Ultrastructural changes during zoospore formation in phytophthora parasitica

Cleavage of the multinucleate sporangial cytoplasm begins by a rearrangement and subsequent fusion of randomly dispersed cleavage vesicles. The vesicles line up in planes equidistant from neighboring nuclei and along the sporangial wall. In addition, they contribute to the enlargement of the central vacuole. Fusion of these vesicles with themselves and with the central vacuole cleaves the cytoplasm into uninucleate zoospores, each with two flagella. The sporangial wall consists of two layers, an outer thin one which is continuous over the plug of the discharge pore and an inner thick one which tapers off near the plug. The plug consists of fibrillar material and is ejected upon release of the zoospores. A plug-like structure separating the forming sporangium from the hypha has a homogeneous matrix pervaded with an anastomosing network of fine electron-dense channels. In addition, glycogen-like granules occur within mitochondria and paired structures are interpreted as procentrioles.     

An ultrastructural study of zoosporogenesis inPythium proliferum de Bary

Summary Zoosporogenesis in the oomycete,Pythium proliferum de Bary initially involves a condensation of cytoplasm at certain hyphal tips and the subsequent enlargement of these hyphal tips to form sporangia. Deposition of a septum at the base of the sporangium and initiation of an apical papilla are followed by cleavage of the sporangial cytoplasm. Packets of presumptive mastigonemes as well as flagella are recognized in the cytoplasm at this time. Subsequently, cleavage vesicles at the periphery of the sporogenic cytoplasm fuse with the plasmalemma thereby emptying their fibrous contents into the space between the sporogenic cytoplasm and the sporangial wall. It is felt that this fibrous material is instrumental in developing the internal pressure necessary within the sporangium to cause discharge of the sporogenic cytoplasm into an evanescent vesicle wherein delimitation of the zoospores is completed. The formation of the spore vesicle from the multilayered apical cap of the papilla is described here for the first time in the Oomycetes and a new term, vesiculogen, is suggested for this structure. Aspects of centriole replication within vegetative hyphae, papilla formation, and morphogenesis of various vesicular inclusions are also described in this study.     

Development of the discharge apparatus in the fungus Entophlyctis

Correlative light and electron microscopic observations were used to reconstruct the morphological events involved in the development of the discharge apparatus of Entophlyctis zoosporangia. A discharge plug formed as vesicles containing fibrillar material fused with the plasma membrane and deposited their matrices between the plasma membrane and zoosporangial wall. At the apex of the enlarging plug, the zoosporangial wall lost its microfibrillar appearance, became diffuse, and left an inoperculate discharge pore. The discharge plug exuded through this pore and then expanded into a sphere which rested at the tip of the discharge papilla or tube. After the release of the discharge plug, the number of fibrilla containing vesicles decreased and abundant endoplasmic reticulum appeared in the cytoplasm below the plug. Granular material then accumulated at the interface of the discharge plug and the plasma membrane. This was the endo-operculum. A single layer of endoplasmic reticulum subtended the area of plasma membrane which the endo-operculum covered. Later, dictyosomes appeared in the cytoplasm below the endo-operculum. Fusion of Golgi vesicles with the plasma membrane below the endo-operculum coincided with the initiation of cytoplasmic cleavage. This sequence of events indicates that, unlike the discharge plug, the endo-operculum does not originate by vesicular addition of preformed material.     

