the H2O2 content and the O2- content and production rate were then further detected. The results showed that compared with the CK-HS group, the H2O2 content and O2- content and production rate of mycelia under HS were significantly increased by different concentrations of BHAM (Figs. 9B-9D). These results indicated that exogenous BHAM could promote the production and accumulation of ROS in mycelia under HS. The effects of exogenous BHAM on the expression of key antioxidant enzyme-encoding genes in mycelia under HS are shown in Figs. 9E-9L. As illustrated in the figures, the addition of exogenous BHAM significantly increased the expression of antioxidant enzyme-encoding genes under HS. Specifically, the expression levels of cat1 (Fig. 9E), cat2 (Fig. 9F), TrxR (Fig. 9G), GSH-PX (Fig. 9H), SOD2 (Fig. 9J) and SOD4 (Fig. 9L) increased with increases in the BHAM concentration, and the highest expression levels of these genes, which were 47.25-fold, 17.76-fold, 1.99-fold, 6.86-fold, 4.76-fold and 3.98-fold higher than those found in the CK-HS group, respectively, were detected with a BHAM concentration of 200 μM. In addition, the highest expression levels of SOD1 and SOD3, which were 1.90-fold and 2.42-fold higher than the control levels, respectively, were obtained with the exogenous addition of 100 μM BHAM (Figs. 9I and 9K).
In conclusion, exogenous BHAM can promote the production and accumulation of ROS under HS and then regulate the expression of antioxidant enzyme-encoding genes.