LbSakA-mediated phosphorylation of the scaffolding protein LbNoxR in the ectomycorrhizal basidiomycete Laccaria bicolor regulates NADPH oxidase activity, ROS accumulation and symbiosis development.

Ectomycorrhizal symbiosis, which involves mutually beneficial interactions between soil fungi and tree roots, is essential for promoting tree growth. To establish this symbiotic relationship, fungal symbionts must initiate and sustain mutualistic interactions with host plants while avoiding host defense responses. This study investigated the role of reactive oxygen species (ROS) generated by fungal NADPH oxidase (Nox) in the development of Laccaria bicolor/Populus tremula × alba symbiosis. Our findings revealed that L. bicolor LbNox expression was significantly higher in ectomycorrhizal roots than in free-living mycelia. RNAi was used to silence LbNox, which resulted in decreased ROS signaling, limited formation of the Hartig net, and a lower mycorrhizal formation rate. Using Y2H library screening, BiFC and Co-IP, we demonstrated an interaction between the mitogen-activated protein kinase LbSakA and LbNoxR. LbSakA-mediated phosphorylation of LbNoxR at T409, T477 and T480 positively modulates LbNox activity, ROS accumulation and upregulation of symbiosis-related genes involved in dampening host defense reactions. These results demonstrate that regulation of fungal ROS metabolism is critical for maintaining the mutualistic interaction between L. bicolor and P. tremula × alba. Our findings also highlight a novel and complex regulatory mechanism governing the development of symbiosis, involving both transcriptional and posttranslational regulation of gene networks.

GlPP2C1 Silencing Increases the Content of Ganodermalingzhi Polysaccharide (GL-PS) and Enhances Slt2 Phosphorylation.

Polysaccharides have attracted much attention in the food industry due to their diverse biological activities. To date, research on the mechanism of polysaccharide synthesis has mainly focused on the role of crucial enzymes in the polysaccharide synthesis pathway, but other genes that regulate polysaccharide synthesis have not been well explored. In this study, the GlPP2C1 gene, encoding a phosphoprotein type 2C phosphatase, was cloned, and PP2C-silenced strains (PP2C1i-1 and PP2C1i-3) were screened. Measurements of the polysaccharide content and cell wall tolerance revealed that GlPP2C1 silencing increased the polysaccharide content and enhanced cell wall resistance in Ganoderma lingzhi. The contents of intracellular polysaccharides (IPS), extracellular polysaccharides (EPS) and ß-1,3-D-glucan in PP2C-silenced strains were increased by 25%, 33% and 36%, respectively, compared with those in the WT strains and strains transformed with an empty vector. Further mechanistic studies showed that GlPP2C1 silencing increased the content of Ganoderma lingzhi polysaccharides (GL-PS) through Slt2. In summary, this study revealed the mechanism through which protein phosphatase regulates GL-PS biosynthesis for the first time.

GlSwi6 Positively Regulates Cellulase and Xylanase Activities through Intracellular Ca2+ Signaling in Ganoderma lucidum.

Ganoderma lucidum is a white-rot fungus that produces a range of lignocellulolytic enzymes to decompose lignin and cellulose. The mitogen-activated protein kinase (MAPK) pathway has been implicated in xylanases and cellulases production. As the downstream transcription factor of Slt2-MAPK, the function of Swi6 in G. lucidum has not been fully studied. In this study, the transcription factor GlSwi6 in G. lucidum was characterized and shown to significantly positively regulate cellulases and xylanases production. Knockdown of the GlSwi6 gene decreased the activities of cellulases and xylanases by approximately 31%~38% and 54%~60% compared with those of the wild-type (WT) strain, respectively. Besides, GlSwi6 can be alternatively spliced into two isoforms, GlSwi6A and GlSwi6B, and overexpression of GlSwi6B increased the activities of cellulase and xylanase by approximately 50% and 60%, respectively. Further study indicates that the existence of GlSwi6B significantly increased the concentration of cytosolic Ca2+. Our study indicated that GlSwi6 promotes the activities of cellulase and xylanase by regulating the Ca2+ signaling. These results connected the GlSwi6 and Ca2+ signaling in the regulation of cellulose degradation, and provide an insight for further improvement of cellulase or xylanase activities in G. lucidum as well as other fungi.

