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Exciton-polaritons are half-light, half-matter bosonic quasiparticles formed by strong exciton-photon coupling in semiconductor microcavities. These hybrid particles possess the strong nonlinear interactions of excitons and keep most of the characteristics of the underlying photons. As bosons, above a threshold density they can undergo Bose-Einstein condensation to a polariton condensate phase and exhibit a rich variety of exotic macroscopic quantum phenomena in solids. Recently, organic semiconductors have been considered as a promising material platform for these studies due to their room-temperature stability, good processability, and abundant photophysics and photochemistry. Herein, recent advances of exciton-polaritons and their Bose-Einstein condensates in organic semiconductor microcavities are summarized. First, the basic physics is introduced, and then their emerging applications are highlighted. The remaining questions are also discussed and a personal viewpoint about the potential directions for future research is given.

We present herein an innovative host–guest method to achieve induced molecular chirality from an achiral stilbazolium dye (DSM). The host–guest system is exquisitely designed by encapsulating the dye molecule in the molecule-sized chiral channel of homochiral lanthanide metal–organic frameworks (P-(+)/M-(−)-TbBTC), in which the P- or M-configuration of the dye is unidirectionally generated via a spatial confinement effect of the MOF and solidified by the dangling water molecules in the channel. Induced chirality of DSM is characterized by solid-state circularly polarized luminescence (CPL) and micro-area polarized emission of DSM@TbTBC, both excited with 514 nm light. A luminescence dissymmetry factor of 10−3 is obtained and the photoluminescence quantum yield (PLQY) of the encapsulated DSM in DSM@TbTBC is ∼10%, which is close to the PLQY value of DSM in dilute dichloromethane. Color-tuning from green to red is achieved, owing to efficient energy transfer (up to 56%) from Ln3+ to the dye. Therefore, this study for the first time exhibits an elegant host–guest system that shows induced strong CPL emission and enables efficient energy transfer from the host chiral Ln-MOF to the achiral guest DSM with the emission color tuned from green to red., Homochiral Ln-MOFs are synthesized to encapsulate achiral dyes to induce strong circularly polarized luminescence with a luminescence dissymmetry factor of 10−3.

Porous organic frameworks have emerged as the promising platforms to construct tunable microlasers. Most of these microlasers are achieved from metal-organic frameworks via meticulously accommodating the laser dyes with the sacrifice of the pore space, yet they often suffer from the obstacles of either relatively limited gain concentration or sophisticated fabrication techniques. Herein, we reported on the first hydrogen-bonded organic framework (HOF) microlasers with color-tunable performance based on conformation-dependent stimulated emissions. Two types of HOF microcrystals with the same gain lumnogen as the building block were synthesized via a temperature-controlled self-assembly method. The distinct frameworks offer different conformations of the gain building block, which lead to great impacts on their conjugation degrees and excited-state processes, resulting in remarkably distinct emission colors (blue and green). Accordingly, blue/green-color lasing actions were achieved in these two types of HOFs based on well-faceted assembled wire-like cavities. These results offer a deep insight on the exploitation of HOF-based miniaturized lasers with desired nanophotonics performances.

Nitrogen limitation has been widely reported to affect the growth and development of fungi, and the transcription factor GCN4 (general control nonderepressible 4) is involved in nitrogen restriction. Here, we found that nitrogen limitation highly induced the expression of GCN4 and promoted the synthesis of ganoderic acid (GA), an important secondary metabolite in Ganoderma lucidum. The activated GCN4 is involved in regulating GA biosynthesis. In addition, the accumulation of reactive oxygen species (ROS) also affects the synthesis of GA under nitrogen restrictions. The silencing of the gcn4 gene led to further accumulation of ROS and increased the content of GA. Further studies found that GCN4 activated the transcription of antioxidant enzyme biosynthesis genes gr, gst2, and cat3 (encoding glutathione reductase, glutathione S-transferase, and catalase, respectively) through direct binding to the promoter of these genes to reduce the ROS accumulation. In conclusion, our study found that GCN4 directly interacts with the ROS signaling pathway to negatively regulate GA biosynthesis under nitrogen-limiting conditions. This provides an essential insight into the understanding of GCN4 transcriptional regulation of the ROS signaling pathway and enriches the knowledge of nitrogen regulation mechanisms in fungal secondary metabolism of G. lucidum. IMPORTANCE Nitrogen has been widely reported to regulate secondary metabolism in fungi. Our study assessed the specific nitrogen regulatory mechanisms in Ganoderma lucidum. We found that GCN4 directly interacts with the ROS signaling pathway to negatively regulate GA biosynthesis under nitrogen-limiting conditions. Our research highlights a novel insight that GCN4, the nitrogen utilization regulator, participates in secondary metabolism through ROS signal regulation. In addition, this also provides a theoretical foundation for exploring the regulation of other physiological processes by GCN4 through ROS in fungi.

