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.

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.

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.

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.

Heat Stress Modulates Mycelium Growth, Heat Shock Protein Expression, Ganoderic Acid Biosynthesis, and Hyphal Branching of Ganoderma lucidum via Cytosolic Ca2.

UNLABELLED: Heat stress (HS) influences the growth and development of organisms. Thus, a comprehensive understanding of how organisms sense HS and respond to it is required. Ganoderma lucidum, a higher basidiomycete with bioactive secondary metabolites, has become a potential model system due to the complete sequencing of its genome, transgenic systems, and reliable reverse genetic tools. In this study, we found that HS inhibited mycelium growth, reduced hyphal branching, and induced the accumulation of ganoderic acid biosynthesis and heat shock proteins (HSPs) in G. lucidum Our data showed that HS induced a significant increase in cytosolic Ca(2+) concentration. Further evidence showed that Ca(2+) might be a factor in the HS-mediated regulation of hyphal branching, ganoderic acid (GA) biosynthesis, and the accumulation of HSPs. Our results further showed that the calcium-permeable channel gene (cch)-silenced and phosphoinositide-specific phospholipase gene (plc)-silenced strains reduced the HS-induced increase in HSP expression compared with that observed for the wild type (WT). This study demonstrates that cytosolic Ca(2+) participates in heat shock signal transduction and regulates downstream events in filamentous fungi. IMPORTANCE: Ganoderma lucidum, a higher basidiomycete with bioactive secondary metabolites, has become a potential model system for evaluating how environmental factors regulate the development and secondary metabolism of basidiomycetes. Heat stress (HS) is an important environmental challenge. In this study, we found that HS inhibited mycelium growth, reduced hyphal branching, and induced HSP expression and ganoderic acid biosynthesis in G. lucidum Further evidence showed that Ca(2+) might be a factor in the HS-mediated regulation of hyphal branching, GA biosynthesis, and the accumulation of HSPs. This study demonstrates that cytosolic Ca(2+) participates in heat shock signal transduction and regulates downstream events in filamentous fungi. Our research offers a new way to understand the mechanism underlying the physiological and metabolic responses to other environmental factors in G. lucidum This research may also provide the basis for heat shock signal transduction studies of other fungi.

The pH-responsive transcription factor PacC regulates mycelial growth, fruiting body development, and ganoderic acid biosynthesis in Ganoderma lucidum.

Ganoderma lucidum is a medicinal macrofungus that is widely used in traditional Chinese medicine. Nonetheless, the scarcity of basic biological studies of this organism has hindered the further development of its commercial value. The pH-responsive transcription factor PacC/Rim101 governs the adaptation to environmental pH, the development and the secondary metabolism of many fungi. In this study, a homologue of PacC/Rim101 that encodes GlPacC was identified in the higher basidiomycete G. lucidum. GlPacC is composed of 807 amino acids and contains three typical C2H2 zinc-finger domains, two potential PEST domains, a putative PKA phosphorylation site, and a putative nuclear localization signal (NLS). GlPacC was transcribed at a high level when the fungus was under neutral and alkaline conditions, and silencing of GlPacC impaired the fungal response to ambient pH. The distance between the hyphal branches (of vegetative hyphae and aerial hyphae) was significantly increased in the GlPacC-silenced strains. The GlPacC-silenced strains grew abnormally or became sickly on solid culture medium and were unable to form primordia and fruiting bodies. The ganoderic acid content, levels of the sqs and ls transcripts, and contents of the metabolic intermediates squalene and lanosterol were all up-regulated in the GlPacC-silenced strains. Our results indicate that GlPacC is functional and plays complex roles in mycelial growth, fruiting body development and ganoderic acid biosynthesis in G. lucidum.

Molecular characterization and expression analysis of GlHMGS, a gene encoding hydroxymethylglutaryl-CoA synthase from Ganoderma lucidum (Ling-zhi) in ganoderic acid biosynthesis pathway.

A hydroxymethylglutaryl-CoA synthase gene, designated as GlHMGS (GenBank accession No. JN391469) involved in ganoderic acid (GA) biosynthesis pathway was cloned from Ganoderma lucidum. The full-length cDNA of GlHMGS (GenBank accession No. JN391468) was found to contain an open reading frame of 1,413 bp encoding a polypeptide of 471 amino acid residues. The deduced amino acid sequence of GlHMGS shared high homology with other known hydroxymethylglutaryl-CoA synthase (HMGS) enzymes. In addition, functional complementation of GlHMGS in a mutant yeast strain YSC1021 lacking HMGS activity demonstrated that the cloned cDNA encodes a functional HMGS. A 1,561 bp promoter sequence was isolated and its putative regulatory elements and potential specific transcription factor binding sites were analyzed. GlHMGS expression profile analysis revealed that salicylic acid, abscisic acid and methyl jasmonate up-regulated GlHMGS transcript levels over the control. Further expression analysis revealed that the developmental stage and carbon source had significant effects on GlHMGS transcript levels. GlHMGS expression peaked on day 16 before decreasing with prolonged culture time. The highest mRNA level was observed when the carbon source was maltose. Overexpression of GlHMGS enhanced GA content in G. lucidum. This study provides useful information for further studying this gene and on its function in the ganoderic acid biosynthetic pathway in G. lucidum.

