Wall texture in the spore of a vesicular-arbuscular mycorrhizal fungus

Summary InGlomus epigaeum Daniels and Trappe, a vesicular-arbuscular mycorrhizal fungus, the mature spore has a complex multi-layered wall containing a regular pattern of wall subunits.The outer wall (2–4 m thick) consists of a simple layer of parallel microfibrils. The inner wall (5–6 m thick) is built from two layers possessing different organization. The innermost layer, near the plasmalemma has a texture of apparently dispersed fibrils, whereas the second layer is regularly organized with an arced texture. Ten to twelve bundles of fibrils connected by apparently bow-shaped fibrils are consistently observed. The appearance of this arced organization depends on the section plane and on the angle of observation in the electron microscope as confirmed by tilting experiments. Wall subunits are evident as straight electron transparent fibrils; particularly well-defined in negatively stained frozen sections: their diameter is about 3.5nm.The regular pattern of wall subunits in this fungal cell wall is compared with the textures shown by cellulose fibrils in algae or higher plants and by chitin fibrils in arthropod cuticle.Research work supported by CNR, Italy. Special grant I.P.R.A.—Sub-project 1. Paper No. 55.     

Architecture of the cell wall of a green alga,Oocystis apiculata

Summary The cell wall of a green alga,Oocystis apiculata, was visualized by electron microscopy after preparation of samples by rapid-freezing and deep-etching techniques. The extracellular spaces clearly showed a random network of dense fibrils of approximately 6.4 nm in diameter. The cell wall was composed of three distinct layers: an outer layer with a smooth appearance and many protuberances on its outermost surface; a middle layer with criss-crossed cellulose microfibrils of approximately 15–17 nm in diameter; and an inner layer with many pores between anastomosing fibers of 8–10 nm in diameter. Both the outer and the inner layer seemed to be composed of amorphous material. Cross-bridges of approximately 4.2 nm in diameter were visualized between adjacent microfibrils by the same techniques. The cross-bridges were easily distinguished from cellulose microfibrils by differences in their dimensions.     

The fine structure of Micrococcus radiophilus and Micrococcus radioproteolyticus

The radiation resistant bacteria Micrococcus radiophilus and M. radioproteolyticus were studied by thin sectioning and freeze-etching techniques and the two species were found to be similar in the fine structure. The only significant difference was in the appearance of the surfaces of the cell walls in freeze-etched preparations.Since the two species, together with M. radiodurans, possess a unique cell wall structure and a cell wall peptidoglycan, which is different from that of other micrococci and Gram-positive cocci, it is recommended that they be reclassified into a new genus.     

Reassembly of the Regularly Arranged Subunits in the Cell Wall of Lactobacillus brevis and Their Reattachment to Cell Walls

The reassembly of tetragonally arranged subunits in the cell wall of Lactobacillus brevis and the reattachment of the subunits to cell wall fragments were investigated by electron microscopy. The subunits dissociated from the cell wall with guanidine hydrochloride (GHCl) reassembled into the same regular array as seen in the native cell wall after dialysis against neutral buffer even in the absence of specific cations. The subunits could also reattach to the cell wall fragments from which they had been removed by treatment with GHCl, sodium dodecyl sulfate or cold trichloroacetic acid but not to those treated with hot formamide. Heterologous reattachment of the subunits occurred on cell wall fragments obtained from L. fermentum but not on those from L. plantarum or L. casei subsp. casei. On the basis of these observations and chemical analyses of the cell wall fragments, the subunits of L. brevis appeared to be bound by hydrogen bonds to a neutral polysaccharide moiety in the cell wall but not to peptidoglycan or teichoic acid.     

Applying the digital image correlation method to estimate the mechanical properties of bacterial biofilms subjected to a wall shear stress

A digital image correlation (DIC) method was applied to characterize the mechanical behavior of Pseudomonas aeruginosa biofilms in response to wall shear stress using digital video micrographs taken from biofilm flow cells. The appearance of the biofilm in the transmitted light photomicrographs presented a natural texture which was highly conducive to random encoding for DIC. The displacement fields were calculated for two biofilm specimens. The DIC method concurred with previous analysis showing that biofilms exhibit viscoelastic behavior, but had the advantage over simple length measurements of longitudinal strain that it could precisely measure local strains in length (x) and width (y) within biofilm clusters with a 2 μm resolution as a function of time and wall shear stress. It was concluded that DIC was more accurate at measuring elastic moduli than simple length measurements, but that time-lapse 3D images would enable even more accurate estimates to be performed.     