Putrescine regulates nitric oxide accumulation in Ganoderma lucidum partly by influencing cellular glutamine levels under heat stress.

When fungi are subjected to abiotic stresses, the polyamines (PAs) level alter significantly. Here, we reveal that the polyamine putrescine (Put) could play an important role in alleviating heat stress(HS)-induced accumulation of nitric oxide (NO). Ornithine decarboxylase (ODC)-silenced mutants that were defective in Put biosynthesis exhibited significantly lower NO levels than the wild type (WT) when subjected to HS. With addition of 5 mM exogenous Put, the ODC-silenced mutant endogenous Put obviously increased under HS. At the same time, the contents of NO in the ODC-silenced mutants recovered to approximately WT levels after the administration of exogenous Put. However, the elevated NO content in the ODC-silenced mutants disappeared when exogenous Put and carboxy-PTIO (PTIO is a specific scavenger of NO) were added. Intriguingly, the content of glutamine (Gln) was significantly increased in the ODC-silenced strains. When exogenous Put was added to the WT, the Gln content was significantly decreased. The appearance of a high level of Gln was accompanied by nitrate reductase (NR) activity reduction. Further studies showed that Put influenced ganoderic acids (GAs) biosynthesis by regulating NO content, possibly through NR, under HS. Our work reported that Put regulates HS-induced NO accumulation by changing the cellular Gln level in filamentous fungi. IMPORTANCE: In our present work, it was HS as an ubiquitous environmental stress that affects the important pharmacological secondary metabolite (GAs) content in G. lucidum. Afterwards, we began to explore the network formed between multiple substances to jointly reduce the massive accumulation of GAs content caused by HS. We firstly focused on Put, a substance that enhances resistance to multiple stresses. Further, we discovered an influence on Put could changing the NO content, which has been shown to decrease the accumulation of GAs via HS. Then, we also found the change of NO content may be due to Put level that would affect intracellular Gln content. It has never been reported. And ultimately, it is Put related network that could reduce HS-inducing secondary metabolite mess in fungi.

Glsnf1-mediated metabolic rearrangement participates in coping with heat stress and influencing secondary metabolism in Ganoderma lucidum.

The AMP-activated protein kinase (AMPK)/Sucrose-nonfermenting serine-threonine protein kinase 1 (Snf1) plays an important role in metabolic remodelling in response to energy stress. However, the role of AMPK/Snf1 in responding to other environmental stresses and metabolic remodelling in microorganisms was unclear. Heat stress (HS), which is one important environmental factor, could induce the production of reactive oxygen species and the accumulation of ganoderic acids (GAs) in Ganoderma lucidum. Here, the functions of AMPK/Snf1 were analysed under HS condition in G. lucidum. We observed that Glsnf1 was rapidly and strongly activated when G. lucidum was exposed to HS. HS significantly increased intracellular H2O2 levels (by approximately 1.6-fold) and decreased the dry weight of G. lucidum (by approximately 45.6%). The exogenous addition of N-acetyl-l-cysteine (NAC) and ascorbic acid (VC), which function as ROS scavengers, partially inhibited the HS-mediated reduction in biomass. Adding the AMPK/Snf1 inhibitor compound C (20 µM) under HS conditions increased the H2O2 content (by approximately 2.3-fold of that found in the strain without HS treatment and 1.5-fold of that found in the strain under HS treatment without compound C) and decreased the dry weight of G. lucidum (an approximately 28.5% decrease compared with that of the strain under HS conditions without compound C). Similar results were obtained by silencing the Glsnf1 gene. Further study found that Glsnf1 meditated metabolite distribution from respiration to glycolysis, which is considered a protective mechanism against oxidative stress. In addition, Glsnf1 negatively regulated the biosynthesis of GA by removing ROS. In conclusion, our results suggest that Glsnf1-mediated metabolic remodelling is involved in heat stress adaptability and the biosynthesis of secondary metabolites in G. lucidum.

