Carboxylierungsreaktionen in Agaricus bisporus

Zusammenfassung In Gegenwart von ATP, Mn^2ü (oder Mg^2ü), CoA und Glutathion fixieren zellfreie Extrakte aus Sporophoren von Agaricus bisporus selbst in Abwesenheit exogener CO₂-Acceptoren in erheblichem Maße Kohlendioxyd. Entsprechend der Hemmbarkeit dieser CO₂-Fixierungsreaktionen durch Avidin und p-Chloromercuribenzoat handelt es sich bei diesen zum mindesten teilweise um durch Biotin-Enzyme katalysierte Vorgänge. Einer der endogenen CO₂-Acceptoren ist sehr washrscheinlich mit aus Isovaleriansäure gebildetem -Methylcrotonyl-CoA identisch.Die experimentellen Ergebnisse werden im Zusammenhang mit den bekannten physiologischen Effekten von CO₂ auf Fruktifikation und Morphogenese des Kulturchampignons diskutiert. Es scheint, daß Biotin-abhängige Carboxylierungsreaktionen eine der biochemischen Grundlagen für den Kontrolleinfluß von Kohlendioxyd im Lebenscyclus von A. bisporus darstellen.

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Carboxylation reactions in Agaricus bisporus I. The endogenous CO₂-acceptor

Summary Cell-free extracts of sporophores of Agaricus bisporus incorporate considerable amounts of carbon dioxide into organic acids even without added substrate, provided that ATP, Mn^2ü or Mg^2ü, Coenzyme A and glutathione are present. These CO₂-fixation reactions are inhibited by avidin and p-chloromercuribenzoate. It is therefore concluded that at least part of these reactions are katalyzed by biotin enzymes. One of the endogenous CO₂-acceptors is probably identical with -methylerotonyl-CoA, formed from isovaleric acid.The experimental results are diseussed in relation to the well-known physiological effects of carbon dioxide on fructification and morphogenesis in the cultivated mushroom. It is suggested that biotin-dependent carboxylations might represent the biochemical basis for the centrolling function of CO₂ in the life cycle of A. bisporus.

     

Fruiting of Agaricus bisporus

Several enzymes were assayed in extracts from mycelium-colonised compost during growth and fruiting of Agaricus bisporus (Lange) Imbach. Comparison of changes of enzyme levels in axenic and nonaxenic cultures and in cultures of non-fruiting strains indicated that they were associated directly with the fungal mycelium. Large changes were found in the amounts of laccase and cellulase which were correlated with fruit body development. Laccase concentration increased during mycelial growth and then declined rapidly at the start of fruiting. Cellulase activity could be detected throughout growth but increased at fruiting. No such changes were observed in xylanase, alkaline protease, laminarinase and acid and alkaline phosphatases. Activities of laccase and cellulase were measured in axenic cultures arrested at various stages of fruiting development. Such cultures showed that the changes in concentration of laccase and cellulase were associated with the enlargement of fruit bodies.     

Spore lipids and germination in Agaricus bisporus

Summary The effects of a number of organic compounds on the germination of spores of Agaricus bisporus (J. Lange) Pilat has been investigated and a preliminary analysis of spore lipids carried out. Germination was stimulated by isocaproic acid but not by straight-chain C₅ to C₁₁ fatty acids or by the amino acids leucine and iso-leucine. Cholesterol at a concentration of 1 ppm was inhibitory. The lipid reserve of the spore comprised mono-, di- and tri-glycerides, free fatty acids and sterols. The phospholipid fraction was unusually small and contained a lecithin and cephalin fraction, phosphatidylinositol and cardiolipin phosphatidic acid; phosphatidylcholine being the most prominent component. The role of lipids and various germination stimulants in the physiology of A. bisporus spores is discussed.     

Double-stranded ribonucleic acid in Agaricus bisporus.

Double-stranded ribonucleic acid present in virus-infected mushrooms of Agaricus bisporus has been resolved through polyacrylamide gel electrophoresis into six molecular-weight forms. Identification of these six double-stranded ribonucleic acids in mushrooms by this procedure has proven to be a useful and diagnostic method for viral infection in the cultivated mushroom.     

2-amino-2-deoxy-D-erythrose in Agaricus bisporus

2-Amino-2-deoxy-D-erythrose was isolated from the cell wall of the fruit body of Agaricus bisporus. The structure of the amino sugar was determined by mass spectrography and 1H-NMR spectrography of its acetylated derivative and by paper chromatographic comparisons with authentic 2-amino-2-deoxy-D-erythrose. This amino sugar is a component of the glycoprotein fraction from the cell wall. Its content in the glycoprotein increased markedly, especially during the ripening stage of the fruit body.     