Cadmium replaces calcium in the cell wall ofUlva lactuca

Electron microscopy, in conjunction with X-ray microanalysis, was used to investigate the effects of exposure to cadmium on the elemental composition of the macroalgaUlva lactuca. The cell wall was the only region of the cell to show any marked change in chemical composition as a result of exposure to cadmium, with less calcium evident in cadmium-treated thallus compared with untreated thalli. The cell wall ofU. lactuca is a complex structure made up of polysaccharides consisting of many-branched chains composed mostly of rhamnose and galactose subunits. Some of the hydroxyl groups on the subunits are substituted by sulphate groups. Borate is associated with the rhamnose subunits, which contain no sulphate groups, and calcium binds to borate, cross-linking the rhamnose groups. The borate-calcium complex adds rigidity to the cell wall; the replacement of calcium by cadmium will, therefore, influence the rigidity of the thallus. The ecological significance of this work is discussed with respect to the ability of the alga to withstand grazing or emersion.     

Observations on the ultrastructure of the thickened sieve cell wall inPinus strobus L.

Summary The thickened sieve cell wall of white pine is shown to comprise a crossed-helical polylamellate structure in which the predominant microfibrillar orientation is greater than 45° with respect to the cell axis. The previously reported observation that microfibrils may be oriented other than parallel to the plane of the cell wall is disputed and it is demonstrated that such an appearance may derive from appropriately oblique sectioning of the wall.     

The glucuronoxylans and the helicoidal shift in cellulose microfibrils in linden wood: Cytochemistryin muro and on isolated molecules

Summary In fibres of wood, the classical S₁ and S₂ layers are connectedvia a transition zone where a helicoidal texture occurs. In order to understand the actual mechanism of cellulose microfibril rotation in this zone, the study of relationship between cellulose and matrix was undertaken cytochemically at the ultrastructural level.Glucuronoxylans,i.e., the main hemicellulose component of hardwood, were studied in cell walls of linden tree. Xylanase-gold complexes were used as a new cytochemical tool to directly and specifically label glucuronoxylans within the wall of fibres. Subtractive localization (KOH or DMSO extraction and PATAg test or shadowing) associated with chemical analysis was carried out as control. The study of isolated glucuronoxylan molecules was undertaken in parallel.Both from direct (xylanase-gold labeling) and indirect techniques (extractions), glucuronoxylans appear preferentially concentrated in the transition zone which overlaps the layers S₁ and S₂. A comparison between KOH and DMSO extraction indicates a difference in accessibility of glucuronoxylans distributed across the whole wall and those located in the transition zone. Isolated molecules have a rodlike aspect and show a tendancy to spatially organize in parallel alignment. Cytochemical labeling of the isolated molecules concerns covalent linkages, vic-glycol groups and acid side groups along the main chain.The preferential localization indicates that in the helicoidal zone glucuronoxylans constitute a thick matrix embedding the cellulose microfibrils in the course of rotation. This data leads to a discussion of how these localized matrix molecules could intervene in the assembly and the twisted morphogenesis of the fibre cell wall.     

Novel actin ring structure in sporulation of Zygosaccharomyces rouxii

The filamentous actin (F-actin) during sporulation of Zygosaccharomyces rouxii was visualized with rhodamine-phalloidin, and then the behavior was observed using confocal laser scanning microscopy. During spore formation, we found a novel actin ring structure that has not been reported in other yeasts and molds in sporulation. The ring surrounded each meiotic nucleus at the peripheral regions of spores. Three-dimensional observation suggested that the ring was not an artificial structure produced by spherical structure sectioning. The period and location of the rings appearance suggest that the ring may have some relation to the spore membrane or wall development. In addition, this ring structure was more stable than other F-actin structures against latrunculin A, an F-actin disrupting agent.     

THE CELL WALL OF COSMARIUM BOTRYTIS12

The cell wall of Cosmarium botrytis was studied through the use of the freeze-etch technique. The cell wall consists of many thin layers. Fracturing along one layer reveals the positioning of the wall sculpturing, wall pores, and wall microfibrils. The individual microfibrils are grouped together in bands of parallel oriented fibrils. The different bands of parallel microfibrils were apparently arranged at random angles with regard to each other. Small particles may also be present in the cell walls. The cell wall pore unit of Cosmarium botrytis was studied through the use of scanning, freeze-etching, and thin sectioning techniques. The pore sheaths, on the outside of the cell wall, form a collar around the mouth of each pore. The pore sheath is composed of needle-like fibrils radiating outward from the pore. A pore channel traverses the cell wall and leads to a complex pore bulb region between the cell wall and the plasmalemma. The pore bulb contains many small fibrils which radiate toward the plasmalemma from a number of net-like fibril layers which in turn merge into a very electron dense region near the base of the pore.     

