Exogenous l-ascorbic acid regulates the antioxidant system to increase the regeneration of damaged mycelia and induce the development of fruiting bodies in Hypsizygus marmoreus

Hypsizygus marmoreus is an important commercial edible fungus, but the lack of basic studies on this fungus has hindered further development of its commercial value. In this study, we found that the treatment of damaged vegetative mycelia with 1 mM l-ascorbic acid (ASA) significantly increased the antioxidant enzyme activities (GPX, GR, CAT and SOD) and antioxidant contents (GSH and ASA) and reduced the ROS levels (H₂O₂ and O₂^-) in mechanically damaged mycelia. Additionally, this treatment increased mycelial biomass. At the reproductive stage, our results demonstrated that the treatment of damaged H. marmoreus mycelia with 2.24 mM ASA significantly increased the antioxidant enzyme activities (GPX, GR, GST, TRXR and CAT), endogenous ASA contents and GSH/GSSG ratios in different developmental stages and significantly decreased the MDA and H₂O₂ contents. Furthermore, this study showed that the expression levels of the antioxidant enzyme genes were consistent with the enzyme activities. Damaged mycelia treated with ASA regenerated 2–3 d earlier than the control group and showed significantly enhanced fruiting body production. These results suggested that exogenous ASA regulated mycelia intracellular ASA content to increase mycelial antioxidant abilities, induce the regeneration of damaged mycelia and regulate the development of fruiting bodies in H. marmoreus.     

Biological Activity of the Alternative Promoters of the Dictyostelium discoideum Adenylyl Cyclase A Gene

Amoebae of the Dictyostelium discoideum species form multicellular fruiting bodies upon starvation. Cyclic adenosine monophosphate (cAMP) is used as intercellular signalling molecule in cell-aggregation, cell differentiation and morphogenesis. This molecule is synthesized by three adenylyl cyclases, one of which, ACA, is required for cell aggregation. The gene coding for ACA (acaA) is transcribed from three different promoters that are active at different developmental stages. Promoter 1 is active during cell-aggregation, promoters 2 and 3 are active in prespore and prestalk tip cells at subsequent developmental stages. The biological relevance of acaA expression from each of the promoters has been studied in this article. The acaA gene was expressed in acaA-mutant cells, that do not aggregate, under control of each of the three acaA promoters. acaA expression under promoter 1 control induced cell aggregation although subsequent development was delayed, very small fruiting bodies were formed and cell differentiation genes were expressed at very low levels. Promoter 2-driven acaA expression induced the formation of small aggregates and small fruiting bodies were formed at the same time as in wild-type strains and differentiation genes were also expressed at lower levels. Expression of acaA from promoter 3 induced aggregates and fruiting bodies formation and their size and the expression of differentiation genes were more similar to that of wild-type cells. Expression of acaA from promoters 1 and 2 in AX4 cells also produced smaller structures. In conclusion, the expression of acaA under control of the aggregation-specific Promoter 1 is able to induce cell aggregation in acaA-mutant strains. Expression from promoters 2 and 3 also recovered aggregation and development although promoter 3 induced a more complete recovery of fruiting body formation.     

Complete Mitochondrial Genome of the Medicinal Mushroom Ganoderma lucidum

Ganoderma lucidum is one of the well-known medicinal basidiomycetes worldwide. The mitochondrion, referred to as the second genome, is an organelle found in most eukaryotic cells and participates in critical cellular functions. Elucidating the structure and function of this genome is important to understand completely the genetic contents of G. lucidum. In this study, we assembled the mitochondrial genome of G. lucidum and analyzed the differential expressions of its encoded genes across three developmental stages. The mitochondrial genome is a typical circular DNA molecule of 60,630 bp with a GC content of 26.67%. Genome annotation identified genes that encode 15 conserved proteins, 27 tRNAs, small and large rRNAs, four homing endonucleases, and two hypothetical proteins. Except for genes encoding trnW and two hypothetical proteins, all genes were located on the positive strand. For the repeat structure analysis, eight forward, two inverted, and three tandem repeats were detected. A pair of fragments with a total length around 5.5 kb was found in both the nuclear and mitochondrial genomes, which suggests the possible transfer of DNA sequences between two genomes. RNA-Seq data for samples derived from three stages, namely, mycelia, primordia, and fruiting bodies, were mapped to the mitochondrial genome and qualified. The protein-coding genes were expressed higher in mycelia or primordial stages compared with those in the fruiting bodies. The rRNA abundances were significantly higher in all three stages. Two regions were transcribed but did not contain any identified protein or tRNA genes. Furthermore, three RNA-editing sites were detected. Genome synteny analysis showed that significant genome rearrangements occurred in the mitochondrial genomes. This study provides valuable information on the gene contents of the mitochondrial genome and their differential expressions at various developmental stages of G. lucidum. The results contribute to the understanding of the functions and evolution of fungal mitochondrial DNA.     

