Responses of agaric fruit-bodies to light and gravity: growth straight downward in response to light from below

Fruit-bodies of Agaricales are known to show positive phototropism during the early stage of development, but negative gravitropism at the later stage after the onset of basidiospore formation. However, when exposed to light from below, the fruit-bodies ofTephrocybe tesquorum andCoprinus spp. grew downward through all stages of development, even after the onset of basidiospore formation. Primordium formation, fruit-body development and basidiospore formation were not disturbed under such conditions. In these downward-growing fruit-bodies, gills stood straight upward. InT. tesquorum, caps often became swollen and stipes sometimes became twisted anticlockwise, contrary to those in light from above, while such behaviours were not observed inCoprinus spp.     

Polyol metabolism in the mycelium and fruit-bodies during development ofFlammulina velutipes

Changes of polyol contents in the mycelium and fruit-bodies ofFlammulina velutipes were measured. The results suggested that arabinitol is accumulated in the fruit-bodies as the end-product after its translocation from the mycelium, while mannitol in the fruit-bodies is converted into fructose by the action of mannitol dehydrogenase (MDH). The development of fruit-bodies was promoted by feeding of mannitol to the mycelial colony. A¹⁴C tracer experiment indicated that half of mannitol translocated from mycelium to fruit-bodies was utilized for fruit-body development. NAD-linked MDH andd-arabinitol dehydroganase (D-ADH) were detected in both mycelium and fruit-bodies. The activities of MDH and ADH in the mycelium reached their maximum levels in the inital stage of fruit-body development and decreased thereafter. In contrast, the activity of MDH in the fruit-bodies showed a peak in the middle stage of development. The activity of ADH in the fruit-bodies was less than half of that of MDH. MDH showed a lower Km value for mannitol (1.3 ×10⁻³M) than for fructose (6.0×10⁻² M). The Km value of ADH for arabinitol was extremely high (1.3×10⁻¹M).     

Metabolic function of glycogen phosphorylase and trehalose phosphorylase in fruit-body formation ofFlammulina velutipes

Glycogen phosphorylase in the vegetative mycelium ofFlammulina velutipes converts glycogen to α-glucose 1-phosphate (G1P) in the colony during fruit-body development. Glycogen may contribute to the synthesis of trehalose as the starting material in the vegetative mycelium during the fruiting process of the colony, and the trehalose produced is translocated into the fruit-bodies as the main carbohydrate substrate for their development. Trehalose phosphorylase activity in the vegetative mycelium was at a relatively high level until fruit-body initiation, suggesting the turnover of this disaccharide during the vegetative stage of the colony development. Trehalose phosphorylase activity in the stipes showed a peak level at the early phase of fruit-body development, suggesting the continuing phosphorolysis of trehalose by this enzyme. The stipes also showed a high specific activity of phosphoglucomutase at a sufficient level to facilitate the conversion of G1P to α-glucose 6-phosphate (G6P). In the pilei a large amount of G1P remained until the growth of the fruit-bodies ceased. Trehalase activities in the stipes and pilei were at a very low level, and this enzyme may not contribute to the catabolism of trehalose in the fruit-body development.     

Characterization of proteins expressed abundantly in the fruit-body ofFlammulina velutipes

Chronological changes of protein expression in the vegetative mycelium ofFlammulina velutipes and expression of these proteins in the fruit-body were investigated by two-dimensional polyacrylamide gel electrophoresis. Four proteins (FBA 1-4) expressed abundantly in the fruit-body were found to have different expression patterns in the vegetative mycelium after the fruiting treatment. FBA 1-4 had similar amino acid sequences and displayed a high similarity with the deduced amino acid sequence of theC1 cDNA, which has an Arg-Gly-Asp (RGD) cell-attachment sequence. This suggests that FBA 1-4 may have cell-to-cell attachment activity.     