Cellulose is a by-product of agricultural production and an abundant waste. As a carbon source, cellulose can be degraded and utilized by fungi. Carbon sources, which act as nutrients, not only provide energy but also serve as regulators of gene expression, metabolism and growth, through various signalling networks that enable cells to sense and adapt to varying environmental conditions. Nutrient-sensing pathways prioritize the use of preferred carbon sources and regulate the production of cellulose-degrading enzymes when necessary. Understanding the regulation of the fungal cellulolytic response will become increasingly important because we strive to increase the efficiency of the utilization of these renewable energy sources. Here, we show that Glsnf1, a sucrose-nonfermenting serine-threonine-protein kinase 1 (Snf1)/AMP-activated protein kinase homologue in medicinal macro basidiomycete Ganoderma lucidum, actively responds to carbon alterations and positively regulates cellulase activity and cellulase-related gene transcription. The carbon catabolite repressor CreA, a zinc binuclear cluster transcription factor that mediates the sensing of nutrients and suppression of the transcription of a number of genes necessary for the consumption of a less preferred carbon source, participates in the Glsnf1-mediated regulation of cellulases. Glsnf1 not only negatively regulates the transcription level of the CreA gene but also hinders its localization in the nucleus. Overall, our findings reveal a key nutrient-sensing mechanism that is critical for the modulation of carbon source adaptation in G. 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 β-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 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 β-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 β-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.

Phellinus igniarius is a very important, high-value medicinal fungus; flavonoids are one of its most important components. Methods to improve the content of flavonoids in Ph. igniarius deserve attention. Moreover, because of the low production of Ph. igniarius, it is also important to increase biomass. This study demonstrated that exogenous salicylic acid (SA) treatment can significantly promote the accumulation of total flavonoids and biomass in Ph. igniarius. After treatment with different concentrations of SA, the flavonoid content and biomass of Ph. igniarius increased by 20.54-93.15% and 31.90-43.92%, respectively, compared with the control, with flavonoid content and biomass reaching maximum values of 300 μM and 200 μM SA, respectively. Additionally, we found that SA induced the biosynthesis of flavonoids not only in a concentration-dependent manner but also in a time-dependent manner. The total flavonoid content reached a maximum at 24 h after SA treatment, which was 118.29% higher than the control. Furthermore, the biomass reached maximum values at 24 h and 36 h, which were 26.11% and 23.76% higher than the control, respectively. The total flavonoid content could be increased up to 15.02 μg rutin equivalents per mg mushroom biomass with the optimum conditions determined by response surface methodology. In this treatment, the biomass was also increased by 26.48%. This approach provides an efficient strategy for improving flavonoid accumulation in Ph. igniarius, with potential value in other applications.

Highly sensitive photodetectors play significant roles in modern optoelectronic integrated circuits. Constructing p-n junctions has been proven to be a particularly powerful approach to realizing sensitive photodetection due to their efficient carrier separation. Recently, p-n-junction photodetectors based on organic-inorganic hybrid perovskites, which combine favorable optoelectronic performance with facile processability, hold great potential in practical applications. So far, these devices have generally been made of polycrystalline films, which exhibit poor carrier-transport efficiency, impeding the further improvement of their photoresponsivities. Here, a type of ultrasensitive photodetector based on single-crystalline perovskite p-n-junction nanowire arrays is demonstrated. The single-crystalline perovskite p-n-junction nanowire arrays not only possess high crystallinity that enables efficient carrier transport but also form a built-in electric field facilitating effective carrier separation. As a result, the devices show excellent photosensitivity over a wide spectral range from 405 to 635 nm with an outstanding responsivity of 2.65 × 10

Heat stress (HS) is inescapable environmental stress that can induce the production of ganoderic acids (GAs) in Ganoderma lucidum. Our previous studies found that putrescine (Put) played an inhibitory role in GAs biosynthesis, which appeared to be inconsistent with the upregulated transcription of the Put biosynthetic gene GlOdc under HS. To uncover the mechanism underlying this phenomenon, two spermidine (Spd) biosynthetic genes, GlSpds1 and GlSpds2, were identified and upregulated under HS. Put and Spd increased by 94% and 160% under HS, respectively, suggesting that HS induces polyamine biosynthesis and promotes the conversion of Put to Spd. By using GlSpds knockdown mutants, it is confirmed that Spd played a stimulatory role in GAs biosynthesis. In GlOdc-kd mutants, Put decreased by 62-67%, Spd decreased by approximately 34%, and GAs increased by 15-22% but sharply increased by 75-89% after supplementation with Spd. In GlSpds-kd mutants, Put increased by 31-41%, Spd decreased by approximately 63%, and GAs decreased by 24-32% and were restored to slightly higher levels than a wild type after supplementation with Spd. This result suggested that Spd, rather than Put, is a crucial factor that leads to the accumulation of GAs under HS. Spd plays a more predominant and stimulative role than Put under HS, possibly because the absolute content of Spd is 10 times greater than that of Put. GABA and H2O2, two major catabolites of Spd, had little effect on GAs biosynthesis. This study provides new insight into the mechanism by which environmental stimuli regulate secondary metabolism via polyamines in fungi. KEY POINTS: • HS induces polyamine biosynthesis and promotes the conversion of Put to Spd in G. lucidum. • Put and Spd played the inhibitory and stimulatory roles in regulating GAs biosynthesis, respectively. • The stimulatory role of Spd was more predominant than the inhibitory role of Put in GAs biosynthesis.

The activity of mitochondrial pyruvate carrier (MPC) can be modulated to regulate intracellular metabolism under different culture conditions. In Ganoderma lucidum, the role of MPC in regulating carbon sources remains unknown. By knocking down MPC genes (MPC1 and MPC2), this research found that the loss of MPC increased the growth rate of G. lucidum by ~30% in a medium with wood chips as a carbon source. Then cellulase and laccase activities were tested. Endoglucanase and laccase activity increased by ~50% and ~35%, respectively, in MPC knockdown mutants compared with that in the wild type strain. Finally, the expression levels of genes related to glycolysis were assayed, and the transcription levels of these enzymes were found to be increased by ~250% compared with the wild type strain. In conclusion, the regulation of intracellular metabolism by MPC provides a new way to improve the use of nondominant carbon sources such as lignocellulose.