An Electron Microscope Study of the Rat Ovum

This paper reports on the fine structure of rat oocytes at stages before ovulation, during maturation, fertilization, and early cleavage. The study includes parallel observations on light and electron microscope preparations with attempted correlations. The follicular cells of the ovarian egg are described as sending long processes through the zona pellucida to the egg surface where they mingle with thin projections from the egg itself. No open communication between follicle cell cytoplasm and egg cytoplasm was observed. During maturation and fertilization both types of processes are withdrawn from the zona. The germinal vesicle and later the pronuclei of the fertilized egg are characterized by numerous large nucleoli. These have the form of thick walled vesicles with diameters as great as 8 to 10 µ. The wall is dense in the EM image and appears to consist in part of small granules. The cytoplasm shows several inclusions including mitochondria of usual form and a Golgi component which has the typical fine structure and the distribution described by earlier light studies. Small dense particles, presumably RNP particles, are distributed throughout the cytoplasmic matrix and show no preference for membranes. The endoplasmic reticulum of the oocyte is represented by a scattering only of vesicles, but begins a more extensive and elaborate development with the onset of segmentation. One inclusion of the ooplasm, similar in size to mitochondria, receives special attention. It is a vesicular structure, containing a large number of small vesicles (10 to 50 mµ in diameter) and frequently a central density or nucleoid. They are referred to as multivesicular bodies. Such bodies are found in small number in the ovarian egg, but increase greatly in number during maturation and fertilization. It appears from the micrographs of eggs in these latter stages that these vesicular bodies break down and liberate their content of small vesicles to the surrounding ooplasm. Comments are provided on the apparent significance of the various observations.     

CHLORTETRACYCLINE-INDUCED FORMATION OF WALL APPOSITIONS (CALLOSE PLUGS) IN INTERNODAL CELLS OF NITELLA FLEXILIS (CHARACEAE)1

The formation of chlortetracycline (CTC)-induced wall appositions or plugs in internodal cells of Nitella flexilis (L.) Ag. was studied with light, fluorescence and electron microscopy. These plugs contain callose and pectin. A few minutes after CTC addition plug formation starts by fusion of polysaccharide-containing vesicles (glycosomes) with the plasmalemma. Plug growth is continued by incorporation of glycosome-endoplasmic reticulum (ER) complexes. The cytoplasm near the plug appears dense because of the accumulation of glycosomes and the increased electron density of plasma matrix and organelles. About 1 h after CTC addition plug growth ceases, the cytoplasm recovers to its pretreatment appearance, and a few glycosomes fuse singly with the plug membrane. Crystalline inclusions which consist of hexagonally packed rods are found near the plug. Coated vesicles and coated pits are clearly seen only in very early and late stages of plug formation. Callose is also found in parts of wound plugs produced after mechanical injury. No callose is present in the underlying, highly ordered wound wall. The failure to produce a wound wall beneath CTC-induced plugs appears to be related to the lower number of coated vesicles during plug formation. The possible significance of the partially coated reticulum in plug and wound wall formation is discussed.     

Budding in the Dimorphic Fungus Phialophora dermatitidis

Ultrastructural comparisons of yeast and hyphal bud formation in Phialophora dermatitidis reveal that bud initiation is characterized by a blastic rupture of the outer portion of the yeast or hyphal wall and the emergence of a bud protuberance through the resulting opening. The wall of the emerging bud is continuous, with only an inner wall layer of the parental yeast or hypha. The outer, ruptured portion of the parental wall typically forms a collar around the constricted emergence region of the developing bud. The cytoplasm within the very young emerging bud invariably contains a small number of membrane-bound vesicles. The septum formed between the daughter bud and the parental yeast or hypha is a complete septum devoid of a septal pore, septal pore plug, or any associated Woronin bodies characteristic of simple septa of the moniliform or true hyphae. These observations suggest that yeast bud formation and lateral hyphal bud formation in the dimorphic fungus P. dermatitidis involve a growth process which occurs identically in both the yeast and mold phase of this human pathogenic organism.     

The relationship of echinate inclusions and coated vesicles on wound healing inNitella flexilis (Characeae)

Summary Wound healing in internodal cells of the freshwater algaNitella flexilis (Characeae) was studied in the light and electron microscope. Immediately after punctation of the cell wall a wound plug is formed which stops outflow of cytoplasm. The plug consists of echinate inclusions which are normally located in the central vacuole. A wound wall consisting of pectin and cellulose microfibrils is formed beneath the plug within one to several hours. During that time the wound shows intensive fluorescence when treated with chlorotetracycline indicating transmembrane Ca²⁺ fluxes. Numerous coated pits and vesicles are found at the plasmalemma. The glycosomes undergo pronounced structural changes. Neither plug nor wound wall formation depend on actin filaments or microtubules as shown by inhibitor experiments with cytochalasin and amiprophos-methyl. The function of the coated vesicles and their interrelationship with other cell organelles is discussed.     