Influence of PacC on the environmental stress adaptability and cell wall components of Ganoderma lucidum.

The transcription factor PacC/Rim101 participates in environmental pH adaptation, development and secondary metabolism in many fungi, but whether PacC/Rim101 contributes to fungal adaptation to environmental stress remains unclear. In our previous study, a homologous gene of PacC/Rim101 was identified, and PacC-silenced strains of the agaricomycete Ganoderma lucidum were constructed. In this study, we further investigated the functions of PacC in G. lucidum and found that PacC-silenced strains were hypersensitive to environmental stresses, such as osmotic stress, oxidative stress and cell wall stress, compared with wild-type (WT) and empty-vector control (CK) strains. In addition, transmission electron microscopy images of the cell wall structure showed that the cell walls of the PacC-silenced strains were thinner (by approximately 25-30%) than those of the WT and CK strains. Further analysis of cell wall composition showed that the ß-1,3-glucan content in the PacC-silenced strains was only approximately 78-80% of that in the WT strain, and the changes in ß-1,3-glucan content were consistent with downregulation of glucan synthase gene expression. The ability of PacC to bind to the promoters of glucan synthase-encoding genes confirms that PacC transcriptionally regulates these genes.

Integrated Proteomics and Metabolomics Analysis Provides Insights into Ganoderic Acid Biosynthesis in Response to Methyl Jasmonate in Ganoderma Lucidum.

Ganoderma lucidum is widely recognized as a medicinal basidiomycete. It was previously reported that the plant hormone methyl jasmonate (MeJA) could induce the biosynthesis of ganoderic acids (GAs), which are the main active ingredients of G. lucidum. However, the regulatory mechanism is still unclear. In this study, integrated proteomics and metabolomics were employed on G. lucidum to globally identify differences in proteins and metabolites under MeJA treatment for 15 min (M15) and 24 h (M24). Our study successfully identified 209 differential abundance proteins (DAPs) in M15 and 202 DAPs in M24. We also identified 154 metabolites by GC-MS and 70 metabolites by LC-MS in M24 that are involved in several metabolic pathways. With an in-depth analysis, we found some DAPs and metabolites that are involved in the oxidoreduction process, secondary metabolism, energy metabolism, transcriptional and translational regulation, and protein synthesis. In particular, our results reveal that MeJA treatment leads to metabolic rearrangement that inhibited the normal glucose metabolism, energy supply, and protein synthesis of cells but promoted secondary metabolites, including GAs. In conclusion, our proteomics and metabolomics data further confirm the promoting effect of MeJA on the biosynthesis of GAs in G. lucidum and will provide a valuable resource for further investigation of the molecular mechanisms of MeJA signal response and GA biosynthesis in G. lucidum and other related species.

Hydrogen sulfide, a novel small molecule signalling agent, participates in the regulation of ganoderic acids biosynthesis induced by heat stress in Ganoderma lucidum.

Hydrogen sulfide (H2S), an emerging small-molecule signalling agent, was recently shown to play a significant role in many physiological processes, but relatively few studies have been conducted on microorganisms compared with mammals and plants. By studying the pretreatment of H2S donor sodium hydrosulfide (NaHS) and the scavenger hypotaurine (HT) and Cystathionine ß-synthase silenced strains, we found that H2S could alleviate the HS-induced ganoderic acids (GAs) biosynthesis. Our transcriptome results also showed that many signaling pathways and metabolic pathways, such as the glycolysis, TCA, oxidative phosphorylation and pentose phosphate pathway, are influenced by H2S. Further experimental results indicated that H2S could affect the physiological process of Ganoderma lucidum by interacting with multiple signals, including ROS, NO, AMPK, sphingolipid, mTOR, phospholipase D and MAPK, and physiological and pharmacological analyses showed that H2S might alleviate the biosynthesis of GAs by inhibiting the intracellular calcium in G. lucidum.