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.

Identification of Reference Genes and Analysis of Heat Shock Protein Gene Expression in Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum, after Exposure to Heat Stress.

Ganoderma lucidum has been considered an emerging model species for studying how environmental factors regulate the growth, development, and secondary metabolism of Basidiomycetes. Heat stress, which is one of the most important environmental abiotic stresses, seriously affects the growth, development, and yield of microorganisms. Understanding the response to heat stress has gradually become a hotspot in microorganism research. But suitable reference genes for expression analysis under heat stress have not been reported in G. lucidum. In this study, we systematically identified 11 candidate reference genes that were measured using reverse transcriptase quantitative polymerase chain reaction, and the gene expression stability was analyzed under heat stress conditions using geNorm and NormFinder. The results show that 5 reference genes-CYP and TIF, followed by UCE2, ACTIN, and UBQ1-are the most stable genes under our experimental conditions. Moreover, the relative expression levels of 3 heat stress response genes (hsp17.4, hsp70, and hsp90) were analyzed under heat stress conditions with different normalization strategies. The results show that use of a gene with unstable expression (SAND) as the reference gene leads to biased data and misinterpretations of the target gene expression level under heat stress.

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.

Profiling and quantifying differential gene transcription provide insights into ganoderic acid biosynthesis in Ganoderma lucidum in response to methyl jasmonate.

Ganoderma lucidum is a mushroom with traditional medicinal properties that has been widely used in China and other countries in Eastern Asia. Ganoderic acids (GA) produced by G. lucidum exhibit important pharmacological activities. Previous studies have demonstrated that methyl jasmonate (MeJA) is a potent inducer of GA biosynthesis and the expression of genes involved in the GA biosynthesis pathway in G. lucidum. To further explore the mechanism of GA biosynthesis, cDNA-Amplified Fragment Length Polymorphism (cDNA-AFLP) was used to identify genes that are differentially expressed in response to MeJA. Using 64 primer combinations, over 3910 transcriptionally derived fragments (TDFs) were obtained. Reliable sequence data were obtained for 390 of 458 selected TDFs. Ninety of these TDFs were annotated with known functions through BLASTX searching the GenBank database, and 12 annotated TDFs were assigned into secondary metabolic pathways by searching the KEGGPATHWAY database. Twenty-five TDFs were selected for qRT-PCR analysis to confirm the expression patterns observed with cDNA-AFLP. The qRT-PCR results were consistent with the altered patterns of gene expression revealed by the cDNA-AFLP technique. Additionally, the transcript levels of 10 genes were measured at the mycelium, primordia, and fruiting body developmental stages of G. lucidum. The greatest expression levels were reached during primordia for all of the genes except cytochrome b2 reached its highest expression level in the mycelium stage. This study not only identifies new candidate genes involved in the regulation of GA biosynthesis but also provides further insight into MeJA-induced gene expression and secondary metabolic response in G. lucidum.

A zinc-dependent nuclear endonuclease is responsible for DNA laddering during salt-induced programmed cell death in root tip cells of rice.

DNA laddering is one of the biochemical processes characteristic of programmed cell death (PCD) both in animals and plants. However, the mechanism of DNA laddering varies in different species, even in different tissues of one organism. In the present study, we used root tip cells of rice, which have been induced by NaCl stress to undergo PCD, to analyze the endonuclease activities of cytoplasmic and nuclear extracts. Two endonucleases, a cytoplasmic of 20kDa (OsCyt20) and a nuclear of 37kDa (OsNuc37), were identified as PCD related. Our results indicated that OsCyt20 is a Ca(2+)/Mg(2+)-dependent nuclease, which is most active at neutral pH, and that OsNuc37 is Zn(2+)-dependent, with a pH optimum of 4.5-6. Both nucleases were induced at the early stage of PCD (2h salt treatment) and exhibited the highest activity approximately 4h after exposure to NaCl, paralleling with the occurrence of DNA laddering. In vitro assays of endonuclease activities further revealed that OsNuc37, a glycoprotein localized in the nucleus, is the executor for DNA laddering. The different effects of both endonucleases on DNA degradation during salt-induced PCD are discussed.