Promoting effect of wood vinegar compounds on fruit-body formation ofPleurotus ostreatus

The promoting effect of wood vinegar compounds on the fruiting ofPleurotus ostreatus (Japanese name, Hiratake) was investigated. Not only crude wood vinegar but its components, 3,5-dimethylphenol, 2-methoxyphenol, butanoic acid and 1-pentanol, had the ability to promote fruit-body formation on liquid medium. For use of these promoters industrially, a test for practical cultivation was carried out using a commercial sawdust medium. The addition of 100 µg/ml butanoic acid and 100 µg/ml 2-methoxyphenol into the sawdust medium after removal of the surface mycelial layer (kinkaki in Japanese) produced 29 and 23% higher yields of fruit-bodies than the control cultures (137.2 g/bottle), respectively. The addition of the crude wood vinegar as a medium component into sawdust substrates in the concentration range of 0.1–6% increased yields of fruit-bodies by 21–42% over the control.     

Responses of Hebeloma radicosum fruit-bodies to light and gravity: negatively gravitropic and nonphototropic growth

Responses of the long-rooting agaric Hebeloma radicosum fruit-bodies to light and gravity were studied. In light from below or obliquely below, fruit-bodies grew straight downward with gills tilted and cap swollen and waved if they had emerged downward from the culture medium, or bent upward from the beginning if they had emerged obliquely downward. In light from above or obliquely above, they grew upward if they had emerged upward. Thus, they did not grow toward unilateral light from obliquely below or obliquely above, and hence their growth was nonphototropic and negatively gravitropic from the beginning of development. Even the straight downward growth seems to be latently negative-gravitropic. In the dark, fruit-bodies grew upward, forming pseudorhizas, but they remained immature; they matured only in the light. These characteristics may be related to the growth habits of the fungus colonizing deep in the ground, forming primordia there, and developing mature fruit-bodies on the ground. Received: March 26, 2001 / Accepted: July 12, 2001     

Alterations in the composition of membrane glycero-and sphingolipids in the course of <Emphasis Type="Italic">Flammulina velutipes</Emphasis> surface culture development

Qualitative and quantitative characteristics of the alterations in the lipid composition of the membrane of the basidial fungus Flammulina velutipes in the course of surface culture development were investigated. Modifications of the lipid composition were shown to be timed to specific ontogeny stages, such as changes in the growth rate of the colonies, the appearance of differentiated vegetative cells, and the formation of generative structures. A slowdown of growth correlated with an alteration in the ratio of major classes of phospholipids, namely, with a decrease of phosphatidylcholine relative content and an increase in phosphatidylethanolamines. The differentiation of vegetative cells of the mycelium proceeded along with modifications of molecular composition of glycoceramides. In the course of the first week of growth, the surface culture of F. velutipes produced monohexosylceramides with epoxidized methyl sphingadienine as a sphingoid base. Later on, along with culture growth and specialization of mycelium cells, molecular species with methyl sphingadienine, common for basidiomycetes, start to prevail among the fungal glycoceramides. The formation of fruit bodies is accompanied by enrichment of molecules of phospholipids, mainly, the phosphatidylcholines, with unsaturated fatty acids.     

Influence of light on the morphological changes that take place during the development of the <Emphasis Type="Italic">Flammulina velutipes</Emphasis> fruit body

We show that fruit bodies of Flammulina velutipes can be induced in complete darkness after a sharp temperature reduction (23° to 16°C). However, the fruit bodies that form in complete darkness have a long stipe with an undeveloped pileus on the top (pinhead fruit bodies) and are thinner and whiter than the normal fruit bodies which are formed in the light. This finding suggests that F. velutipes fruit bodies cannot mature in complete darkness. However, when we irradiated the fruit bodies that had formed in complete darkness, a pileus developed immediately, and 4 days later the separation between the stipe and the pileus could be observed. Immediately after light exposure, the stipe also thickened and became increasingly pigmented. The stipe elongation was inhibited until 8 days after light exposure, although stipe elongation progressed very quickly thereafter. Basidospores were also visible in the gills 8 days after light exposure. We consider that the basidiospore development is involved in this rapid stipe elongation, which aids the effective dispersal of basidiospores.     