The Slt2-MAPK pathway is involved in the mechanism by which target of rapamycin regulates cell wall components in Ganoderma lucidum.

The fungal cell wall is very important for cell growth and survival during stress, and the target of rapamycin (TOR) pathway plays a major role in regulating cell growth in response to environmental cues. Ganoderma lucidum is an important edible and medicinal fungus, and the function of TOR in this organism remains unclear. As shown in the present study, the TOR pathway regulates cell wall integrity (CWI) in G. lucidum. Inhibition of TOR signaling by RNA interference (RNAi) or rapamycin treatment reduced the growth of G. lucidum mycelia, increased contents of the cell wall components chitin and ß-1,3-glucan, and increased cell wall thickness. Furthermore, inhibition of TOR signaling enhanced the relative level of phosphorylated Slt2, a member of the MAPK cascade involved in CWI signaling. Moreover, when treated with rapamycin, significantly lower chitin and ß-1,3-glucan contents were observed in Slt2-silenced strains than in WT strains, indicating that TOR regulates the synthesis of these cell wall components through the Slt2-MAPK pathway. These results indicate a potential relationship between TOR signaling and CWI signaling. Additionally, participation of Slt2-MAPK in TOR-mediated regulation of cell wall component production has not previously been reported in a microorganism.

Conversion of phosphatidylinositol (PI) to PI4-phosphate (PI4P) and then to PI(4,5)P2 is essential for the cytosolic Ca2+ concentration under heat stress in Ganoderma lucidum.

How cells drive the phospholipid signal response to heat stress (HS) to maintain cellular homeostasis is a fundamental issue in biology, but the regulatory mechanism of this fundamental process is unclear. Previous quantitative analyses of lipids showed that phosphatidylinositol (PI) accumulates after HS in Ganoderma lucidum, implying the inositol phospholipid signal may be associated with HS signal transduction. Here, we found that the PI-4-kinase and PI-4-phosphate-5-kinase activities are activated and that their lipid products PI-4-phosphate and PI-4,5-bisphosphate are increased under HS. Further experimental results showed that the cytosolic Ca2+ ([Ca2+ ]c ) and ganoderic acid (GA) contents induced by HS were decreased when cells were pretreated with Li+ , an inhibitor of inositol monophosphatase, and this decrease could be rescued by PI and PI-4-phosphate. Furthermore, inhibition of PI-4-kinases resulted in a decrease in the Ca2+ and GA contents under HS that could be rescued by PI-4-phosphate but not PI. However, the decrease in the Ca2+ and GA contents by silencing of PI-4-phosphate-5-kinase could not be rescued by PI-4-phosphate. Taken together, our study reveals the essential role of the step converting PI to PI-4-phosphate and then to PI-4,5-bisphosphate in [Ca2+ ]c signalling and GA biosynthesis under HS.

SA inhibits complex III activity to generate reactive oxygen species and thereby induces GA overproduction in Ganoderma lucidum.