The growth of the pollen tube wall in Oenothera organensis.

The growth of the pollen tube wall of Oenothera is effected by the expulsion of fibrillar material from the cytoplasm into the developing wall. This material may also be seen in the cytoplasm, contained in membrane-bound vesicles. It is not clear how the content of the vesicles is discharged, but it appears not to involve the participation of microtubules. The source of the cytoplasmic fibrillar bodies depends upon the stage of development of the pollen tube. The earilest growth is derived from the inclusion into the wall of vesicles containing pre-formed materials present in the grain on pollination. During the next stage of growth the wall is derived from the content of double-membraned inclusions also present in the pollen. The content of the former vesicles is not so similar to the wall as the latter, but intermediates between the 2 types of vesicle may be seen in the cytoplasm, indicating that the former are formed from the latter. Most of the tube wall is derived from the products of dictyosomes in the pollen grain or tube. These dicytosomes are few in number and they must be exceedingly active. This, and the observation that dictyosome vesicles are frequently associated with banked complexes of mitochondria, indicates that some steps in the metabolism of the vesicular content, perhaps phosphorylation, take place distant from the dicytosomes. These different sources of fibrillar material presumably permit the rapid starting of tube growth, without any attendant metabolism. However, it would be impossible to include enough pre-formed wall material in the grain to enable the full growth of the tube, so once started, it seems that the tube then relies on the elaboration of simple reserves for the contruction of its wall. These reserves are likely to be held in the pollen, and may be the large numbers of starch grains characteristic of the pollen cytoplasm.     

Ultrastructure of Wall Swellings of Germinating Sporangia of Phytophthora palmivora (Butl.) Butl.

Germ tubes of directly germinating sporangia of P. palmivoraincubated in yeast extract solution at 30 ?C usually developedinto prominent swellings from which hyphae later emerged. Thegerm tubes arose as an extension of a new germination wall formedinternal to the sporangial wall prior to germination. The germtube swellings contained typical hyphal organelles. The germtube swelling possessed a thicker wall than both hyphae growingout of it and germ tubes that did not form swellings.     

Fine-structural Correlates of Growth in Hyphae of Ascodesmis sphaerospora

Mycelial mats of Ascodesmis sphaerospora were fixed and embedded for electron microscopy, and thin sections of 1-mm blocks, taken from the 1st to the 7th mm behind the hyphal tips, were cut parallel to the long axis of the hyphae. The hyphal tip region is characterized by an outer zone of electron-transparent vesicles, 500 to 1,000 A in diameter, and is apparently associated with wall elaboration. Immediately behind this region, dense granules become evident along convoluted membrane systems and along the plasma membrane; in the same region are numerous small lomasomes in the lateral wall. As the hypha grows, septa are laid down at 3- to 7-min intervals at a distance of 200 to 250 μ behind the hyphal tip. A cylinder of endoplasmic reticulum is intimately involved in cross-wall deposition from its earliest stages; as the wall grows in, it becomes increasingly constricted in the pore region, finally assuming a torus-like configuration. Woronin bodies are shown to have a crystalline substructure and to originate in pouch-like membrane systems. Cross-walls from a 7- to 13-hr-old mycelium frequently show highly ordered structures in the vicinity of the pore. These structures may appear either as laminar stacks of discs to one side of the pore or as series of stubby concentric rings within the pore area itself. In the latter case, a mass of granular material is frequently seen plugging the pore. Other unusual organelles and inclusions in 7- to 13-hr hyphae are vesicles containing swirls of beaded or dilated membrane, membrane-enclosed rods, and stacks of unit membranes associated with spherical, electron-transparent vesicles.     