Electron microscope investigation of sieve-element ontogeny and structure inUlmus americana

Summary During advanced stages of sieve-element differentiation inUlmus americana L., dispersal of the P-protein (slime) bodies results in formation of a peripheral network of strands consisting of aggregates of P-protein components having a striated, fibrillar appearance. The tonoplast is present throughout the period of P-protein body dispersal. Perforation of the sieve plates is initiated during early stages of P-protein body dispersal.Small P-protein bodies consist of tubular components, most of which measure about 180 Å in diameter. With increase in size of the P-protein bodies narrower components appear. At the time of initiation of P-protein body dispersal, most of the components comprising the bodies are of relatively narrow diameters (most 130–140 Å) and have a striated, fibrillar appearance. Both wide and narrow P-protein components are present throughout the period of sieve-element differentiation and in the mature cell as well, and a complete intergradation in size and appearance exists between the two extremes. Both extremes of P-protein component have a similar substructure: an electron-transparent lumen and an electronopaque wall composed of subunits, apparently in helical arrangement. The distribution of P protein in mature sieve elements was quite variable.The parietal layer of cytoplasm in matureUlmus sieve elements consists of plasmalemma, endoplasmic reticulum cisternae in two forms (as a complex network closely applied to the plasmalemma and in stacks along the wall), mitochondria, and plastids.     

Fine structure of swarmers of Cladophora and Chaetomorpha

Summary Naked swarmers of Cladophora have been collected and wall synthesis and development have been followed using the techniques of freeze-etching and sectioning. Swarmers frozen after 9 hours liberation have lost their flagella, developed the characteristic fibrous layer and show the initial stages of wall production. Both the first formed (randomly oriented) and the later (more ordered) microfibrils appear to have a distinct granular texture. Occasionally linear arrays of granules up to 4 m long may be seen. After 5 days settling a thick wall composed of almost transversely oriented microfibrils is present and a rhizoid is pushed out. Also characteristic of this stage is the central localisation of cell components and peripheral vacuolar distribution. Longitudinally oriented microtubules also reappear at this stage having been absent during carlier wall formation.A possible relationship between the cortical microtubules of the motile swarmer and the development of the fibrous layer is suggested.     

AN ELECTRON-MICROSCOPIC STUDY OF SPORE WALL STRUCTURE IN THE MUSCI

Spores of 9 species of mosses were embedded in plastic and sectioned for light and electron microscopy. The presence of 4 spore wall layers was found to be of common occurrence. A comparison with pollen wall structure was made. Variations in the layers contributing to the ornamentation were noted. A multi-layered intine was observed in the genus Archidium. The results indicate that thin sectioning should yield additional sporoderm characteristics of systematic value.     

Cell wall assembly in vitro from Chlamydomonas reinhardi

Summary The conditions for in vitro dissociation and reassembly of the cell wall of Chlamydomonas reinhardi have been investigated. The cell wall dissociates in aqueous 8 M lithium chloride to two homogeneous components (6.8 S₂₀w and 9.3 S₂₀w)and dialysis of the cell wall subunits against water causes reassembly of a product having the same overall morphology and chemically similar to the original cell walls. Cell wall subunits in 8 M lithium chloride alone do not reassemble on dialysis against water to form cell walls but a nucleation centre has to be provided.     

Ultrastructure of square bacteria from a brine pool in Southern Sinai.

The square bacterium discovered by Walsby (Nature [London] 283:69-71, 1980) has been shown to possess the ultrastructural features of a typical halophile. The cell wall is comprised of regularly arranged subunits demonstrated by thin sectioning, shadowed replicas, and negative staining. Optical diffraction confirms the existence of both hexagonal and tetragonal arrangements of the cell wall subunits and also of different lattice constants and suggests a mixed population of bacteria.     