Ganoderic acid (GA) is an important bioactive metabolite of G. lucidum, whose biosynthesis pathway has been elucidated. We reviewed the studies on the biosynthetic pathway of GA and its regulation. The following are described: (1) Biosynthetic pathway of GA; (2) Regulation of GA biosynthesis by environmental factors; (3) Role of signaling in GA biosynthesis regulation; (4) Crosstalk of different signals for GA biosynthesis; and (5) Key genes involved in the signaling pathway regulation of GA biosynthesis. In particular, the mechanisms of different environmental factors that regulate GA biosynthesis are described in detail, including nutritional conditions, physical means, and single chemical or biological signals. More and more evidence indicates that various signaling molecules (such as ROS, Ca2+, NO, H2S, cAMP, and so on) are involved in GA biosynthesis regulation in response to environmental factors. Some key signaling molecules lead to crosstalk of different pathways. Also, under different environmental stress, these signaling molecules may interact differently, demonstrating the signaling regulatory networks’ complexity. Lastly, the future direction are described in this area.

In the postgenomic era, proteomics is an important part of systems biology research. Two-dimensional electrophoresis, iTRAQ, and mass spectrometry were used to analyze thousands of proteins in cells and tissues comprehensively. Based on the examples of the application, this chapter reviews the researches on the complex system of G. lucidum using proteomics technology. The main techniques and applications of proteomics in the G. lucidum complex system research were reviewed and discussed here. The conventional proteomics analysis process usually includes sample preparation, chromatographic separation, mass spectrometry detection, and data analysis. However, with the increasing demands for large-scale proteomics analysis, high-coverage, short-time, and fast ‘high-throughput’ proteomics analysis is becoming one of the critical technical challenges that need to be resolved in G. lucidum. In this part, the research strategy and future research direction of proteomics of G. lucidum are proposed and discussed, which will provide some ideas for researchers engaged in proteomics of G. lucidum.

MOF-based one-dimensional materials have received increasing attention in the nanophotonics field, but it is still difficult in the flexible shape evolution of MOF micro/nanocrystals for desired optical functionalities due to the susceptible solvothermal growth process. Herein, we report on the well-controlled shape evolution of pure-MOF microcrystals with optical waveguide and lasing performances based on a bottom-up and top-down synergistic method. The MOF microcrystals from solvothermal synthesis (bottom-up) enable the evolution from microrods via microtubes to nanowires through a chelating agent-assisted etching process (top-down). The three types of MOF 1D-microstructures with high crystallinity and smooth surfaces all exhibit efficient optical waveguide performance. Furthermore, MOF nanowire with lowest propagation loss served as low-threshold pure-MOF nanolasers with Fabry-Perot resonance. These results advance the fundamental understanding on the controlled MOF evolution mechanism, and offer a valuable route for the development of pure-MOF-based photonic components with desired functionalities.

The heme oxygenase gene has antioxidant and cytoprotective effects in organisms, but no related research has been conducted in Ganoderma lucidum. For the first time, we cloned the HMX1 gene in G. lucidum. The CDS is 1092 bp in length and encodes 363 amino acids. The HMX1 protein was prokaryotically expressed and purified, and the enzyme activity of the purified protein was measured. The value of Km was 0.699 μM, and Vm was 81.9 nmol BV h-1 nmol-1 protein. By constructing the silencing vector pAN7-dual-HMX1i, the transformants HMX1i1 and HMX1i2 were obtained. Compared with the wild-type (WT), the average growth rate of HMX1i1 and HMX1i2 decreased by 31% and 23%, respectively, and the mycelium biomass decreased by 53% and 48%, respectively. Compared with the WT, the extracellular polysaccharide content of HMX1i1 and HMX1i2 increased by 59% and 51%, and the intracellular polysaccharide content increased by 24% and 22%, respectively. These results indicate that the HMX1 gene affects mycelial growth and polysaccharide synthesis in G. lucidum.

Glutamine synthetase (GS), a central nitrogen metabolic enzyme, plays important roles in the nitrogen regulation network and secondary metabolism in fungi. However, the mechanisms by which external nitrogen sources regulate fungal GS activity have not been determined. Here, we found that GS activity was inhibited under nitrate conditions in Ganoderma lucidum. By constructing gs-silenced strains and adding 1 mM GS inhibitor to inhibit GS activity, we found that a decrease in GS activity led to a decrease in ganoderic acid biosynthesis. The transcription of gs increased approximately 5-fold under nitrate conditions compared with that under ammonia. Electrophoretic mobility shift and yeast one-hybrid assay assays showed that gs was transcriptionally regulated by AreA. Although both gs expression and GS protein content increased under nitrate conditions, the GS activity still decreased. Treatment of recombinant GS with SIN-1 (protein nitration donor) resulted in a strengthened nitration accompanied by a 71% decrease in recombinant GS activity. Furthermore, intracellular GS could be nitrated from mycelia cultivated under nitrate conditions. These results indicated that GS activity could be inhibited by NO-mediated protein nitration. Our findings provide the first insight into the role of transcriptional and posttranslational regulation of GS activity in regulating secondary metabolism in fungi. This article is protected by copyright. All rights reserved.