Structure and composition of tubular aggregates of skeletal muscle fibres

Unusual regions of densely packed membranous tubules known as tubular aggregates (TAs) have been observed in skeletal muscle fibres of mammals under numerous pathological conditions but also in health. Their causality is unclear. It is neither known whether TAs are destructive and should be treated or whether they have a compensating function in an endangered muscle. In spite of many similarities, the histochemical, immunocytochemical and ultrastructural characteristics of tubular aggregates do vary. Histochemistry provided an overall characteristic of TAs as membranous inclusions with a variety of enzymatic activities. Immunocytochemical evidence revealed that tubular aggregates contain miscellaneous proteins and that derive from membranes of sarcoplasmic reticulum and mitochondria. No evidence for the presence of contractile and cytoskeletal proteins in TAs was found. Ultrastructurally, TAs are characterized as more or less densely packed aggregates of vesicular or tubular membranes of variable forms and sizes that may contain amorphous material, filaments or inner tubules. Various reported types of tubular aggregates, namely, proliferating terminal cisterns, vesicular membrane collections, TAs with double-walled tubules, TAs with single-walled tubules, aggregates of dilated tubules with inner tubules, aggregates of tubulo-filamentous structures, filamentous tubules, riesentubuli, and related membranous structures including cylindrical spirals are sumarized and analyzed here in detail.     

Traffic of chitin synthase 1 (CHS-1) to the Spitzenkörper and developing septa in hyphae of Neurospora crassa: actin dependence and evidence of distinct microvesicle populations

We describe the subcellular location of chitin synthase 1 (CHS-1), one of seven chitin synthases in Neurospora crassa. Laser scanning confocal microscopy of growing hyphae showed CHS-1–green fluorescent protein (GFP) localized conspicuously in regions of active wall synthesis, namely, the core of the Spitzenkörper (Spk), the apical cell surface, and developing septa. It was also present in numerous fine particles throughout the cytoplasm plus some large vacuoles in distal hyphal regions. Although the same general subcellular distribution was observed previously for CHS-3 and CHS-6, they did not fully colocalize. Dual labeling showed that the three different chitin synthases were contained in different vesicular compartments, suggesting the existence of a different subpopulation of chitosomes for each CHS. CHS-1–GFP persisted in the Spk during hyphal elongation but disappeared from the septum after its development was completed. Wide-field fluorescence microscopy and total internal reflection fluorescence microscopy revealed subapical clouds of particles, suggestive of chitosomes moving continuously toward the Spk. Benomyl had no effect on CHS-1–GFP localization, indicating that microtubules are not strictly required for CHS trafficking to the hyphal apex. Conversely, actin inhibitors caused severe mislocalization of CHS-1–GFP, indicating that actin plays a major role in the orderly traffic and localization of CHS-1 at the apex.     

Electron microscopy of vesicular-arbuscular mycorrhizae of yellow poplar. II. Intracellular hyphae and vesicles.

Intracellular hyphae and vesicles in mycorrhizal roots of yellow poplar were examined by electron microscopy. An investing layer of host wall material and cytoplasm enclosed the endophyte within the cells. Young developing hyphae contained abundant cytoplasm and few vacuoles. As hyphae matured, they became highly vacuolated and accumulated carbohydrate (glycogen) and lipid reserves. Mature vesicles were engorged with lipid droplets, possessed a trilaminate wall and were also enclosed by host wall material and cytoplasm. Compared with uninfected cells, infected cortical cells showed an increase in cytoplasmic volume, enlarged nuclei, and a reduction of starch reserves. Host nuclei were always proximal to the hyphae during hyphal development and deterioration. While other cytoplasmic components of infected and uninfected cells were comparable large electron-dense bodies occurred in vacuoles of most cells containing hyphae. Deterioration of intracellular hyphae occurred throughout the samples examined. Septa separated functional and degenerating portions of the hyphae. Hyphal deterioration involved degeneration and ultimate disappearance of fungal cytoplasm as well as collapse of hyphal walls. Based on these observations, the authors hypothesize that deterioration of the endophyte may release significant quantities of mineral nutrients, via hyphal contents, which are absorbed by the host.     