Genetic immobilization of cellulase on the cell surface of Saccharomyces cerevisiae

We tried genetically to immobilize cellulase protein on the cell surface of the yeast Saccharomyces cerevisiae in its active form. A cDNA encoding FI-carboxymethylcellulase (CMCase) of the fungus Aspergillus aculeatus, with its secretion signal peptide, was fused with the gene encoding the C-terminal half (320 amino acid residues from the C terminus) of yeast α-agglutinin, a protein involved in mating and covalently anchored to the cell wall. The plasmid constructed containing this fusion gene was introduced into S. cerevisiae and expressed under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter from S. cerevisiae. The CMCase activity was detected in the cell pellet fraction. The CMCase protein was solubilized from the cell wall fraction by glucanase treatment but not by sodium dodecyl sulphate treatment, indicating the covalent binding of the fusion protein to the cell wall. The appearance of the fused protein on the cell surface was further confirmed by immunofluorescence microscopy and immunoelectron microscopy. These results proved that the CMCase was anchored on the cell wall in its active form. Received: 19 March 1997 / Received revision: 19 May 1997 / Accepted: 1 June 1997     

Investigation of structure and antigenic capacities of Thermococcales cell envelopes and reclassification of "Caldococcus litoralis" Z-1301 as Thermococcus litoralis Z-1301

Fourteen strains of hyperthermophilic organotrophic anaerobic marine Archaea were isolated from shallow water and deep-sea hot vents, and four of them were characterized. These isolates, eight previously published strains, and six type strains of species of the order Thermococcales were selected for the study of cell wall components by means of thin sectioning or freeze-etching electron microscopy. The cell envelopes of most isolates were shown to consist of regularly arrayed surface protein layers, either single or double, with hexagonal lattice (p6) symmetry, as the exclusive constituents outside the cytoplasmic membrane. The S-layers studied differed in center-to-center spacing and molecular mass of the constituent protein subunits. Polyclonal antisera raised against the cells of 10 species were found to be species-specific and allowed 12 new isolates from shallow water hot vents to be identified as representatives of the species Thermococcus litoralis, Thermococcus stetteri, Thermococcus chitonophagus, and Thermococcus pacificus. Of the 7 deep-sea isolates, only 1 was identified as a T. litoralis strain. Thus, hyperthermophilic marine organotrophic isolates obtained from deep-sea hot vents showed greater diversity with regard to their S-layer proteins than shallow water isolates. Received: February 5, 1999 / Accepted: May 11, 1999     

The effect of protease digestion (in situ) on the slime substance of mature sieve tubes

Summary The sieve tube slime of Tetragonia and Lycopersicum petiolar phloem was subjected to proteolytic enzymes both before and after embedding and sectioning. Subsequent electron microscopical examination confirmed that the fibrillar material is protein in nature, and provided evidence that the protein is acidic rather than basic.     

SPORE WALL ULTRASTRUCTURE IN THE LIVERWORT ATHALAMIA HYALINA

Mature spores of Athalamia hyalina (Marchantiales, Cleveaceae) were examined with both scanning and transmission electron microscopes. Single, hollow, dome-like projections, sometimes having small pores and a coarsely granular surface texture, stud the spore surface, usually in a pattern of concentric circles. In section, the spore wall has an intine and two-layered exine. Intine-like material separates some lamellae of the inner exine, which is joined to the outer exine around the dome bases. Inner exine lamellae are composed of thin (5–6 nm), closely parallel membrane-like subunits. The outer exine is formed from a single large highly modified and doubly-coated lamella, the undulations of which form the surface domes. Dome cavities often are filled with a loose network of granular material.     

Ultrastructural study of the relationship between generative and vegetative cells inMagnolia ×soulangeana Soul.-Bod. pollen grains

Summary InMagnolia ×soulangeana pollen grains the generative cell (GC) does not become totally free within the vegetative cell (VC), at least until the pollen tube emergence. Due to a deviation in its detachment process from the sporoderm, the opposing ends of the VC plasmalemma do not fuse themselves when the GC moves away from the intine. Consequently, the interplasmalemmic space surrounding the GC does not become isolated but rather maintains continuity with the sporoderm through a complex formation that we have called plasmalemmic cord. The real existence of this formation was confirmed through serial sectioning showing the plasmalemmic cord to consist of the VC plasmalemma. In its initial portion it is occupied by a reasonably accentuated wall ingrowth of the inner layer of the intine (intine 3). In the remainder portion, neither of the cytochemical tests used in this work have revealed the presence of a significant amount of wall material. However, ultrathin sections of samples processed either chemically or by cryofixation showed the existence of an intricate system of tubules and vesicles, some of which are evaginations of the VC plasmalemma. The hypothesis that the plasmalemmic cord may have a role in the complex interactions between the two pollen cells is discussed.