Ganoderma lucidum, which contains numerous biologically active compounds, is known worldwide as a medicinal basidiomycete. Because of its application for the prevention and treatment of various diseases, most of artificially cultivated G. lucidum is output to many countries as food, tea, and dietary supplements for further processing. Methyl jasmonate (MeJA) has been reported as a compound that can induce ganoderic acid (GA) biosynthesis, an important secondary metabolite of G. lucidum. Herein, MeJA was found to increase the intracellular level of nitric oxide (NO). In addition, upregulation of GA biosynthesis in the presence of MeJA was abolished when NO was depleted from the culture. This result demonstrated that MeJA-regulated GA biosynthesis might occur via NO signaling. To elucidate the underlying mechanism, we used gene-silenced strains of nitrate reductase (NR) and the inhibitor of NR to illustrate the role of NO in MeJA induction. The results indicated that the increase in GA biosynthesis induced by MeJA was activated by NR-generated NO. Furthermore, the findings indicated that the reduction of NO could induce GA levels in the control group, but NO could also activate GA biosynthesis upon MeJA treatment. Further results indicated that NR silencing reversed the increased enzymatic activity of NOX to generate ROS due to MeJA induction. Importantly, our results highlight the NR-generated NO functions in signaling crosstalk between reactive oxygen species and MeJA. These results provide a good opportunity to determine the potential pathway linking NO to the ROS signaling pathway in fungi treated with MeJA. KEY POINTS: • MeJA increased the intracellular level of nitric oxide (NO) in G. lucidum. • The increase in GA biosynthesis induced by MeJA is activated by NR-generated NO. • NO acts as a signaling molecule between reactive oxygen species (ROS) and MeJA.

Identifying new and economical means to utilize diverse lignocellulosic biomass is an urgent task. Ganoderma lucidum is a well-known edible and medicinal basidiomycete with an excellent ability to degrade a wide range of cellulosic biomass, and its nutrient use efficiency is closely related to the activity of extracellular cellulase. Intracellular nicotinamide adenine dinucleotide (NAD

Background: There is an urgent need to identify new and economical ways to utilize diverse types of lignocellulosic biomass. Ganoderma lucidum is well-known edible medicinal fungus that has an excellent ability to degrade a wide range of cellulosic biomass, and its nutrient utilization is closely related to the production of extracellular cellulase. Nicotinamide adenine dinucleotide (NAD+), a nutritional sensor molecule, can respond to nutritional states and regulate cellular metabolism, and nicotinamide mononucleotide adenyltransferase (nmnat) is the key enzyme that catalyses the biosynthesis of NAD+.Results: In this study, a homologue of the gene encoding nmnat was cloned from G. lucidum. The Agrobacterium tumefaciens-mediated transformation (ATMT) method was used to construct two G. lucidum overexpression strains, OE::nmnat4 and OE::nmnat19, in which the nmnat gene transcript levels and the NAD+ content were significantly increased. Glnmnat overexpression strains showed dramatically increased colony growth on different carbon sources, and the intracellular Ca2+ concentration was increased by 3.95- and 2.10-fold in the G. lucidum OE::nmnat4 and OE::nmnat19 strains, respectively, compared with that observed in the WT strain. The CMCase activity increased by approximately 2.8- and 3-fold, and that of pNPGase increased by approximately 1.9- and 2.1-fold in the G. lucidum OE::nmnat4 and OE::nmnat19 strains, respectively, compared with that observed in the WT strain. Furthermore, NAD+ was observed to potentially induce cellulase production by regulating the cytosolic Ca2+ concentration.Conclusions: Taken together, for the first time, our results revealed that NAD+ can stimulate cellulase production and increase the transcript levels of cellulase genes via increasing the intracellular Ca2+ concentration in G. lucidum. This research also provides a theoretical basis for conducting cellulase-related work in other basidiomycetes.

Background: There is an urgent need to search for new and economical ways to utilize diverse lignocellulose. Ganoderma lucidum is well-known edible medicinal fungus that has a strong ability to degrade a wide variety of cellulosic biomass and its nutrient utilization is closely related to extracellular cellulase. Nicotinamide adenine dinucleotide (NAD+), a nutritional sensor molecule, can respond to nutritional states and regulate cellular metabolism. Nicotinamide mononucleotide adenyltransferase (nmnat) is the key enzyme that catalyses the biosynthesis of NAD+.Result: In this study, a homologue of the gene encoding nmnat was cloned from G. lucidum. The Agrobacterium tumefaciens -mediated transformation (ATMT) method was used to construct the two overexpression strains OE:: nmnat4 and OE:: nmnat19 in G. lucidum. In the overexpression strains, the transcript levels of the nmnat gene and the NAD+ content were significantly increased. Gl nmnat overexpression strains showed dramatically stronger colony growth on different carbon sources, and the intracellular Ca 2+ concentration increased 3.95-fold and 2.10-fold in OE:: nmnat4 and OE:: nmnat19 , respectively, compared with WT strains. The CMCase activity increased by approximately 2.8-fold and 3-fold, and p NPGase activity increased by approximately 1.9-fold and 2.1-fold in OE:: nmnat4 and OE:: nmnat19 strains compared with the WT strains, respectively. Furthermore, it was found that NAD+ might induce cellulase production by regulating cytosolic Ca 2+ concentration.Conclusions: Taken together, our results revealed for the first time that NAD + could stimulate cellulase production and demonstrated that NAD + could increase the transcript levels of cellulase genes via the intracellular Ca 2+ concentration in G. lucidum. This research also provides a theoretical basis for conducting cellulase-related work on other basidiomycetes.