Ganoderma lucidum has high commercial value because it produces many active compounds, such as ganoderic acids (GAs). Salicylic acid (SA) was previously reported to induce the biosynthesis of GA in G. lucidum. In this study, we found that SA induces GA biosynthesis by increasing ROS production, and further research found that NADPH oxidase-silenced strains exhibited a partial reduction in the response to SA, resulting in the induction of increased ROS production. Furthermore, the localization of ROS shows that mitochondria are sources of ROS production in response to SA treatment. An additional analysis focused on the relationship between SA-induced ROS production and mitochondrial functions, and the results showed that inhibitors of mitochondrial complexes I and II exert approximately 40-50% superimposed inhibitory effects on the respiration rate and H2O2 content when co-administered with SA. However, no obvious superimposed inhibition effects were observed in the sample co-treated with mitochondrial complex III inhibitor and SA, implying that the inhibitor of mitochondrial complex III and SA might act on the same site in mitochondria. Additional experiments revealed that complex III activity was decreased 51%, 62% and 75% after treatment with 100, 200, and 400 µM SA, respectively. Our results highlight the finding that SA inhibits mitochondrial complex III activity to increase ROS generation. In addition, inhibition of mitochondrial complex III caused ROS accumulation, which plays an essential role in SA-mediated GA biosynthesis in G. lucidum. This conclusion was also demonstrated in complex III-silenced strains. To the best of our knowledge, this study provides the first demonstration that SA inhibits complex III activity to increase the ROS levels and thereby regulate secondary metabolite biosynthesis.

Heat stress-induced reactive oxygen species participate in the regulation of HSP expression, hyphal branching and ganoderic acid biosynthesis in Ganoderma lucidum.

Heat stress (HS) is an important environmental factor that affects the growth and metabolism of edible fungi, but the molecular mechanism of the heat stress response (HSR) remains unclear. We previously reported that HS treatment increased the length between two hyphal branches and induced the accumulation of ganoderic acid biosynthesis and the gene expression of heat shock proteins (HSPs) in Ganoderma lucidum. In this study, we found that HS induced a significant increase in the cytosolic ROS concentration, and exogenously added ROS scavengers NAC, VC and NADPH oxidase (Nox) inhibitor DPI reduce the cytosolic ROS accumulation in G. lucidum. In addition, the phenomena of the increased gene expression and increased length between the two hyphal branches and the accumulation of GA biosynthesis induced by HS were mitigated. Furthermore, we investigated the effects of HS on Nox-silenced strains (NoxABi-10, NoxABi-11 and NoxRi-4, NoxRi-7) and found that the level of ROS concentration was lower than that in wild-type (WT) strains treated with HS. Additionally, Nox silenced strains reduced the HS-induced increase in HSP expression, the length between two hyphal branches and GA biosynthesis compared with the WT strain. These data indicate that HS-induced ROS participate in the regulation of HSP expression, hyphal branching and ganoderic acid biosynthesis in G. lucidum. In addition, these findings identified potential pathways linking ROS networks to HSR, physiological and metabolic processes in fungi and provide a valuable reference for studying the role of ROS in HSR, mycelium growth and secondary metabolites.

Cross Talk between Nitric Oxide and Calcium-Calmodulin Regulates Ganoderic Acid Biosynthesis in Ganoderma lucidum under Heat Stress.

We previously reported that high temperature impacts ganoderic acid (GA) biosynthesis in Ganoderma lucidum via Ca2+ Therefore, to further understand the signal-regulating network of the organism's response to heat stress (HS), we examined the role of nitric oxide (NO) under HS. After HS treatment, the NO level was significantly increased by 120% compared to that under the control conditions. The application of a NO scavenger resulted in a 25% increase in GA compared with that found in the sample treated only with HS. Additionally, the application of a NO donor to increase NO resulted in a 30% lower GA content than that in the sample treated only with HS. These results show that the increase in NO levels alleviates HS-induced GA accumulation. Subsequently, we aimed to detect the effects of the interaction between NO and Ca2+ on GA biosynthesis under HS in G. lucidum Our pharmacological approaches revealed that the NO and Ca2+ signals promoted each other in response to HS. We further constructed the silenced strain of nitrate reductase (NR) and calmodulin (CaM), and the results are in good agreement with the silenced strain and pharmacological experiment. The cross-promotion between NO and Ca2+ signals is involved in the regulation of HS-induced GA biosynthesis in G. lucidum, and this finding is supported by studies with NR-silenced (NRi) and CaM-silenced (CaMi) strains. However, Ca2+ may have a more direct and significant effect on the HS-induced GA increase than NO. These data indicate that NO functions in signaling and has a close relationship with Ca2+ in HS-induced GA biosynthesis.IMPORTANCE HS is an important environmental stress affecting the growth and development of organisms. We previously reported that HS modulates GA biosynthesis in G. lucidum via Ca2+ However, the signal-regulating network of the organism's response to HS has not yet been elucidated. In this study, we found that NO relieved HS-induced GA accumulation, and NO and Ca2+ could exert promoting effects on each other in response to HS. Further research on the effect of NO and Ca2+ on the production of GAs in response to HS indicated that Ca2+ has a notably more direct and significant effect on the HS-induced GA increase than NO. Our results improve our understanding of the mechanism of HS signal transduction in fungi. A greater understanding of the regulation of secondary metabolism in response to environmental stimuli will provide clues regarding the role of these products in fungal biology.