Comparative studies on the diversity of the edible mushroom Pleurotus eryngii: ITS sequence analysis, RAPD fingerprinting, and physiological characteristics

Verification of Pleurotus eryngii strains was assessed using ITS sequence analysis and RAPD fingerprinting. Sequence analysis of the ITS1–5.8S rDNA–ITS2 region of 24 strains of Pleurotus sp., which consisted of 22 strains of P. eryngii and the control strains P. ostreatus and P. ferulae, demonstrated that the DNA regions share mostly 99 % sequence identity, indicating that sequence-based analysis is not applicable for the verification of closely related mushroom strains. To verify the mushroom strains using RAPD, we amplified DNA fragments from the total cellular DNA of 24 mushroom strains with 18 different random primers, yielding 538 distinct DNA fragments ranging from 200–4000 bp. Analysis of the DNA fragment pattern showed that the 22 P. eryngii strains were clearly distinguished from the control strains P. ostreatus and P. ferulae, and could be categorized into five subgroups. Subsequent physiological studies on the development of fruiting bodies demonstrated the close correlation of the RAPD-based grouping with the phenotypical characteristics of mushroom fruiting bodies.     

Isolation and characterization of extra- and intra-cellular metal proteinases produced in the spawn-running process ofHypsizygus marmoreus

Isolation and characterization of extra-(PE-1) and intra-cellular (PE-2) metal proteinases produced during the spawn-running process ofHypsizygus marmoreus were carried out. These enzymes were the most active toward Hammarsten casein at pH 7.0 (PE-1) and pH 6.5–7.5 (PE-2). The molecular weight and pl value of PE-1 were 29,500, 8.8 and those of PE-2 were 21,500, 8.4. Km values against the synthetic peptide substrate Z-Gly-l-Leu-NH₂ were 0.9×10⁻³M (PE-1) and 1.2×10⁻³M (PE-2). PE-1 was strongly inhibited by phosphoramidon, whereas PE-2 was weakly inhibited. These enzymes are considered to play an important role in providing nitrogenous substrates during fruit-body formation.     

Application of the equilibrium concept to the development of agaric fruit-bodies, with special reference to their straight downward growth in light from below

Equilibrium, a concept of dynamics, is found to be applicable to the phototropic and gravitropic growth in agaric fruit-bodies. The fruit-bodies exposed to light from below grow straight downward without bending upward, and those exposed to light from obliquely below grow first downward and then upward by negative gravitropism. The fruit-bodies exposed to light from above grow upward. Fruit-bodies growing straight downward or upward do not change the direction of growth; they are in ‘equilibria’. The straight downward growth can be regarded as an ‘unstable equilibrium’ having a higher potential, and the straight upward growth as a ‘stable equilibrium’ having a lower potential. The change in the direction of growth can be explained by the change in the potential; the upward bending in fruit-bodies that have grown obliquely downward can be regarded as a ‘transition’ from the unstable equilibrium to the stable one.     

Purification and characterization of extracellular laccase fromPleurotus ostreatus

Laccase (EC 1.10.3.2) from the culture filtrate of a strain of white rot basidiomycetePleurotus ostreatus was purified using DEAE-Toyopearl 650M and butyl-Toyopearl 650M column chromatographies and Superdex 75 HR 10/30 fast protein liquid chromatography. Molecular weight of the purified laccase was about 55,000, and the isoelectric point was 3.0. The optimum pH for enzyme activity was 6.5, and the optimum temperature was 50°C. This enzyme contained 7.4% sugar and two copper atoms per molecule. The substrate specificity was similar to those of other fungal laccases. Comparison of the N-terminal amino acid sequence of theP. ostreatus laccase with those fromPleurotus ostreatus Florida,Coriolus hirsutus, Phlebia radiata, basidiomycete PM1 (CECT 2971),Trametes villosa, Pycnoporus cinnabarinus, Ceriporiopsis subvermispora, andAgaricus bisporus showed 95, 65, 60, 55, 55, 55, 50, and 35% similarity, respectively, in the first 20 residues. No similarity in this region was detected with laccases fromNeurospora crassa, Aspergillus nidulans, andCryptococcus neoformans.     

