Development and fate of electron-dense microbodies in trap cells of the nematophagous fungusArthrobotrys oligospora

The development of electron-dense microbodies in cells of capture organs of the nematophagous fungus Arthrobotrys oligospora was studied with different ultrastructural techniques. Kinetic experiments revealed that the synthesis of these microbodies started in a very early stage of trap formation; the organelles originated from special regions of endoplasmic reticulum by budding. Mature organelles were surrounded by a single membrane of approximately 9 nm (KMnO4-fixation) and lacked crystalline inclusions. The presence of the electron-dense microbodies was independent of the conditions during which the traps had developed. The organelles remained intact during aging of the trap cells. They were also observed in the trophic hyphae after capture and penetration of nematodes. However, the distribution patterns of these organelles in the trophic hyphae, which were identical to those observed after germination of isolated traps on different cultivation media, suggested that their presence must be explained by dilution of organelles in newly formed cells.     

Significance of electron dense microbodies in trap cells of the nematophagous fungus Arthrobotrys oligospora

We have studied the fate of electron dense microbodies in nematode-trapping organs (traps) of the fungus A. oligospora during the initial hours following nematode capture. The interaction studies were performed with isolated traps which had captured a nematode under conditions where the fungal cells had no access to external energy sources. Video enhanced contrast microscopy showed that under these conditions the number of dense bodies present in the trap cell that formed the penetration tube, rapidly decreased. During subsequent penetration and development of the infection bulb this decrease continued while at this time common cell organelles such as mitochondria and vacuoles were formed. This was confirmed by electron microscopy which also revealed that the dense bodies were degraded by means of an autophagic process. The organelles were degraded individually and finally turned into compartments which, based on ultrastructural criteria, were considered vacuoles. Fusion of such vacuoles into larger organelles frequently occurred. The degradation process was initiated early in the interaction since initial stages were already evident within 15 min after capture. Generally it took 1–2 h before the infection bulb had fully developed and trophic hyphae formation started. During this time the original trap cell, characterized by numerous dense bodies, was transformed into an active vegetative hyphal cell containing typical cell organelles such as nuclei, mitochondria, a strongly proliferated endoplasmic reticulum, vacuoles and normal microbodies but lacked dense bodies. This disappearance of dense bodies was confined to the cell that penetrated the nematode and—less frequently—its two neighbouring cells in the hyphal loop. In the other cells, constituting the trap, the dense bodies remained unaffected. As will be discussed, the present results support our current view that traps of A. oligospora contribute to the survival of the organism in its natural environment.     

Physiological role of microbodies in the yeast Trichosporon cutaneum during growth on ethylamine as the source of energy,carbon and nitrogen

Compartmentation of the metabolism of ethylamine in Trichosporon cutaneum X4 was studied in cells, grown on this compound as the sole source of energy, carbon, and nitrogen. Transfer experiments indicated that an amine oxidase is involved in the early metabolism of ethylamine. The synthesis of this enzyme was induced by primary amines and was subject to partial carbon catabolite repression. Repression by ammonium ions was not observed. Adaptation of glucose-grown cells to growth on ethylamine was associated with the development of many microbodies, which developed from already existing organelles present in the inoculum cells and multiplied by division. Cytochemical experiments indicated that the organelles contained amine oxidase and catalase. Therefore, they were considered to play a key role in the metabolism of ethylamine. The physiological significance of the microbodies was investigated by fractionation studies of homogenized protoplasts from ethylamine-grown cells by differential- and sucrose-gradient centrifugation of subcellular organelles. Intact microbodies were only obtained when the isolation procedure was performed at pH 5.8 in the absence of Mg²⁺-ions. Analysis of the different fractions indicated that the key enzymes of the glyoxylate cycle, namely isocitrate lyase and malate synthase, cosedimented together with catalase and amine oxidase. In addition, activities of malate dehydrogenase, glutamate:oxaloacetate aminotransferase (GOT) and (NAD-dependent) glutamate dehydrogenase were detected in these fractions. Electron microscopy revealed that they mainly contained microbodies. Cytochemical experiments indicated that the above enzymes were all present in the same organelle. These findings suggest that microbodies of ethylamine-grown T. cutaneum X4 produce aspartate, so allowing NADH generated in the oxidation of malate by malate dehydrogenase to be quantitatively reoxidized inside the organelles in a series of reactions involving GOT and glutamate dehydrogenase. Aspartase and fumarase were not detected in the microbodies; activities of these two enzymes were present in the cytoplasm.Abbreviations ABTS 2,2-Azino-di(3-ethylbenzthiazoline sulfonate [6]) - DTT dithiothreitol - GOT glutamate:oxaloacetate aminotransferase - DTNB 5,5-dithiobis-2-nitrobenzoate - DAB diaminobenzidine - BSPT 2-(2-benzothiazolyl)-3-(4-phthalhydrazidyl)-t-styryl-sH-tetrazolium chloride - PF convex fracture face - EF concave fracture face     