Changeability in Retama raetam essential oils chemical composition,antioxidant and antimicrobial properties as affected by the physiological stage

Abstract

Retama raetam essential oils (EOs) composition and biological activities were assessed during three developmental periods. The essential oil yield varied significantly among the developmental stages and the optimal was detected at the fresh fruiting stage (0.34%). In addition, EOs composition varied significantly (p < 0.05) according to the different developmental stages. In fact, 2-Methoxy-4-vinylphenol, linalool, and 1-octen-3-ol were the main compounds in the vegetative, flowering, and the fresh fruiting stages, respectively. Developmental stage had also a strong effect on EOs antioxidant and antimicrobial activities. In fact, during the fresh fruiting stage, IC₅₀ and EC₅₀ values of the antioxidant assays were 2 to 3 times inferior to all others stages. Concerning the determination of the diameter of inhibition, a slight to high antimicrobial activity was revealed against 12 bacteria and 4 yeasts. Once again, EOs from the fresh fruiting stage had higher bactericidal effect than those from the flowering and vegetative ones (IZ varied from 10 to 13 mm). The results of this investigation showed for the first time the high accumulation of EOs at the early stages of fruit development making the fresh fruiting optimal stage for the extraction of powerful antioxidant and antimicrobial EOs.     

Hydrogen-rich water mediates redox regulation of the antioxidant system,mycelial regeneration and fruiting body development in Hypsizygus marmoreus

Hypsizygus marmoreus is an important industrialized mushroom, yet the lack of basic research on this fungus has hindered further development of its economic value. In this study, mycelia injured by scratching were treated with hydrogen-rich water (HRW) to investigate the involvement of the redox system in fruiting body development. Compared to the control group, damaged mycelia treated with HRW regenerated earlier and showed significantly enhanced fruiting body production. Antioxidant capacity increased significantly in damaged mycelia after HRW treatment, as indicated by higher antioxidant enzyme activities and antioxidant contents; the levels of reactive oxygen species (ROS) and malondialdehyde (MDA) were also reduced at the mycelial regeneration stage after treatment with HRW. Furthermore, genes involved in ROS, Ca²⁺, MAPK and oxylipin signaling pathways were up-regulated by HRW treatment. In addition, laccase and manganese peroxidase activities and mycelial biomass were higher after HRW treatment, suggesting that HRW might enhance the substrate-degradation rate to provide more carbon sources for fruiting body production.     

Biochemical constituents of a wild strain of Schizophyllum commune isolated from Achanakmar-Amarkantak Biosphere Reserve (ABR), India

A wild strain of Schizophyllum commune (MTCC 9670) isolated from Achanakmar-Amarkantak Biosphere Reserve of Central India was evaluated for the production of bioactive compounds. The chemical constituents of wild and in vitro grown cultures were compared. Under optimized conditions, different organic and aqueous extracts from mycelia and fruiting bodies were used to extract chemical components from the cultures grown in vitro. The gas chromatography combined wih mass spectrometry analysis of extracts identified two phenolic compounds, namely Phenyl benzoate (C₁₃H₁₀O₂) and 4-(phenyl methoxy) phenol (C₁₃H₁₂O₂) in the ethanolic extract of in vitro grown fruiting bodies and one antibacterial compound Pyrrolo (1, 2-a) piperazine-3, 6-dione (C₇H₁₀O₂N₂) in the methanolic extract of mycelia. High-performance liquid chromatography analysis revealed that the gallic acid and l-ascorbic acid were identifiable antioxidant components in the extracts possessing high free radical scavenging activity. The findings suggest that the wild strain of S. commune may serve as the source of novel bioactive compounds with effective antimicrobial and antioxidant activities.     