Fermentation optimization and characterization of the laccase from Pleurotus ostreatus strain 10969

Laccase is a widespread group of multi-copper enzymes which can catalyze the oxidation of a variety of organic compounds, with concomitant reduction of molecular oxygen to water. It has a wide application in industrial processes, particularly in renewable bio-energy industry. In this study, Pleurotus ostreatus strain 10969 with high yield of laccase, previously isolated from edible fungus growing on Juncao, was applied for optimization of fermentation media and growth parameters for the maximal enzyme production through response surface methodology and further characterization of the laccase activity. The results show that glucose and Mg²⁺ are the key ingredients for laccase production with the optimum concentration of 0.0988 g/mL and 7.3 mmol/L respectively. Compared to the initial medium, the highest laccase yield observed is approximately increased by 2.5 times under the optimized conditions. Extracellular laccase was then purified and its characters were analyzed. The molecular weight of the laccase is about 40 kDa, and the optimum pH and temperature for its activity is 4.0 and 50 °C with the corresponding Km and Vmax of 0.31 mmol/L and 303.25 mmol/min respectively. DTT, β-mercaptoethanol and NaN₃ nearly inhibit all activity of the laccase, as well as the metal ions especially Ag⁺. In summary, our results will facilitate the utilization of plant lignin in biomass energy industry.     

Partial characterization of a hydrophobin protein Po.HYD1 purified from the oyster mushroom <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis>

Hydrophobins fulfill various physiological functions in fungal development and growth, based on their mechanism of self-assembly at hydrophilic–hydrophobic interfaces into nano-scale, amphipathic membranes. One hydrophobin with an approximate molecular weight of 15 kDa, designated Po.HYD1, was purified from aerial hyphae of Pleurotus ostreatus strain Pm039. Ultrastructures of self-assembled films formed by Po.HYD1 on hydrophobic and hydrophilic substrates were revealed by atomic force microscopy (AFM). A monomolecular adsorption layer, thickness ranging from 3.2 to 3.8 nm, was observed on the surface of highly oriented pyrolytic graphite (HOPG), while a typical rodlet layer with uniform thickness of 4.2 ± 0.1 nm formed on the mica surface. Comparison of CD spectra showed significant secondary structural changes between monomeric and self-assembled states. The spectrum of monomeric Po.HYD1 had a maximum ellipticity at 190 nm and a minimum at 209 nm, and that of assemblage showed the maximum at 195 nm and the minimum shifted to 215–218 nm. Po.HYD1 showed high surface activity, based on the dramatic drop of surface tension through self-assembly at the water–air interface. Moreover, Po.HYD1 is capable of stabilizing the emulsion consisting of water and hexane.     

A heteropolysaccharide from aqueous extract of an edible mushroom, Pleurotus ostreatus cultivar: structural and biological studies

A water soluble polysaccharide isolated from the hot aqueous extract of Pleurotusostreatus cultivar was found to contain d-glucose and d-galactose in a molar ratio of nearly 7:1. Structural investigation was carried out using acid hydrolysis, methylation analysis, periodate oxidation, Smith degradation, and NMR studies (¹H, ¹³C, DEPT-135, TOCSY, DQF-COSY, NOESY, ROESY, HMQC, and HMBC). On the basis of the above mentioned experiments the structure of the repeating unit of the polysaccharide was established as:This heteroglycan stimulates macrophages, splenocytes, and thymocytes.     

Intracellular substrates of a heme-containing ascorbate oxidase in <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis>

A novel heme-containing ascorbate oxidase isolated from oyster mushroom, Pleurotus ostreatus, catalyzes oxidation of ascorbic acid (Kim et al., 1996). In this report, we describe the identification of intracellular substrates of the enzyme in the mushroom. Six compounds, which can serve as substrate of the heme-containing ascorbate oxidase, were identified as L-ascorbic acid, D-erythroascorbic acid, 5-O-(α-D-glucopyranosyl)-D-erythroascorbic acid, 5-O-(α-D-xylopyranosyl)-D-erythroascorbic acid, 5-methyl-5-O-(α-D-gluco-pyranosyl)-D-erythroascorbic acid, and 5-methyl-5-O-(α-D-xylopyranosyl)-D-erythroascorbic acid. All of the compounds were oxidized at a significant rate by the heme-containing ascorbate oxidase. Oxidation of the compounds produced equimolar amounts of hydrogen peroxide per mole of substrate.     