The occurrence and fine structural characterization of microbodies inEuglena gracilis

Summary The fine structure of an organelle morphologically similar to microbodies found in higher plants and animals was studied in cells ofEuglena gracilis fixed simultaneously in glutaraldehyde and osmium tetroxide. These organelles were 0.4 to 0.8 microns in diameter, bounded by a single membrane, and frequently observed in close spatial association with both endoplasmic reticulum and mitochondria. Their finely granular matrices frequently contained membranous cores. Though these organelles were relatively abundant in acetate- and ethanolgrown cells, they were rarely observed in glucose-grown cells, an indication that they play the same role in the metabolism of 2-carbon substrates as do glyoxysomes in higher plants. The presence of these organelles, assumed to be microbodies, is also of considerable interest since catalase, an enzyme characteristic of microbodies from a variety of sources, was not detected.This work was supported in part by grant GY 3804 from the National Science Foundation to L.B.G.     

The structure of microbodies and their associations with other organelles in zoosporangia ofEntophlyctis variabilis

Summary The ultrastructure of microbodies in developing zoosporangia ofEntophlyctis variabilis was studied by three dimensional reconstructions from serial sections and by cytochemical localization of catalase activity. The morphology of microbodies and the spatial association of microbodies with other organelles varied during fungal development. In incipient zoo-sporangia, granular dilations resembling microbodies arose from rough ER. Young, enlarging zoosporangia contained elongate, contorted microbodies continuous with ER and aligned along bundles of microtubules. Oval, paired microbodies, lying on each side of an ER cisternae, were found in all zoosporangia, but in older zoosporangia this configuration of microbodies predominated. Analysis of serial sections revealed that these oval, paired microbodies were sometimes continuous with each other, with ER, and also apparently with the ER cisterna interposed between them. Other paired, oval microbodies were clearly discrete. Constrictions were found along the length of elongate microbodies and at junctions between oval microbodies. These constrictions may represent stages in fragmentation of microbodies from pre-existing microbodies. These observations suggest that microbodies originated in three ways: 1. as local dilations in tubular ER, 2. as lateral buds from opposite sides of ER cisternae, and 3. as fragments from elongate microbodies.Microbodies were consistently spatially associated with ER, nuclear envelopes, and mitochondria. The cisterna of ER passing between paired microbodies sometimes extended into a branching, tubular system of ER which curved around the side of one microbody and lay between this microbody and the forming face of a dictyosome. The cytochemical localization of thiamine pyrophosphatase activity in this cisterna when it is not associated with dictyosomes suggests a role in metabolic control. These spatial associations indicate that the microbody assemblage with other organelles represents functional units where propinquity to other organelles and intraluminal continuities insure a system for transport of substrates and products.     

Biogenesis and metabolic significance of microbodies in urate-utilizing yeasts

Growth of Candida famata and Trichosporon cutaneum on uric acid as the sole source of carbon and nitrogen was associated with the development of a number of microbodies in the cells. Cytochemical staining experiments showed that the organelles contained urate oxidase, a key enzyme of uric acid metabolism, and catalase. Transfer of cells, precultured on glucose or glycerol, into uric acid-containing media indicated that these microbodies originated from the organelles, originally present in the inoculum cells, by growth and division. In urate-grown C. famata the microbodies were frequently observed in large clusters; in both organisms they existed in close association with mitochondria and strands of ER. The organelles lacked crystalline inclusions. In freeze-fractured cells their surrounding membranes showed smooth fracture faces.Exposure of urate-grown cells to glucose-excess conditions led to a rapid inactivation of urate oxidase activity but catalase was only slightly inactivated. Glucose-induced enzyme inactivation was not associated with the degradation of the microbodies present in the cells. Similarly, repression of urate oxidase synthesis by ammonium ions also did not lead to the degradation of peroxisomes.     