14-3-3 proteins are involved in growth, hyphal branching, ganoderic acid biosynthesis, and response to abiotic stress in Ganoderma lucidum.

Ganoderma lucidum, which contains many pharmacologically active compounds, is regarded as a traditional medicinal fungus. Nevertheless, the scarcity of basic research limits the commercial value and utilization of G. lucidum. As a class of highly conserved, phosphopeptide-binding proteins present in all eukaryotes, 14-3-3 proteins play vital roles in controlling multiple physiological processes, including signal transduction, primary metabolism, and stress responses. However, knowledge of the roles of 14-3-3 proteins in Basidiomycetes is sparse. In this article, two homologs of 14-3-3 proteins, encoded by the two distinct genes GlBmh1 and GlBmh2, were distinguished in G. lucidum. We found that GlBmh1 and GlBmh2 were expressed at various developmental stages, including in vegetative mycelium cultivated on solid medium and in primordia and fruiting bodies. Moreover, we constructed GlBmh1 single-silenced strains, GlBmh2 single-silenced strains, and 14-3-3 double-silenced mutants for further study. When GlBmh1 and GlBmh2 were inhibited by RNA interference, the growth rate of mycelia was decreased, and the distance between the aerial hyphal branches was reduced; responses to various abiotic stresses such as oxidants and cell wall and osmotic stressors were also changed. Furthermore, the contents of secondary metabolite ganoderic acids (GAs) were increased after GlBmh1 and GlBmh2 were simultaneously silenced. Taken together, we provide evidence that implicates potential roles for the two 14-3-3 proteins in affecting growth and GA biosynthesis, thereby providing new insights into the basic functions of 14-3-3 proteins in G. lucidum.

Phospholipase D and phosphatidic acid mediate heat stress induced secondary metabolism in Ganoderma lucidum.

Phospholipid-mediated signal transduction plays a key role in responses to environmental changes, but little is known about the role of phospholipid signalling in microorganisms. Heat stress (HS) is one of the most important environmental factors. Our previous study found that HS could induce the biosynthesis of the secondary metabolites, ganoderic acids (GA). Here, we performed a comprehensive mass spectrometry-based analysis to investigate HS-induced lipid remodelling in Ganoderma lucidum. In particular, we observed a significant accumulation of phosphatidic acid (PA) on HS. Further genetic tests in which pld-silencing strains were constructed demonstrated that the accumulation of PA is dependent on HS-activated phospholipase D (PLD) hydrolysing phosphatidylethanolamine. Furthermore, we determined the role of PLD and PA in HS-induced secondary metabolism in G. lucidum. Exogenous 1-butanol, which decreased PLD-mediated formation of PA, reverses the increased GA biosynthesis that was elicited by HS. The pld-silenced strains partly blocked HS-induced GA biosynthesis, and this block can be reversed by adding PA. Taken together, our results suggest that PLD and PA are involved in the regulation of HS-induced secondary metabolism in G. lucidum. Our findings provide key insights into how microorganisms respond to heat stress and then consequently accumulate secondary metabolites by phospholipid remodelling.