With the scale of grid-tied renewable energy generation systems increasing significantly, the grid stability and safety have to be ensured by new strict grid code. The low voltage ride through (LVRT) regulation which requires the renewable energy generation systems should connect and provide reactive power to support during grid fault and the islanding detection which ensures grid safety are two focuses in the grid code of many countries. This paper proposes a power perturbation method, which consists of dual second order generalized integrator (DSOGI) PLL, power perturbation module and islanding detection module. This method can meet the requirement of LVRT and islanding detection simultaneously by adjusting proportion of active power current and reactive power current generated by grid-tied renewable energy generation systems. Finally, validity and accuracy of the proposed method was verified by simulation results.

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., Highlights • Mitochondria as a source of salicylic acid (SA) induced reactive oxygen species (ROS) production in Ganoderma lucidum. • SA induces the accumulation of ganoderic acids in Ganoderma lucidum by mitochondria ROS overproduction. • SA inhibits mitochondrial complex III activity to increase ROS and thereby induces ganoderic acids biosynthesis.

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.

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.

The APSES transcription factors have been identified as key regulators of fungal development and other biological processes in fungi. In the present study, the function of Ganoderma lucidum GlSwi6, a homolog of Saccharomyces cerevisiae Swi6, was characterized. RNAi was used to examine the function of GlSwi6 in G. lucidum. Silencing GlSwi6 resulted in multiple developmental defects, including reduced fungal growth and increased hyphal branching, and the GlSwi6-silenced strains did not exhibit primordium or fruiting body formation. In addition, the H2O2 and ganoderic-acid (GA) levels of the GlSwi6-silenced strains decreased approximately 50% and 25%, respectively, compared with those of the WT strain. Furthermore, the addition of H2O2 led to the recovery of the GA levels of GlSwi6-silenced strains, implying that GlSwi6 might regulate GA biosynthesis by regulating the intracellular ROS levels. Taken together, these results indicate that GlSwi6 is involved in fungal growth, development and GA biosynthesis in G. 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.

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.

The mitogen-activated protein kinases (MAPKs) are crucial signaling instruments in eukaryotes that play key roles in regulating fungal growth, development, and secondary metabolism and in adapting to the environment. In this study, we characterized an Slt2-type MAPK in Ganoderma lucidum, GlSlt2, which was transcriptionally induced during the primordium and fruiting body stages. RNA interference was used to examine the function of GlSlt2. Knockdown of GlSlt2 caused defects in growth and increased hyphal branching as well as hypersensitivity to cell wall-disturbing substances. Consistently, the chitin and β-1,3-d-glucan contents and the expression of cell wall biosynthesis genes were decreased and down-regulated, respectively, in GlSlt2 knockdown strains compared with those in the wild type (WT). In addition, no primordium or fruiting body could be observed in GlSlt2 knockdown strains. Furthermore, the intracellular reactive oxygen species (ROS) content and ganoderic acid biosynthesis also decreased in GlSlt2 knockdown strains. Addition of H2O2 could recover the decreased ganoderic acid content in GlSlt2 knockdown strains, indicating that GlSlt2 might regulate ganoderic acid biosynthesis via the intracellular ROS level. Overall, GlSlt2 is involved in hyphal growth, fruiting body development, cell wall integrity, oxidative stress and ganoderic acid biosynthesis in G. lucidum.

This study aimed to establish an RNAi system based on a dual promoter construct to interfere with the expression of target genes in Flammulina velutipes . In this study, the endogenous laccase gene, which was cloned as a silencing reporter, was introduced into F. velutipes through electroporation-mediated transformation. Our data unequivocally indicate that the dual promoter silencing system significantly reduced the expression and activity of lac6 . Additionally, reductions in lac6 mRNA levels and enzyme activity were correlated (minimum of a 50% reduction). The molecular tools developed in this study should facilitate the functional characterization of genes in this important species.

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.

Flammulina velutipes is a well-known edible mushroom cultivated all over the world. However, because of the low transformation frequency, the expensive instruments required, and the complicated, time-consuming procedures necessary, there is insufficient genetic research on F. velutipes. In this study, we report a liposome-mediated transformation (LMT) system for the genetic transformation of F. velutipes. Using the LMT system, we obtained 82 ± 4 stable F. velutipes transformants per 105 protoplasts, which is a clear increase in transformation frequency compared to the other methods used. We were able to detect the expression of an EGFP reporter gene in the F. velutipes transformants using fluorescence imaging assays. Furthermore, we used this method to transfer the laccase gene into F. velutipes and found that the transcriptional level and enzymatic activity increased in these transformants. Mitotic stability analysis showed that all of the selected transformants remained mitotically stable, even after five successive rounds of sub-culturing. These results demonstrate a new transgenic approach that will facilitate F. velutipes research.