Peroxisomes in intestinal and gallbladder epithelial cells of the stickleback,Gasterosteus aculeatus L. (Teleostei)

Summary The occurrence of microbodies in the epithelial cells of the intestine and gallbladder of the stickleback, Gasterosteus aculeatus L., is described. In the intestine the organelles are predominantly located in the apical and perinuclear zone of the cells and may contain small crystalline cores. In gallbladder epithelial cells the microbodies are distributed randomly. The latter organelles are characterized by the presence of large crystalloids. Cytochemical and biochemical experiments show that catalase and D-amino acid oxidase are main matrix components of the microbodies in both the intestinal and gallbladder epithelia. These organelles therefore are considered peroxisomes. In addition, in intestinal mucosa but not in gallbladder epithelium a low activity of palmitoyl CoA oxidase was detected biochemically. Urate oxidase and L- hydroxy acid oxidase activities could not be demonstrated.     

Cytochemical localization of glucose oxidase in peroxisomes of Aspergillus niger

Summary The subcellular localization of glucose oxidase (E.C. 1.1.3.4) in mycelia of Aspergillus niger has been investigated using cytochemical staining techniques. Mycelia from fermenter cultures, which produced gluconic acid from glucose, contained elevated levels of glucose oxidase and catalase. Both enzymes were located in microbodies. In addition, when the organism was grown on glucose with methylamine as a nitrogen source, amine oxidase activity was detected in the microbodies. These organelles can therefore be designated as peroxisomes.     

Occurrence and metabolic significance of microbodies in trophic hyphae of the nematophagous fungus Arthrobotrys oligospora

This paper describes the results of an ultrastructural study on the subcellular events occurring in nematode-infecting (trophic) hyphae of the nematophagous fungus Arthrobotrys oligospora. In early stages of the infection process (30 min-4 h), the infection bulb and developing trophic hyphae are characterized by a highly proliferated endoplasmic reticulum (ER). Its membranes often appeared vesiculated and occur in close association with the cell membrane of the cells. Upon further invasion of the nematode, lipid droplets developed in the trophic hyphae; these droplets were first observed 4–5 h after the infection but were abundantly present after 24–36 h. Along with the formation of lipid droplets proliferation of microbodies was observed. These organeles were characterized by the presence of catalase and thiolase and were frequently observed in close association with the lipid droplets. Later on the lipid droplets disappeared. During this period new vegetative mycelium developed from the trap that had originally captured the nematode. Our results suggest that part of the nutrients released from the nematode are first converted into lipids by the fungus which in turn are degraded via the -oxidation pathway and further metabolized to support growth of new vegetative hyphae.     

Ultrastructure of methanotrophic yeasts.

The cellular structure of two yeast strains capable of growth on methane was investigated by electron microscopy. Microbodies were observed in cells of Sporobolomyces roseus strain Y and Rhodotorula glutinis strain CY when grown on methane but rarely when grown on glucose. The size of the microbodies and the number observed per cell in a thin section did not increase with culture age. No crystalline organization was observed within these organelles. Similar microbodies were also observed in cells of R. glutinis CY grown on hexadecane. The plasma membranes of both methane and hexadecane-grown cells exhibited increased invagination compared to that of glucose-grown cells. Catalase activity was detected in the microbodies of alkane-grown cells by using 3,3'-diaminobenzidine as a cytochemical stain. The data presented suggest that microbodies, and the catalase contained within them, play a role in eucaryotic methane metabolism.     