Germination of basidiospores of Agaricus bisporus

The type of dormancy and conditions necessary for germination of Agaricus bisporus basidiospores (BS) were studied. BS failed to germinate on starvation agar and required the presence of carbon and nitrogen (asparagine and/or glucose) sources in the medium. Upon 3-week storage, BS germinated after 4-5 days. Heat shock (20 min at 45 degrees C) and the decreased temperature facilitated activation of germination. Heterocyclic compounds stimulating germination of endogenously dormant spores, such as furfural, failed to activate germination. The data obtained suggested an endogenous dormancy of A. bisporus BS differing from zygospores of Mucorales. BS contained 17-19% lipids with a composition of fatty acids differing from those of pileus and stipe of the fruiting body. The soluble carbohydrates of the cytosol amounted to 12% dry spore weight and consisted of mannitol (74%) and trehalose (26%). Unlike BS stored at 2 degrees C, the BS stored for 5 months at 20 degrees C lost their ability to germinate, which correlated with a decrease in the content of trehalose.     

NMR lipid profile of Agaricus bisporus

Lipids extracted from freeze dried and powdered cultivated Agaricus bisporus by the Bligh and Dwyer method, were subjected to 1D-proton and 2D-COSY NMR analysis. The diacylglycerophospholipids, mono-, di- and tri-glycerides, ether lipids, sphingolipids and steroidal lipids were studied qualitatively and quantitatively. Our findings suggested that (a) ethanolamines and cholines were the predominant diacylphospholipids, (b) sterols, mainly ergosterol, were present in relatively large quantities and (c) the phospholipid fatty acid composition consisted almost exclusively of linoleic acid. This type of detailed data on lipid composition was accurately and rapidly obtained in one step, without chemical modification of the sample. Additional information on four classes of lipid, including their fatty acid composition was obtained after separating the total lipid extract by NH₂-aminopropyl Certify II Bond Elut solid phase chromatography and analysing the NMR spectra of each class of lipids. The results demonstrated the potential of the method for the study of plant metabolism, development and taxonomy.     

The lipids of Agaricus bisporus.

A comparison of the lipid composition of the vegetative and reproductive stages of Agaricus bisporus revealed no major qualitative differences, although quantitative divergence exist. The glycolipids consisted of acylglucoses, acylmannitol, acyltrehalose and a glucosyloxyfatty acid. Two of the acylglucoses corresponded to a tetra-acylglucose and to either a di- or a triacylglucose. The phospholipids were distinctive in that phosphatidylcholine could not be detected. Phosphatidylethanolamine and phosphatidylserine were the major phosphoglycerides. Examination of the neutral lipids revealed the expected array of acylglycerols, free and esterified sterols, and free fatty acids. A substantial amount (26 to 33%) of the fatty acids of the neutral lipids from both sporophore and mycelium were apparently of chain length greater than C18. Linoleic acid was a minor component of the total neutral-lipid fatty acids but comprised about one-half of the total free fatty acids.     

Germination of Basidiospores of Agaricus bisporus

The type of dormancy and conditions necessary for germination of Agaricus bisporus basidiospores were studied. Basidiospores failed to germinate on starvation agar and required the presence of carbon and nitrogen sources (asparagine and/or glucose) in the medium. Upon 3-week storage, basidiospores germinated after 4–5 days. Heat shock (20 min at 45°C) and decreased temperature facilitated activation of germination. Heterocyclic compounds stimulating germination of endogenously dormant spores, such as furfural, failed to activate germination. The data obtained suggested an endogenous dormancy of A. bisporus basidiospores differing from zygospores of Mucorales. Basidiospores contained 17–19% lipids with a composition of fatty acids differing from those of the pileus and stipe of the fruiting body. The soluble carbohydrates of the cytosol amounted to 12% dry spore weight and consisted of mannitol (74%) and trehalose (26%). Unlike basidiospores stored at 2°C, basidiospores stored for 5 months at 20°C lost their ability to germinate, which correlated with a decrease in the content of trehalose.     