Background: It is unavoidable for learners undergoing health professions training to encounter different complex clinical scenarios related to diagnoses, treatment and ethical dilemmas. The lack of standard answers to such clinical challenges can cause uncertainty in the process of teaching, learning and assessment as learners grapple with the need to critically appraise the specific context, past practices and extant literature to arrive at a thoughtful decision. In this aspect, debate can be a useful pedagogical tool to consider multiple, different perspectives regarding these issues. As the use of debate within the health professions education has not been reviewed, we conducted a systematic review of the current literature on the adoption of debate as a pedagogical tool to clarify the specific context of use as well as its effectiveness in the learning of skills and content within the health professions education. Methods: A systematic review was conducted on relevant published literature in English within journal databases until September 2018 that employed debate as a pedagogical tool within health professions education. Results: Out of 626 screened articles, 12 studies were finally included based on inclusion/exclusion criteria. The 12 studies (9 undergraduate, 3 postgraduate) covered a diverse range of disciplines and debate in health professions education was adopted for acquisition of skills (such as critical thinking, communication skills, teamwork), or learning of specific topics (such as patient safety, ethical issues, teaching of new topics) as well as to examine evidence based practice. In the majority of studies (10 of 12 studies, 83.3%), debate has been deemed by the learners to be effective in facilitating the learning of new content and skills such as communication and critical thinking, which are related to processes aligned with adult learning, motivational, reflective and social learning theories. Conclusion: Overall, sparse studies to date with relatively low risk of bias found debate to be effective in enabling the learning of skills and content within health professions education. Future studies may want to incorporate more objective measures of the learning outcomes of learners following the adoption of debate as a teaching tool as well as to examine the combinatorial use of debate with other pedagogical tools and their longitudinal impact on learners and learning., Hintergrund: Für Lernende während der ärztlichen Ausbildung ist es unvermeidbar, verschiedenen komplexen klinischen Szenarien hinsichtlich Diagnose, Behandlung und ethischer Dilemmata zu begegnen. Der Mangel an Standardantworten auf solche klinischen Herausforderungen kann zu Unsicherheit im Prozess des Lehrens, Lernens und der Einschätzung führen, da die Lernenden sich mit dem Bedürfnis nach kritischer Bewertung eines spezifischen Kontextes, vergangenen Praktiken und vorhandener Literatur auseinandersetzen müssen, um zu einer durchdachten Entscheidung zu gelangen. In dieser Hinsicht kann die Debatte ein nützliches pädagogisches Werkzeug sein, um verschiedenartige, multiple Perspektiven bezüglich dieser Probleme zu berücksichtigen. Da der Nutzen von Debatten innerhalb der ärztlichen Ausbildung noch nicht überprüft worden ist, haben wir einen systematischen Überblick über die aktuelle Literatur zur Einbeziehung von Debatten als pädagogisches Werkzeug vorgenommen, um deren spezifischen Nutzungskontext sowie Effektivität beim Erlernen von Fähigkeiten und Inhalten innerhalb der ärztlichen Ausbildung zu klären. Methoden: Es wurde eine systematische Überprüfung der relevanten, bis September 2018 veröffentlichten englischsprachigen Literatur in Zeitschriftendatenbanken durchgeführt, die sich mit Debatten als pädagogischem Werkzeug innerhalb der Ausbildung von Gesundheitsberufen befassten. Ergebnisse: Von 626 angezeigten Artikeln wurden schließlich 12 Studien basierend auf Inklusions-/Exklusionskriterien einbezogen. Diese 12 Studien (9 universitäre, 3 postgraduierte) deckten ganz verschiedene Bereiche und Disziplinen ab, und die Debatte in der Ausbildung von Gesundheitsberufen wurde zum Erwerb von Fähigkeiten (wie z. B. kritisches Denken, kommunikative Fähigkeiten, Teamwork) oder zum Erlernen spezifischer Themen (wie z. B. Patientensicherheit, ethische Probleme, Unterrichten neuer Themenbereiche) sowie zur Überprüfung Evidenz-basierter Praxis genutzt. Bei der Mehrheit der Studien (10 von 12 Studien, 83,3%) wurden Debatten seitens der Lernenden als effektiv bei der Erleichterung des Erlernens neuer Inhalte und Fähigkeiten, wie z. B. bei der Kommunikation und dem kritischen Denken, eingeschätzt, die verbunden sind mit Prozessen, die am Lernen im Erwachsenenalter, an Motivations-, Reflexions- und sozialen Lerntheorien ausgerichtet sind. Schlussfolgerung: Insgesamt schätzten die bislang nur spärlich vorhandenen Studien mit einem relativ niedrigen Risiko der systematischen Verzerrung Debatten als effektiv bei der Ermöglichung des Erlernens von Fähigkeiten und Inhalten innerhalb der Ausbildung von Gesundheitsberufen ein. Zukünftige Studien sollten eventuell objektivere Maßstäbe bzgl. der Lernergebnisse nach Einbeziehung von Debatten als Lehrwerkzeug anlegen, sowie die kombinierte Nutzung von Debatten zusammen mit anderen pädagogischen Werkzeugen und deren langfristigen Einfluss auf Lernende und Lernen untersuchen., GMS Journal for Medical Education; 36(4):Doc37

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 β-1,3-glucan content in the PacC-silenced strains was only approximately 78-80% of that in the WT strain, and the changes in β-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.

The secondary metabolites of fungi are often produced at very low concentrations, and until recently the regulatory mechanisms of secondary metabolite biosynthesis have been unclear. Ganoderma lucidum is a macrofungus that is widely used as a traditional Chinese medicine or medicinal mushroom: ganoderic acid (GA) is one of the main active ingredients. Here, we review research from the last decade on which and how environmental factors regulate GA biosynthesis. These environmental factors are mainly three components: a single chemical/biological or biochemical signal, physical triggers, and nutritional conditions. Because G. lucidum is a non-model Basidiomycete, a combination of physiological and genetic research is needed to determine how those environmental factors regulate GA biosynthesis. The regulation of GA biosynthesis includes ROS, Ca2+, cAMP and phospholipid signaling, and cross-talk between different signaling pathways. The regulatory mechanisms for the synthesis of this secondary metabolite, from the perspective of physiology and genetics, in G. lucidum will provide ideas for studying the regulation of fungal secondary metabolism in other non-model species, especially those fungi with limitations in genetic manipulation.