Mitochondrial morphology in Basidiobolus haptosporus

Young hyphal cells of the potentially zoopathogenic fungus Basidiobolus haptosporus characteristically exhibit unusual proportions of annulate views of mitochondria in the two-dimensional perspective of thin sections. Such views exhibit a central space containing cytoplasmic ground substance and often profiles of other cytoplasmic organelles (lipid bodies, other mitochondrial forms, and especially crystalloid-containing microbodies). Three-dimensional projections are presented to suggest that these mitochondria have assumed the form of a goblet-shaped enclosure, and that the various annulate views are the consequence of plane of section viewed by electron microscopy. Their frequent occurrence and consistent morphology argues against their being random expressions of mitochondrial plasticity, but rather for close spatial associations amongst cytoplasmic organelles of young hyphae. When the fungus is grown on xanthine or its catabolites as sole sources of nitrogen, there is a proliferation of crystalloid-containing microbodies, double-membraned vesicles, and ovate to ellipsoidal mitochondria. Annulate views of mitochondria then are no longer observed, but microbodies again frequently appear in close association with mitochondria and at times in intimate contact with the mitochondrial outer membrane.     

Characterization of ψM1, a virulent phage of Methanobacterium thermoautotrophicum Marburg

We have studied the biogenesis and enzymic composition of microbodies in different yeasts during adaptation of cells to a new growth environment. After a shift of cells of Candida boidinii and Hansenula polymorpha from glucose to methanol/methylamine-containing media, newly synthesized alcohol oxidase and amine oxidase are imported in one and the same organelle together with catalase; as a consequence the cells contain one class of morphologically and enzymatically identical microbodies. Similar results were obtained when Candida utilis cells were transferred from glucose to ethanol/ethylamine-containing media upon which all cells formed microbodies containing amine oxidase and catalase.However, when methanol-limited cells of H. polymorpha were transferred from media containing ammonium sulphate to those with methylamine as the nitrogen source, newly synthesized amine oxidase was incorporated only in part of the microbodies present in these cells. This uptake was confined to the few smaller organelles generally present at the perimeter of the cells, which were considered not fully developed (immature) as judged by their size. Essentially similar results were obtained when stationary phase cells of C. boidinii or C. utilis — grown on methanol and ethanol plus ammonium sulphate, respectively — were shifted to media containing (m)ethylamine as the nitrogen source. These results indicate that mature microbodies may exist in yeasts which no longer are involved in the uptake of matrix proteins. Therefore, these yeasts may display heterogeneities in their microbody population.     

Microbodies in fungi: a review

Summary Microbodies are ubiquitous organelles in fungal cells, occurring in both vegetative hyphae and spores. They are bounded by a single membrane and may contain a crystalloid inclusion with subunits spaced at regular intervals. Typically, they contain catalase which reacts with the cytochemical stain 3,3-diaminobenzidine to yield an electron-opaque product, urate oxidase,l--hydroxy acid oxidase andd-amino acid oxidase. Their fragility and the necessity to disrupt the tough fungal cell wall before isolating them make them difficult to isolate. Analysis of enzymes in purified or partially purified microbodies from fungi indicates that they participate in fatty acid degradation, the glyoxylate cycle, purine metabolism, methanol oxidation, assimilation of nitrogenous compounds, amine metabolism and oxalate synthesis. In organisms where microbodies are known to contain enzymes of the glyoxylate cycle, they are known as glyoxysomes; where they are known to contain peroxidatic activity, they are known as peroxisomes. In some cases microbodies contain enzymes for only a portion of a pathway or cycle. Thus, they must be involved in metabolic cooperation with other organelles, particularly mitochondria. The number, size and shape of microbodies in cells, their buoyant density and their enzyme contents may vary with the composition of the medium; their proliferation in cells is regulated by the growth environment. The isolation from the same organism of microbodies with different buoyant densities and different enzymes suggests strongly that more than one type of microbody can be formed by fungi.     