Waste-reducing cultivation of Pleurotus ostreatus and Pleurotus pulmonarius on coffee pulp: changes in the production of some lignocellulolytic enzymes

Fruiting body production for one strain of Pleurotus ostreatus and three strains of P. pulmonarius was evaluated on coffee pulp pasteurized at 80 °C for 1 h. Based upon three harvests per strain, the single P. ostreatus line was found to display a 40-day culture cycle, whereas the three P. pulmonarius strains completed their cycles after more than 50 days of incubation. These time periods were notably shorter than those observed in previous studies using other growth substrates. Nevertheless, yields expressed as biological efficiencies were not significantly different among strains, fluctuating between 125 and 138%. Extracellular enzymatic activity was also monitored for P. ostreatus and P. pulmonarius (one strain only). To do this, samples of mycelium-bearing substrate were taken every 4 days throughout the incubation period. Care was taken to represent all developmental stages, including primordial and fruiting bodies. Samples were either lyophilized and then analysed or, in some cases, analysed immediately without lyophilization. Hydrolase activity (i.e. endoglucanase (CMC) and cellobiohydrolase (CBH)) was found to depend on developmental stage, showing peak production during fruiting body formation. On the other hand, oxidase activity-(i.e. laccase (LAC) and Mn-peroxidase (MnP)) was associated with phenol degradation. Nevertheless, in the case of oxidases developmental timing differences were also observed. Specifically, LAC activity was detected as early as 8 days after inoculation in non-lyophilized samples, whereas MnP appeared near the end of the incubation period. No LAC activity was observed in lyophilized samples. This study concludes that coffee pulp might be successfully employed in the cultivation of mushrooms, not only because important extracellular enzymes are produced by mushrooms when grown upon this substrate, but also because the abbreviated cultivation cycle associated with this medium favours commercial processes. Commercialization might be further improved if strains specifically adapted to this novel substrate are selected.     

Purification and some properties of an isoform of metal proteinases fromHypsizygus marmoreus grown on sawdust culture

Purification and some properties of an isoform of metal proteinase fromHypsizygus marmoreus are described. This enzyme was purified 711-fold with 5.44% recovery. The molecular weight and pl value were 41,500 and pH 7.7, respectively. The highest activity was observed against milk casein as the substrate. This enzyme was strongly inhibited by metal proteinase inhibitors such as phosphoramidon, EDTA, ando-phenanthroline.     

Desensitization by red and blue light of phototropism in maize coleoptiles

The effects of pretreatments with red and blue light (RL, BL) on the fluence-response curve for the phototropism induced by a BL pulse (first positive curvature) were investigated with darkadapted maize (Zea mays L.) coleoptiles. A pulse of RL, giving a fluence sufficient to saturate phytochrome-mediated responses in this material, shifted the bell-shaped phototropic fluence-response curve to higher fluences and increased its peak height. A pulse of high-fluence BL given immediately prior to this RL treatment temporarily suppressed the phototropic fluence-response curve, and shifted the curve to higher fluences than induced by RL alone. The shift by BL progressed rapidly compared to that by RL. The results indicate (1) that first positive curvature is desensitized by both phytochrome and a BL system, (2) that desensitization by BL occurs with respect to both the maximal response and the quantum efficiency, and (3) that the desensitization responses mediated by phytochrome and the BL system can be induced simultaneously but develop following different kinetics. It is suggested that theses desensitization responses contribute to the induction of second positive curvature, a response induced by prolonged irradiation.Abbreviations BL blue light - RL red light CIW-DPB Publication No. 1001     

Purification and characterization of an aflatoxin degradation enzyme from Pleurotus ostreatus

Nineteen fungi were tested for their ability to degrade aflatoxin B1 (AFB1). An extracellular enzyme from the edible mushroom Pleurotus ostreatus showed afaltoxin-degradation activity detected by thin-layer chromatography (TLC). An enzyme with this activity was purified by two chromatographies on DEAE-Sepharose and Phenyl-Sepharose. The apparent molecular mass of the purified enzyme was estimated to be 90 kDa by SDS-PAGE. Optimum activities were found in the pH range between 4.0 and 5.0 and at 25 degrees C. Also, degradation activity of several dyes in the presence of H2O2 was tested, resulting in the detection of bromophenol blue-decolorizing activity. Based on these data, we suggest this enzyme is a novel enzyme with aflatoxin-degradation activity. Fluorescence measurements suggest that the enzyme cleaves the lactone ring of aflatoxin.