Nitrogen metabolism repression (NMR) has been well studied in filamentous fungi, but the molecular mechanism of its effects on fungal secondary metabolism has been generally unexplored. Ganoderic acid (GA) biosynthesis in Ganoderma lucidum differs between ammonia and nitrate nitrogen sources. To explain the functions of NMR in secondary metabolism, AreA, which is a core transcription factor of NMR, was characterized in G. lucidum. The transcription level of AreA was dramatically increased (approximately 4.5-folds), with the nitrate as the sole nitrogen source, compared with that with ammonia as the source. In addition, the expression of related genes involved in NMR was changed (upregulated of MeaB and downregulated of Nmr and GlnA) when AreA was knockdown. Yeast one-hybrid and electrophoretic mobility shift assay results showed that AreA could directly bind to the promoter of fps (encoding farnesyl-diphosphate synthase) to activate its expression. However, GA biosynthesis was increased (27% in the ammonia source and 77% in the nitrate source) in AreAi mutant strains versus that in control strains. These results showed that another important factor must participate in regulating GA biosynthesis other than the direct activation of AreA. Furthermore, we found that the content of nitric oxide (NO) was increased approximately 2.7-folds in the nitrate source compared with that in the ammonia. By adding the NO donor (SNP) or scavenger (cPTIO) and using NR-silenced or NR-overexpressed strains, we found that there was a negative correlation between the NO contents and GA biosynthesis. NO generated by nitrate reductase (NR) during the nitrogen utilization burst and could negatively influence GA biosynthesis. As a global transcription factor, AreA could also regulate the expression of NR. Our studies provide novel insight into the dual functions of AreA in GA biosynthesis during nitrogen assimilation.

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.

A novel composite of polyhedral MnO ( p -MnO) nanocrystals and reduced graphene oxide (RGO) nanosheets as a high performance electrode material for lithium ion batteries (LIBs) is prepared by homogeneous precipitation, subsequent reduction with hydrazine under microwave irradiation and final annealing procedure. The characterizations of structure and morphology reveal that p -MnO nanocrystals are homogeneously anchored onto the RGO nanosheets. The synergistic effect of the p -MnO nanocrystals with high crystallinity and RGO nanosheets makes the as-prepared nanocomposite possesses high contact area between electrolyte and electrode, uniform delivery of electrons and ions, short Li + insertion/extraction pathways, superior protection against volume change of electroactive materials and excellent electrical conductivity of the overall electrode during electrochemical processes. As an anode material for LIBs, the p -MnO/RGO nanocomposite exhibits superior electrochemical performances including large reversible capacity, excellent cyclic performance and high rate capability. For example, the p -MnO/RGO electrode shows a reversible capacity of 988.6 mA h g −1 after 120 cycles at a current density of 100 mA g −1 and delivers 591.1 mA h g −1 at a rate of 1600 mA g −1 . Our results highlight the importance of anchoring nanocrystals with special morphology on RGO nanosheets for maximum utilization of electroactive materials to enhance energy storage in high-performance LIBs.

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.

Ceramide synthases (CERSs) catalyse an N-acyltransferase reaction using long-chain base (LCB) and fatty acyl-coenzyme A (CoA) as substrates to synthesize ceramide (Cer), which is the backbone of all complex sphingolipids. In the present study, three CERSs (LAG1, LAG2 and LAG3) form Ganoderma lucidum were analysed. The silencing of lag1 by RNA interference reduced ganoderic acid biosynthesis and Cer and complex sphingolipids contents, which contain long-chain-fatty-acids (LCFAs, including C16 and C18). In contrast, the silencing of lag2 or lag3 did not result in obvious phenotypic and sphingolipid homeostasis changes, although the lag2/lag3 double-silenced mutants exhibited increased ganoderic acid biosynthesis as well as reduced growth, reduced Cer and complex sphingolipids contents, which contain very-long-chain fatty acids (VLCFAs, including C22, C24 and C26). The results of the present study indicate that the three assayed CERSs have distinct physiological functions and substrate specificities in G. lucidum.

Isoforms of 14-3-3 proteins, similar to their highly conserved homologs in mammals and plants, are both transcriptionally and functionally affected by their extracellular and intracellular environments. These proteins bind to phosphorylated client proteins to modulate their functions in fungi. Since phosphorylation regulates a plethora of different physiological responses in organisms, 14-3-3 proteins play roles in multiple physiological functions, including those controlling metabolisms, cell division, and responses to environmental stimulation. These proteins could also modulate signaling pathways that transduce inputs from the environment and downstream proteins that elicit physiological responses. Increasing evidence supports a prominent role for 14-3-3 proteins in regulating development and metabolism at various levels. In this review, we first provide a brief summary of the molecular structure of 14-3-3 proteins. Second, we discuss the potential roles of 14-3-3 proteins in the regulation of development and metabolism. Third, we review the roles of 14-3-3 proteins in the regulation of their binding partners, including receptors, protein kinases, and some protein kinase substrates. Finally, this review examines recent advances that further elucidate the role of 14-3-3 proteins in signaling transduction in response to environmental stress.

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&ndash, MS and 70 metabolites by LC&ndash, 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.