CYTOCHEMICAL AND DEVELOPMENTAL CHANGES IN MICROBODIES (GLYOXYSOMES) AND RELATED ORGANELLES OF CASTOR BEAN ENDOSPERM

Structural changes in endosperm cells of germinating castor beans were examined and complemented with a cytochemical analysis of staining with diaminobenzidine (DAB). Deposition of oxidized DAB occurred only in microbodies due to the presence of catalase, and in cell walls associated with peroxidase activity. Seedling development paralleled the disappearance of spherosomes (lipid bodies) and matrix of aleurone grains in endosperm cells. 6 to 7 days after germination, a cross-section through the endosperm contained cells in all stages of development and senescence beginning at the seed coat and progressing inward to the cotyledons. Part of this aging process involved vacuole formation by fusion of aleurone grain membranes. This coincided with an increase in microbodies (glyoxsomes), mitochondria, plastids with an elaborate tubular network, and the formation of a new protein body referred to as a dilated cisterna, which is structurally and biochemically distinct from microbodies although both apparently develop from rough endoplasmic reticulum (ER). In vacuolate cells microbodies are the most numerous organelle and are intimately associated with spherosomes and dilated cisternae. This phenomenon is discussed in relation to the biochemical activities of these organelles. Turnover of microbodies involves sequestration into autophagic vacuoles as intact organelles which still retain catalase activity. Crystalloids present in microbodies develop by condensation of matrix protein and are the principal site of catalase formerly in the matrix.     

Heavy trap formation by Arthrobotrys oligospora in liquid culture

Abstract The nematode-trapping fungus Arthrobotrys oligospora was grown in liquid culture in modified separatory funnels with vigorous air bubbling. The growth medium was a dilute soya peptone broth supplemented with a trap-inducing substance. The dipeptide l -phenylalanyl- l -valine or its constituents phenylalanine and valine were used as inducers of trap formation. Trap formation started within 2 days after inoculation and the traps increased in number and size during a 7-day period. The traps formed in liquid culture were fully functional in trapping nematodes. At the ultrastructural level they were characterized by the presence of many electron-dense microbodies similar to those in trap cells grown on solid media. Biomass increase, amounting to 6–7 mg dry weight · ml⁻¹, and trap formation were highly synchronized. The mycelial mass was homogeneously dispersed in the growth vessel during the entire growth period. Heavy trap formation in liquid culture by this fungus has not been reported previously.     

The evolutionary origin of glycosomes

The glycolytic pathway of the Kinetoplastida is organized in a unique manner: the majority of its enzymes are contained in organelles called glycosomes. In this article Paul Michels and Fred Opperdoes argue that the glycosomes are equivalent to the microbodies and peroxisomes identified in other eukaryotic cells. They explore the possible evolutionary origin of the glycosome by comparing many of its structural and functional properties with those of other members of the microbody family and with some features of other organelles, the mitochondria and chloroplasts, which have been studied in much more detail.     

Isolation and Characterization of Microbodies from the Alga Chlorogonium elongatum

The alga Chlorogonium elongatum was grown autotrophically or heterotrophically on acetate. Cells harvested in the logarithmic phase of growth were disrupted, and the whole homogenates were fractionated on sucrose gradients. Protein and enzyme determinations carried out on the fractions led to the following conclusions. Chloroplast fragments which represent the major portion of particulate protein in autotrophic cells migrate to density 1.17 g/cm³. In heterotrophic cells, mitochondria comprise most of the particulate protein, and these particles accumulate at density 1.19 g/cm³, as shown by a peak of cytochrome oxidase in this region. Part of the catalase and uricase, two marker enzymes for microbodies, were found in the soluble fractions, but 60% or more of these activities were recovered at density 1.225 g/cm³ from autotrophic cells. Electron micrographs showed that in this region there were microbodies with a diameter of 0.4 micrometer. The isolated microbodies contained no isocitrate lyase, a marker enzyme of glyoxysomes. This enzyme was completely soluble and therefore seems not to be associated with organelles in this organism.     

Fatty acid beta-oxidation system in microbodies of n-alkane-grown Candida tropicalis.

Localization of fatty acid beta-oxidation system in microbodies of Candida tropicalis cells growing on n-alkanes was studied. Microbodies isolated from the yeast cells showed palmitate-dependent activities of NAD reduction, acetyl-CoA formation and oxygen consumption. When sodium azide, an inhibitor of catalase, was added to the system, palmitate-dependent formation of hydrogen peroxide was observed. Stoichiometric study revealed that two moles of NAD were reduced per one mole of oxygen consumed in the absence of sodium azide and the presence of the inhibitor doubled the oxygen consumption by microbodies without an appreciable change in NAD reduction. These results indicate that the yeast microbodies contain beta-oxidation system of fatty acid, and that catalase located in the organelles participates in the degradation of hydrogen peroxide to be formed at the step of dehydrogenation of acyl-CoA.     