Zur stoffwechselphysiologischen bedeutung von mannit und trehalose in Agaricus bisporus (Eine gaschromatographische studie)

Zusammenfassung Es wird eine gaschromatographische Methode zur simultanen Bestimmung von Mannit und Trehalose in Pilzextrakten beschrieben und damit die Zusammensetzung der Neutralfraktionen einiger Basidiomyceten-species untersucht.Die Anwendung des Verfahrens zur Analyse der einzelnen Teile von Sporokarpen verschiedener Entwicklungsstadien von Agaricus bisporus zeigt, daß dieser entgegen früheren Literaturangaben wie die meisten Basidiomyceten ebenfalls Trehalose enthält (0,03–0,3% des Frischgewichtes).Es besteht ein negativer Gradient von Mannit in Richtung der Pilzlängsachse, welcher während der ganzen Lebensdauer des Fruchtkörpers aufrechterhalten wird und sich mit zunehmendem Alter akzentuiert. Für offene, sporulierende Fruchtkörper wurden Mannitgehalte von 4% (des FG) im Stiel, 3,3% im Hutplectenchym und 1% im Hymenium festgestellt. Hingegen waren keine dem Mannit entsprechende oder zu ihm inverse Konzentrationsgradienten von Trehalose nachzuweisen. Das Verhältnis von Mannit zu Trehalose weist zeitliche Variationen und örtliche Unterschiede innerhalb des Basidiokarps auf; der Quotient M/T erreicht in den Pinheads und in den Sporen den kleinsten Wert (4).Die experimentellen Ergebnisse werden in bezug auf die stoffwechselphysiologische Bedeutung von Mannit und Trehalose in höhern Pilzen diskutiert.

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Summary A gaschromatographic method for the simultaneous determination of mannitol and trehalose in fungal extracts is described. It is used to determine the composition of the neutral fractions of some basidiomycetous species.The application of the procedure to the analysis of the individual parts of sporocarps from Agaricus bisporus in different developmental stages shows that this species contains trehalose (0.03–0.3% of the fresh weight) like most basidiomycetes, contrary to the citations in the literature.There exists a negative gradient of mannitol in the direction of the longitudinal axis of the mushroom which is maintained during the whole life of the fruit body and accentuated with increasing age. The mannitol contents of open, sporulating fruit bodies were 4% (of the fresh weight) in the stipe, 3.3% in the pileus and 1% in the hymenium. However, no gradient of trehalose corresponding to or inverse to that of mannitol was found. The ratio of mannitol to trehalose shows temporal and topographic differences within the basidiocarp; in the pinheads and the spores the ratio of M/T is lowest (4).The experimental results are discussed in relation to the physiological rôle of mannito and trehalose in higher fungi.

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Purine degradation in the edible mushroom Agaricus bisporus

Agaricus bisporus is able to use urate, allantoin, allantoate, urea and alloxanate as nitrogen sources for growth. The presence of urate oxidase, allantoinase, ureidoglycolase and urease activities, both in fruit bodies and mycelia, points to a degradative pathway for urate similar to that found in various microorganisms. So far all efforts, to demonstrate the enzyme responsible for allantoate degradation failed. A urease inhibitor appeared to be present in cell-free extracts, from fruit bodies.     

Respiratory pathways in Agaricus bisporus and Scytalidium thermophilum

Abstract The respiratory pathways of Agaricus bisporus and Scytalidium thermophilum were studied. A. bisporus appeared to possess both a cyanide-sensitive and a cyanide-insensitive respiration while in S. thermophilum the cyande-insensitive respiration was absent. Growth experiments showed the ecological advantage for A. bisporus under conditions where cytochrome mediated respiration is inhibited.     

RAPD discrimination of Agaricus bisporus mushroom cultivars

Cultivars of the white button mushroom Agaricus bisporus are difficult to differentiate, which has made strain protection problematic for this crop species. We have used RAPDs to discriminate between 26 strains of A. bisporus, 24 of which were commercial cultivars, and to characterise the genetic relatedness of these strains. Using 20 primers, 211 RAPD markers were identified and used in hierarchical cluster, patristic distance and parsimony analyses. All strains could be differentiated using the aggregated primer data. Although no one primer could differentiate all 26 strains, several individual primers yielded unique fingerprints for a variety of strains. The greatest differences (up to 28% variation) were observed in comparisons with or between two wild collections of A. bisporus. Quondam cultivars, commercial brown and off-white varieties proved more variable than the widely grown 'hybrid' types. Of the 15 hybrid varieties analysed, only one differed substantially (20% or more variable). The patristic and parsimony analyses both demonstrated the gross similarity of the hybrids, many of which appear to be essentially derived varieties from two original hybrid cultivars. RAPD analyses can assist mushroom strain identification and could play a role in the protection of novel cultivars.