Ganoderma lucidum is among the best known medicinal basidiomycetes due to its production of many pharmacologically active compounds. To study the regulatory networks involved in its growth and development, we analyzed the relationship between reactive oxygen species (ROS) and Ca 2+ signaling in the regulation of hyphal branching and ganoderic acid (GA) biosynthesis after Cu 2+ treatment. Our results revealed that Cu 2+ treatment decreased the distance between hyphal branches and increased the GA content and the intracellular levels of ROS and Ca 2+ . Further research revealed that the Cu 2+ -induced changes in hyphal branch distance, GA content, and cytosolic Ca 2+ level were dependent on increases in cytosolic ROS. Our results also showed that increased cytosolic Ca 2+ could reduce cytosolic ROS by activating antioxidases and modulating Cu 2+ accumulation, resulting in feedback to adjust hyphal growth and GA biosynthesis. These results indicated that cytosolic ROS and Ca 2+ levels exert important cross talk in the regulation of hyphal growth and GA biosynthesis induced by Cu 2+ . Taken together, our results provide a reference for analyzing the interactions among different signal transduction pathways with regard to the regulation of growth and development in other filamentous fungi. IMPORTANCE Ganoderma lucidum , which is known as an important medicinal basidiomycete, is gradually becoming a model organism for studying environmental regulation and metabolism. In this study, we analyzed the relationship between reactive oxygen species (ROS) and Ca 2+ signaling in the regulation of hyphal branching and ganoderic acid (GA) biosynthesis under Cu 2+ stress. The results revealed that the Cu 2+ -induced changes in the hyphal branch distance, GA content, and cytosolic Ca 2+ level were dependent on increases in cytosolic ROS. Furthermore, the results indicated that increased cytosolic Ca 2+ could reduce cytosolic ROS levels by activating antioxidases and modulating Cu 2+ accumulation. The results in this paper indicate that there was important cross talk between cytosolic ROS and Ca 2+ levels in the regulation of hyphal growth and GA biosynthesis induced by Cu 2+ .

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 Ca

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.

Phosphoglucomutase (pgm) is an important enzyme in carbohydrate metabolism that is located at the branching point between glycolysis and the Leloir pathway. pgm catalyzes the reversible conversion reaction between glucose-6-phosphate (Glc-6-P) and glucose-1-phosphate (Glc-1-P). The glpgm gene was cloned in Escherichia coli, and the recombinant pgm protein from Ganoderma lucidum was purified in this study. The activity of native pgm was also detected to demonstrate that this predicted gene was functional in G. lucidum. Interestingly, silencing the glpgm gene in the fungus reduced hyphal growth. Moreover, glpgm silencing was associated with declining extracellular polysaccharide (EPS) production (approximately 20-40% of that in the WT strain) and increasing intracellular polysaccharide (IPS) production (approximately 1.7-fold that in the WT strain). Additionally, in our research, cell wall components were also shown to differ according to the glpgmi strain. Compared with WT, chitin significantly increased by 1.5-fold; however, the content of β-1,3-glucan was observably reduced to 60-70% that of the WT. Further research showed that the cell wall component changes were associated with the transcription of related genes. These findings provide references for further study on the potential physiological function of pgm in G. lucidum.

The Hypsizygus marmoreus laccase gene (lcc1) sequence was cloned and analyzed. The genomic DNA of lcc1 is 2336 bp, comprising 13 introns and 14 exons. The 1626-bp full-length cDNA encodes a mature laccase protein containing 542 amino acids, with a 21-amino acid signal peptide. Phylogenetic analysis showed that the lcc1 amino acid sequence is homologous to basidiomycete laccases and shares the highest similarity with Flammulina velutipes laccase. A 2021-bp promoter sequence containing a TATA box, CAAT box, and several putative cis-acting elements was also identified. To study the function of lcc1, we first overexpressed lcc1 in H. marmoreus and found that the transgenic fungus producing recombinant laccase displayed faster mycelial growth than the wild-type (wt) strain. Additionally, primordium initiation was induced 3-5 days earlier in the transgenic fungus, and fruiting body maturation was also promoted approximately five days earlier than in the wt strain. Furthermore, we detected that lcc1 was sustainably overexpressed and that laccase activity was also higher in the transgenic strains compared with the wt strain during development in H. marmoreus. These results indicate that the H. marmoreus lcc1 gene is involved in mycelial growth and fruiting body initiation by increasing laccase activity.

UDP-glucose pyrophosphorylase (UGP) is a key enzyme involved in carbohydrate metabolism, but there are few studies on the functions of this enzyme in fungi. The ugp gene of Ganoderma lucidum was cloned, and enzyme kinetic parameters of the UGP recombinant protein were determined in vitro, revealing that this protein was functional and catalyzed the reversible conversion between Glc-1-P and UDP-Glc. ugp silencing by RNA interference resulted in changes in the levels of the intermediate metabolites Glc-1-P and UDP-Glc. The compounds and structure of the cell wall in the silenced strains were also altered compared with those in the wild-type strains. Moreover, the number of hyphal branches was also changed in the silenced strains. To verify the role of UGP in hyphal branching, a ugp-overexpressing strain was constructed. The results showed that the number of hyphal branches was influenced by UGP. The mechanism underlying hyphal branching was further investigated by adding exogenous Glc-1-P. Our results showed that hyphal branching was regulated by a change in the cytosolic Ca(2+) concentration, which was affected by the level of the intermediate metabolite Glc-1-P, in G. lucidum. Our findings indicate the existence of an interaction between carbon metabolism and Ca(2+) signaling in this fungus.