The Ultrastructural Feature and Distribution of Microbodies in Mature Embryo Cells of Pinus bungeana

The material of pine seeds used in this investigation was collected in 1982 from Peking. The microbodies of mature embryo ceils are very well developed and their diameter averages about 2–3 μm, even up to 4.3 μm. The appearance is usually ovoid or elliptic. The microbodies are essentially glyoxysomes. The microbody matrix is composed of two types of substances, one type is of a finely granular material in a densely arrangement (Plate Ⅲ Fig. 6); the other is of coarsely granular or flocculant in appearance and the elements of the matrix are loosely distributed. These matrices usually contain an amorphous inclusion or crystalline arrays in regular arrangement. The inclusion sometimes occupies a small portion of the microbody matrix (Plate Ⅲ, Figs, 5, 6) and sometimes the inclusion occupies nearly the entire glyoxysome (Plate Ⅱ, Fig. 3). It is interesting that the “pockets” frequently appear in the microbodies of mature embryo cells, and those are actually as a result of invagination in microbodies (Plate Ⅱ, Fig. 4). In addition, an electron-transparent “oil body-like space” occurs occasionally in microbody (Plate Ⅰ, Fig. 1). The periphery of “space” is a constitutive part of matrix or continuing with the matrix. This “space” may be due to the degradation in a part of the matrix. While the periphery of the pocket is membranaceous and an electron-opaque cytoplasmic groundplasm was found within the pocket. The microbodies of mature embryo cells in Pinus are mainly distributed in pericolumn cells of the root cap and cortical cells of the hypocotyl. Besides the dominant organelles of lipid bodies in the cells of above mentioned tissues, there are also microbodies, amyloplasts, mitochondria, plastids, endoplasm reticulum and Golgi apparatus, of which the microbodies are the most aboundant organelles. In contrast, the microbodies and other organelle are rare in the parenchyma of the cotyledons in Pinus. Their common and outstanding characteristics in various tissues of mature embryo is that the entire cytoplasm of the cells is almost full of the lipid bodies, and each organelle is directly surrounded by a number of lipid bodies (Plate Ⅰ—Ⅲ, Figs. 1–6). Because of the other organelles are rare in parenchyma of the cotyledons, the lipid bodies are so appressed with each other that the inlaid periphery of lipid bodies frequently occurs in some degree. To sum up, based upon 'the state of distribution of microbodies in mature embryo tissues, cotyledons of Pinus could be considered as the main storage organ of nutrient substances, while the root cap and hypocotyl are the important sites of glyoxysome metabolism. The function of glyoxysomes is to convert lipid into the carbohydrates and to transfer the latter to embryos for growth.     

Ultrastructural cytochemistry of the diaminobenzidine reaction in the aquatic fungus Entophlyctis variabilis

Ultrastructural localization of peroxidatic activity was investigated in the chytrid Entophlyctis variabilis with the 3,3-diaminobenzidine (DAB) cytochemical prodedure. The subcellular distribution of reaction product varied with changes in pH of the DAB medium and with the developmental stage of the fungus. Incubations in the DAB reaction medium at pH 9.2 produced an electron dense reaction product within single membrane bounded organelles which resembled microbodies but which varied in shapes from elongate to oval. At this pH the cell wall also stained darkly. When the pH of the DAB medium was lowered to pH 8.2 or 7.0, DAB oxidation product was localized within mitochondrial cristae as well as in microbodies and zoosporangial walls. As soon as zoospores were completely cleaved out of the zoosporangial cytoplasm, endoplasmic reticulum (ER) also stained. When the wall appeared around the encysted zoospore, ER staining was no longer found. The influence of the catalase inhibitor, aminotriazole, and the inhibitors of heme enzymes, sodium azide and sodium cyanide, on the staining patterns within cells incubated in the DAB media indicates that microbody staining is due to both catalase and peroxidase, mitochondrial staining is due to cytochrome c, and ER staining is due to peroxidase.Abbreviations DAB 3,3-diaminobenzidine-HCl - ER endoplasmic reticulum