Ultrastructure of Gum-resin Ducts in Cashew (Anacardium occidentale)

In Anacardium occidentale L., the gum-resin ducts in the primaryphloem of the stem develop schizogenously. Appearance of anintercellular space amongst the densely stained cells signalsthe initiation of a duct. During this process, the middle lamella,appears in places, as an electron-opaque area. The epitheialcells bordering the duct are oval and have convex inner tangentialwalls which are without any plasmodesmata, although they areabundant on radial and outer tangential walls. The cytoplasmof the epitheial cells is rich in rough endoplasmic reticulum(ER), free ribosomes, polysomes, mitochondria with swollen cristae,plastids with occasional osmiophilic inclusions, dictyosomesand vesicles. Osmiophilic material has been observed in thedilation of the cisternae of dictyosomes and also in vesicles.Sometimes, the osmiophilic material aggregates and forms largemembrane-bound globules. The globules fuse with the plasmalemmaat the inner tangential wall, and presumably the contents aredeposited in the space between the protoplast and the wall.This material passes through the loose matrix of the wall intothe duct. Some of the epitheial cells of the mature duct show‘dark’ cytoplasm, degraded organelles and occasionallyhigh vacuolation; ultimately they are lysed and their remainscollect in the duct. Anacardium occidentale L., cashew, gum-resin ducts, epithlial cell, dictyosome, osmiophilic material, ultrastructure     

BASIDIOSPOROGENESIS IN BOLETUS RUBINELLUS. I. STERIGMAL INITIATION AND EARLY SPORE DEVELOPMENT

Sterigmal initiation in Boletus rubinellus resembled hyphal tip growth. Four stages in early basidiospore development have been delineated based on gross morphology, and changes in wall layers and cytoplasm. Changes in wall layers and cytoplasm during spore development were stage-specific. During Stage 1 the spore wall consisted of two layers identical to those of the sterigmal wall with occasional pellicle remnants on the outer surface. The onset of wall differentiation began in Stage 2, and during Stage 3 wall layers characteristic of the mature spore developed. At Stage 4 there was a pronounced gradient in wall thickness from the apex to the base of the spore. Small vesicles (30–60 nm diam) were uniformly distributed in the cytoplasm of spherically enlarging spores (Stage 2), but during spore elongation (Stages 3 and 4) numerous larger vesicles as well as small vesicles aggregated at the spore apex. A variety of cytoplasmic organelles entered the spore during Stage 3; however, migration of storage materials and the nucleus to the spore did not occur until late basidiospore development. The hilar appendix body developed in the earliest spore primordium and persisted until Stage 3. Development of wall layers and their differential thickening, distribution of vesicles, and probable function of the hilar appendix body are discussed with reference to the control of spore shape. Systematic implications of the data are considered.     

Fine Structure of Blue-Green Algae and the Cells Lined along the Endophyte Cavity in the Coralloid Root of Cycas

The ultrastructure of vegetative cells of blue-green alga, Anabaena cycadae, in the coralloid root of Cycas revoluta has the general characteristics of the cyanophycean cells. Their heterocysts are characterized by heavy envelope deposition, well developed pore channel with its plug, absence of large granules as inclusions and reduced and flattened photosynthetic thylakoids. By these characteristical features, the frequency of heterocysts occurring in this algal population of the coralloid root may be estimated to ca. 40%. This high heterocyst frequency is a sign of relatively high activity of nitrogen fixation in this symbiont. The ultrastructure of the cells lined along the endophyte cavity in the coralloid root shows that they have the function to maintain vigorous nutritional transport in short distance. These cells are especially characterized by the presence of numerious outgrowths on the cell wall into the endophyte cavity. Correspondingly, there are abundant mitochondria, dictyosomes and numerious vesicles in the cytoplasm. The plasma membrane becomes tortuous along the cell wall and many secretory granules are present between the plasma membrane and cell wall in the cytoplasm amyloplasts and starch granules also occur constantly. The ultrastructure observed above indicates the fact that there is sound structural basis for the metabolic relationship between the host cells and the symbiont.     