Alternative oxidase impacts ganoderic acid biosynthesis by regulating intracellular ROS levels in Ganoderma lucidum.

The alternative oxidase (AOX), which forms a branch of the mitochondrial respiratory electron transport pathway, functions to sustain electron flux and alleviate reactive oxygen species (ROS) production. In this article, a homologous AOX gene was identified in Ganoderma lucidum. The coding sequence of the AOX gene in G. lucidum contains 1038 nucleotides and encodes a protein of 39.48 kDa. RNA interference (RNAi) was used to study the function of AOX in G. lucidum, and two silenced strains (AOXi6 and AOXi21) were obtained, showing significant decreases of approximately 60 and 50 %, respectively, in alternative pathway respiratory efficiency compared to WT. The content of ganoderic acid (GA) in the mutant strains AOXi6 and AOXi21 showed significant increases of approximately 42 and 44 %, respectively, compared to WT. Elevated contents of intermediate metabolites in GA biosynthesis and elevated transcription levels of corresponding genes were also observed in the mutant strains AOXi6 and AOXi21. In addition, the intracellular ROS content in strains AOXi6 and AOXi21 was significantly increased, by approximately 1.75- and 1.93-fold, respectively, compared with WT. Furthermore, adding N-acetyl-l-cysteine (NAC), a ROS scavenger, significantly depressed the intracellular ROS content and GA accumulation in AOX-silenced strains. These results indicate that AOX affects GA biosynthesis by regulating intracellular ROS levels. Our research revealed the important role of AOX in the secondary metabolism of G. lucidum.

Ornithine Decarboxylase-Mediated Production of Putrescine Influences Ganoderic Acid Biosynthesis by Regulating Reactive Oxygen Species in Ganoderma lucidum.

Putrescine is an important polyamine that participates in a variety of stress responses. Ornithine decarboxylase (ODC) is a key enzyme that catalyzes the biosynthesis of putrescine. A homolog of the gene encoding ODC was cloned from Ganoderma lucidum In the ODC-silenced strains, the transcript levels of the ODC gene and the putrescine content were significantly decreased. The ODC-silenced strains were more sensitive to oxidative stress. The content of ganoderic acid was increased by approximately 43 to 46% in the ODC-silenced strains. The content of ganoderic acid could be recovered after the addition of exogenous putrescine. Additionally, the content of reactive oxygen species (ROS) was significantly increased by approximately 1.3-fold in the ODC-silenced strains. The ROS content was significantly reduced after the addition of exogenous putrescine. The gene transcript levels and the activities of four major antioxidant enzymes were measured to further explore the effect of putrescine on the intracellular ROS levels. Further studies showed that the effect of the ODC-mediated production of putrescine on ROS might be a factor influencing the biosynthesis of ganoderic acid. Our study reports the role of putrescine in large basidiomycetes, providing a basis for future studies of the physiological functions of putrescine in microbes.IMPORTANCE It is well known that ODC and the ODC-mediated production of putrescine play an important role in resisting various environmental stresses, but there are few reports regarding the mechanisms underlying the effect of putrescine on secondary metabolism in microorganisms, particularly in fungi. G. lucidum is gradually becoming a model organism for studying environmental regulation and metabolism. In this study, a homolog of the gene encoding ODC was cloned in Ganoderma lucidum We found that the transcript level of the ODC gene and the content of putrescine were significantly decreased in the ODC-silenced strains. The content of ganoderic acid was significantly increased in the ODC-silenced strains. Further studies showed that the effect of the ODC-mediated production of putrescine on ROS might be a factor influencing the biosynthesis of ganoderic acid. Our study reports the role of putrescine in large basidiomycetes, providing a basis for future studies of the physiological functions of putrescine in microbes.

Effects of Exogenous Salicylic Acid on Ganoderic Acid Biosynthesis and the Expression of Key Genes in the Ganoderic Acid Biosynthesis Pathway in the Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (Agaricomycetes).