Tintinnadiol,a sphaeroane diterpene from fruiting bodies of Mycena tintinnabulum

Antifungal metabolites were isolated from fruiting bodies of Mycena tintinnabulum and from mycelia grown in either complex media or on oak wood. Strobilurins were produced in fruiting bodies and in mycelial cultures, whereas a new diterpene, named tintinnadiol, was isolated only from the fruiting bodies. The structure of the new compound was determined by spectroscopic methods. It exhibited cytotoxic effects.     

Cloning and characteristics of the antibacterial peptide gene abaecin in the bumblebee Bombus lantschouensis (Hymenoptera: Apidae)

Among the antimicrobial peptides, abaecin is rich in proline content and plays a vital role in insect innate immune defense. Here, the full-length gene of abaecin from the bumblebee Bombus lantschouensis was cloned, and its expression profiles for different tissues, developmental stages and reproductive statuses were analyzed by RT-qPCR. Meanwhile, the responses of abaecin to a bacterium (Escherichia coli) and a fungus (Beauveria bassiana) were tested. The full length of abaecin cDNA was 470 bp, and the open reading frame (ORF) was 258 bp, encoding a polypeptide of 85 amino acids. The abaecin gene consists of three exons and two introns. Phylogenetic analysis showed that Bombus ignitus was the closest species to B. lantschouensis base on putative Abaecin protein sequence. Expression analysis showed that abaecin was expressed broadly in different tissues, with the highest expression in fat bodies and extremely low expression in antennae. Regarding developmental stage, low expression of abacein was detected in eggs and larvae, and high expression was detected in pupal stages. The highest expression was observed at the Pw pupal stage (pupae with an unpigmented body cuticle and white eyes), and the expression then decreased from the Pp (pupae with pink eyes) to the Pdd (dark-eye pupae with a dark-pigmented cuticle) stages. In addition, the expression of abaecin was higher in egg-laying than in non-egg-laying female bumblebees. Both E. coli and B. bassiana infections induced the expression of abaecin. Our results indicated that the abaecin gene plays important roles in the development, reproduction and immune responses of bumblebees. During the artificial rearing of bumblebees, a good environment should be created to avoid infection with bacteria or fungi.     

Metallic and metalloid elements in various developmental stages of Amanita muscaria (L.) Lam

There is growing evidence that mushrooms (fruiting bodies) can be suitable for biogeochemical prospecting for minerals and as indicators of heavy metal and radioactive contaminants in the terrestrial environment. Apart from the nutritional aspect, knowledge of accumulation dynamics and distribution of elements in fruiting bodies, from emergence to senescence, is essential as is standardization when choosing mushroom species as potential bioindicators and for monitoring purposes. We studied the effect of fruitbody developmental stage on the contents of the elements (Li, K, V, Cr, Mn, Mg, Co, Ni, Cu, Zn, As, Rb, Sr, Ag, Al, Cd, Sb, Cs, Ba, Pb, Tl and U) in the individual parts of the Amanita muscaria fruiting body. Elements such as K, Mg, Mn, Ni, Co, Cu, Zn and Se remained similar throughout all developmental stages studied, however for K, differences occurred in the values of caps and stipes, as expressed by the cap to stipe concentration quotient (index Q_C/S). The other elements quantified, i.e., Li, V, Cr, As, Rb, Sr, Ag, Al, Cd, Sb, Cs, Ba, Pb, Tl and U are considered as nonessential or toxic (with the exception of V in A. muscaria). Their accumulation in the fruiting bodies and their distribution between cap and stipe did not show a uniform pattern. Pb, Sb, Tl, Ba, Sr, Li, Rb and Cs decreased with increasing maturity of the fruitbodies, implying that translocation, distribution and accumulation in stipes and caps was not a continuous process, while V, Cr, As, Ag, Cd, and U remained at the same concentration, similarly to the essential elements. Our results for A. muscaria confirm that elemental distribution in different parts of fruiting bodies is variable for each element and may change during maturation. Soil properties, species specificity and the pattern of fruitbody development may all contribute to the various types of elemental distribution and suggest that the results for one species in one location may have only limited potential for generalization.     