Composition and Ultrastructure of Streptomyces venezuelae

Streptomyces venezuelae is a filamentous bacterium with branching vegetative hyphae embedded in the substrate and aerial hyphae bearing spores. The exterior of the spore is inlaid with myriads of tiny rods which can be removed with xylene. The spore wall is approximately 30 nanometers thick. Occasionally, it can be seen that the plasma membrane and the membranous bodies within a spore are connected. The spore's germ plasm is not separated from the cytoplasm by a nuclear envelope. The cell walls of the vegetative hyphae, which are about 15 nanometers thick, are structurally and chemically similar to those of gram-positive bacteria. The numerous internal membranous bodies, some of which arise from the plasma membrane of the vegetative hypha, may be vesicular, whirled, or convoluted. Membranous bodies are usually prominent at the hyphal apices and are associated with septum formation. The germ plasm is an elongate, contorted, centrally placed area of lower electron density than the hyphal cytoplasm. The spores differ from the vegetative hyphae, not only in fine structure, but also in the arginine and leucine contents of their total cellular proteins.     

Electron microscopic observations of cell regeneration from cultured protoplasts ofAmmi visnaga

Summary Protoplasts ofAmmi visnaga initiated cell wall formation within 2 days in culture; after 13 days the new cells were enclosed by a cell wall similar to the walls on the original cultured cells. Budding occurred in protoplasts with little or no detectable cell wall. No evidence was obtained for direct participation of any organelle in cell wall formation. The cytoplasm of regenerating cells contained numerous organelles and appeared typical of actively growing plant cells; they were easily distinguished from degenerate cells and protoplasts. While coated vesicles were common, spiny vesicles occurred in only a few cells. Sustained cell division yielded multicellular aggregates. Multinucleate protoplasts, formed by spontaneous fusion, did not divide; some of them contained annulate lamellae with few pore complexes.Supported by the National Research Council of Canada, Grant A6304.     

Ultrastructure of Aerial Hyphae in Linderina pennispora

Ultrathin sections of the aerial hyphae of Linderina pennispora,fixed in glutaraldehyde, show the wall to be composed of anouter electron-dense layer and an inner less-dense one. Thefully developed septum is plugged by electrondense material.A similar septum exists at the base of the pseudophialide. Cytoplasmiccontinuity on both sides of the septum is maintained by theplasmalemma which passes through the septal pore around theperiphery of the plug. Membrane-lined vesicles may occur inthe wall, between the wall and the plasmalemma and internalto the plasmalemma. Ribosome-like particles are numerous inthe cytoplasm and endoplasmic reticulum is virtually absent.     

ULTRASTRUCTURE OF CYST FORMATION IN OCHROMONAS TUBERCULATA (CHRYSOPHYCEAE)1

The cysts (statospores) of Ochromonas tuberculata Hibberd are produced within a cytoplasmic silica deposition vesicle (SDV) whose membrane (silicalemma) appears to be formed by the coalescence of golgi vesicles. Silica is first deposited as small nodules and the collar and spines develop by centrifugal growth only after a complete but still thin wall has been laid down. Small vesicles appear to be attached to the SDV only in the region overlying the developing collar; a cap of radially arranged, moderately electron-dense material occurs at the tip of the growing spines. The cyst pore is formed at the anterior end of the flagellate cell, by lack of silica deposition over a small region of the SDV and rupture of the SDV and other membranes crossing this region. When the cyst wall is complete, an extracystic plug is formed in the pore, resulting in the loss of some extracystic cytoplasm and the plasmalemma, and the inner SDV membrane becomes the functional plasmalemma. The plug develops first by coalescence with the cell membrane of golgi-derived vesicles containing dense but apparently nonsiliceous spicules surrounded by amorphous material. During later stages of plug formation only fibrous material is deposited, some of which may be extruded through the pore forcing out some of the spiculate component. Scanning electron micrographs of the mature wall show it is smooth except for the concentrically wrinkled inner face of the flared collar and that the real pore diameter is only ca. half that of the collar. At germination the plug completely disappears in an unknown way and a single cell, similar to a normal vegetative cell emerges through the pore. Chrysophycean cyst formation generally resembles cell wall formation in diatoms, but differs in some details.