We demonstrate herein that salicylic acid (SA) can enhance ganoderic acid (GA) accumulation in the lingzhi or reishi medicinal mushroom Ganoderma lucidum. Following treatment with different concentrations of SA, the GA content was increased 22.72% to 43.04% compared with the control group. When the fungi were treated with 200 µmol/L SA at different times, the GA content was improved 10.21% to 35.24% compared with the control group. By choosing the optimum point based on response surface methodology, the GA content could be increased up to 229.03 µg/100 mg, which was improved 66.38% compared with the control group. When the fungi were treated with 200 µmol/L SA, the transcription levels of key genes in the GA biosynthesis pathway-squalene (SQ) synthase (sqs), lanosterol (Lano; osc), and hydroxy-3-methylglutaryl-coenzyme A reductase (hmgr)-were improved 119.6-, 3.2-, and 4.2-fold, respectively. In addition, following treatment with 100 µmol/L SA, the levels of Lano and SQ, which are intermediate metabolites of GA biosynthesis, were increased 2.8- and 1.4-fold, respectively. These results indicate that SA can regulate the expression of genes related to GA biosynthesis and increases the metabolic levels of Lano and SQ, thereby resulting in the accumulation of GA.

Membrane fluidity is involved in the regulation of heat stress induced secondary metabolism in Ganoderma lucidum.

Ganoderma lucidum has become a potential model system for evaluating how environmental factors regulate the secondary metabolism of basidiomycetes. Heat stress (HS) is one of the most important environmental factors. It was previously reported that HS could induce the biosynthesis of ganoderic acids (GA). In this study, we found that HS increased GA biosynthesis and also significantly increased cell membrane fluidity. Furthermore, our results showed that addition of the membrane rigidifier dimethylsulfoxide (DMSO) could revert the increased GA biosynthesis elicited by HS. These results indicate that an increase in membrane fluidity is associated with HS-induced GA biosynthesis. Further evidence showed that the GA content was decreased in D9des-silenced strains and could be reverted to WT levels by addition of the membrane fluidizer benzyl alcohol (BA). In contrast, GA content was increased in D9des-overexpression strains and could be reverted to WT levels by the addition of DMSO. Furthermore, both membrane fluidity and GA biosynthesis induced by HS could be reverted by DMSO in WT and D9des-silenced strains. To the best of our knowledge, this is the first report demonstrating that membrane fluidity is involved in the regulation of heat stress induced secondary metabolism in filamentous fungi.

Hydrogen-rich water regulates effects of ROS balance on morphology, growth and secondary metabolism via glutathione peroxidase in Ganoderma lucidum.

Ganoderma lucidum is one of the most important medicinal fungi, but the lack of basic study on the fungus has hindered the further development of its value. To investigate the roles of the redox system in G. lucidum, acetic acid (HAc) was applied as a reactive oxygen species (ROS) stress inducer, and hydrogen-rich water (HRW) was used to relieve the ROS stress in this study. Our results demonstrate that the treatment of 5% HRW significantly decreased the ROS content, maintained biomass and polar growth morphology of mycelium, and decreased secondary metabolism under HAc-induced oxidative stress. Furthermore, the roles of HRW were largely dependent on restoring the glutathione system under HAc stress in G. lucidum. To provide further evidence, we used two glutathione peroxidase (GPX)-defective strains, the gpxi strain, the mercaptosuccinic acid (MS, a GPX inhibitor)-treated wide-type (WT) strain, and gpx overexpression strains for further research. The results show that HRW was unable to relieve the HAc-induced ROS overproduction, decreased biomass, mycelium morphology change and increased secondary metabolism biosynthesis in the absence of GPX function. The gpx overexpression strains exhibited resistance to HAc-induced oxidative stress. Thus, we propose that HRW regulates morphology, growth and secondary metabolism via glutathione peroxidase under HAc stress in the fungus G. lucidum. Furthermore, our research also provides a method to study the ROS system in other fungi.