Isolation of genes expressed during the developmental stages of the oyster mushroom, Pleurotus ostreatus, using expressed sequence tags

In order to isolate and identify the developmentally regulated genes during fruiting body development, cDNA libraries were constructed from eight developmental stages of the Oyster mushroom, Pleurotus ostreatus. From these libraries, 11 761 expressed sequence tags (PoESTs) were generated. Of these, 4060 different genes (PoUnigenes) were identified, representing 34.5% of the entire genome. Redundancy analysis of ESTs during the developmental stages identified eight, 13 and two genes that were specifically expressed in mycelia, fruiting body and basidiospore, respectively. RT-PCR was used to confirm the specific expression of nine genes which showed specific redundancy in fruiting body stages, four genes of which were expressed specifically in fruiting body stages as expected in redundancy analysis, and other genes were expressed abundantly in fruiting body stages. The EST database of P. ostreatus generated during this study provides a genetic and biochemical basis for future studies of the developmental stages of basidiomycetes.     

Characterization of Serine Proteinase Expression in Agaricus bisporus and Coprinopsis cinerea by Using Green Fluorescent Protein and the A. bisporus SPR1 Promoter

The Agaricus bisporus serine proteinase 1 (SPR1) appears to be significant in both mycelial nutrition and senescence of the fruiting body. We report on the construction of an SPR promoter::green fluorescent protein (GFP) fusion cassette, pGreen_hph1_SPR_GFP, for the investigation of temporal and developmental expression of SPR1 in homobasidiomycetes and to determine how expression is linked to physiological and environmental stimuli. Monitoring of A. bisporus pGreen_hph1_SPR_GFP transformants on media rich in ammonia or containing different nitrogen sources demonstrated that SPR1 is produced in response to available nitrogen. In A. bisporus fruiting bodies, GFP activity was localized to the stipe of postharvest senescing sporophores. pGreen_hph1_SPR_GFP was also transformed into the model basidiomycete Coprinopsis cinerea. Endogenous C. cinerea proteinase activity was profiled during liquid culture and fruiting body development. Maximum activity was observed in the mature cap, while activity dropped during autolysis. Analysis of the C. cinerea genome revealed seven genes showing significant homology to the A. bisporus SPR1 and SPR2 genes. These genes contain the aspartic acid, histidine, and serine residues common to serine proteinases. Analysis of the promoter regions revealed at least one CreA and several AreA regulatory motifs in all sequences. Fruiting was induced in C. cinerea dikaryons, and fluorescence was determined in different developmental stages. GFP expression was observed throughout the life cycle, demonstrating that serine proteinase can be active in all stages of C. cinerea fruiting body development. Serine proteinase expression (GFP fluorescence) was most concentrated during development of young tissue, which may be indicative of high protein turnover during cell differentiation.     

信息素信号通路基因在斑玉蕈生长发育过程中的差异表达

为了更清楚地了解斑玉蕈菌丝成熟、原基形成和子实体发育的过程,本研究对不同菌丝培养时期的栽培瓶进行出菇实验,并对其不同培养时期和生长发育关键时期的信息素通路基因进行差异表达分析,以期揭示信息素信号通路基因参与调节斑玉蕈菌丝的生长、子实体形成和发育的作用。研究结果表明：斑玉蕈菌丝培养40-80d过程中,子实体产量呈上升的趋势,说明菌丝的成熟程度对产量会产生重要影响。对斑玉蕈基因组中的信息素信号通路基因进行分析鉴定共获得了8个关键基因。信息素通路基因差异表达分析表明：在菌丝培养40-80d过程中,大部分信息素信号通路基因在第60天时表达量最高,其中ste20、cdc24和ste12上调了4-20倍,而在第80天出现下降。从菌丝恢复到扭结形成原基和子实体发育的过程中,大多数基因在原基时期表达量最高,其中ste20、cdc24、ste11和ste12表达量上调最为显著,在子实体成熟期这些基因表达量下降。因此,这说明在菌丝营养生长过程中,在第60天菌丝细胞增殖生长最为旺盛,而在第80天菌丝细胞基本停止生长,菌丝也逐渐达到成熟。同时,在菌丝生殖生长过程中,斑玉蕈持续地上调信息素通路基因表达使菌丝细胞不断地分裂增殖,从而使新生的菌丝扭结形成原基,其中ste3、ste20、cdc24、ste11和ste12基因可能对斑玉蕈菌丝细胞的分裂增殖和诱导子实体形成起到关键的作用。