THE MICROFIBRILLAR STRUCTURE OF THE CELL WALLS OF THE FILAMENTOUS FUNGUS, Allomyces

Cell walls of the fungus, Allomyces, were isolated by chemical procedures, using either potassium permanganate oxidation or glacial acetic acid-hydrogen peroxide treatment followed by dilute mineral acid. The structure of the treated walls was investigated by means of electron microscopy and electron diffraction analysis which showed that rhizoidal walls were especially suitable for observation. Chitin microfibrils exist in the extreme tips of rhizoidal walls, and tend to lie in a preferred longitudinal orientation. Older rhizoidal wall segments show a crossed fibrillar structure under a thin layer of short randomly arranged microfibrils. In the possession of systems of crossed fibrils these walls are like the cell walls of certain green algae. Walls of branch rhizoidal filaments were observed in the early stages of development, in which case the observed microfibrillar orientations are such that it is possible to envisage their origin from pre-existing fibrils that have passively reoriented. With respect to the continued growth of the filaments, however, it is difficult to explain the observed microfibrillar arrangements in terms of the "multi-net" theory. Hyphal walls usually show two layers, the outer consisting of microfibrils arranged randomly, and the inner consisting of well oriented microfibrils running parallel with the longitudinal axis of the hypha. The oriented inner layer appears to be similar in structure to the secondary wall of the Phycomyces sporangiophore.     

Ultrastructural studies on the cell walls in Fusarium sulphureum.

The cell walls of Fusarium sulphureum have a microfibrillar component that is randomly arranged. X-ray-diffraction diagrams of the microfibrils are consistent with a high degree of crystallinity and show that they are chitin. The chitin microfibrils of the peripheral walls envelop the hyphal apex and extend across the septae. During the first 8h in culture, the conversion of conidial cells to chlamydospores is evidenced by a swelling of the cells and the original microfibrils remain randomly arranged. Within 24h new wall material is deposited as the cells expand and the wall thickens. The new microfibrils are indistinguishable from those of the original conidial cells. After 3 days in culture, the chlamydospores are fully developed and have the characteristic thick wall which is a continuous layer of randomly arranged microfibrils. Chlamydospores maintained in a conversion medium for 8 days have microfibrils identical with those in 3-day-old cultures; thus a further change in the microfibril orientation did not occur during that period. Alkaline hydrolysis of the walls removes most of the electron-dense staining constituents from the inner wall layer and leaves the outer wall layer intact. This treatment also reveals some of the wall microfibrils. An additional treatment of the walls with HAc/H2O2 completely removes the wall components that react positively to heavy metal stains. The results are discussed in relation to the structure of other fungal cell walls.     

Structure and differentiation of the cell wall of Phytophthora palmivora: Cysts,hyphae and sporangia

Summary Walls from cysts, hyphae and sporangia of Phytophthora palmivora consist chiefly (ca. 90% dry wt) of -glucans with 1,3-, 1,4- and 1,6-links. The glucans are predominatly -1,3-linked but there are significant differences in the relative proportion of 1,3-, 1,6- and 1,4-linked glucosyl residues among the three wall types. There are also differences in protein content, susceptibility to degradation by various -glucanases, and surface texture. The isolated cyst wall consists solely of a thin fabric of long, tightly interwoven, randomly oriented microfibrils. Both inner and outer surfaces of the cyst wall are distinctly microfibrillar. The hyphal wall has two different textures; the internal surface is distinctly microfibrillar while the external surface is non-fibrillar. In a germinated cyst, there is a zone of demarcation where the microfibrils of the cyst wall disappear into the smooth outer texture of the germ tube wall. An exo--1,3-glucanase preferentially removed the amorphous material of the outer surface of the germ tube leaving exposed a continuous microfibrillar fabric from cyst to hyphal tube. Conceivably, the textural and structural differentiation of the cell wall may play a decisive role in cellular morphogenesis.     

A LIGHT- AND ELECTRON-MICROSCOPIC SURVEY OF ALGAL CELL WALLS. I. PHAEOPHYTA AND RHODOPHYTA

Dawes , Clinton J., Flora M. Scott , and E. Bowler . (U. California, Los Angeles.) A light- and electron-microscopic survey of algal cell walls. I. Phaeophyta and Rhodophyta. Amer. Jour. Bot. 48(10): 925–934. Illus. 1961.—An introductory survey of the structure of the cell walls of brown, red, and green algae, as seen under light and electron microscopes, has been completed. In the present paper (Part I) the structure of the thalli of the Phaeophyta and Rhodophyta is compared, and the occurrence of intercellular spaces, pitting, and microfibrillar patterns is discussed. A detailed comparison of the cell-wall structure and growth of a brown alga, Dictyota flabellata, and of a red alga, Helminthocladia californica, is also presented. In Dictyota, typical of the brown algae, the microfibrillar pattern in the apical cells and in the adjacent cells of the thallus tip is reticulate. In mature cells, the microfibrils are dominantly parallel in orientation. Pits, which are fields of closely set pores, are distinctive. The microfibrils in the pit areas are masked by non-fibrillar material. Helminthocladia, with a cell wall characteristic of the red algae, differs from Dictyota in that the microfibrillar pattern is reticulate at all ages of the cell and throughout the thallus. In the pit areas, the microfibrils are not masked by amorphous material. Pit connections, characteristic of the Florideae, can be divided into 2 major groups. Either the pit connection is an open channel between 2 adjacent cells, or it is composed of numerous plasmodesmata traversing a continuous, loose, microfibrillar wall. The techniques of the survey are emphasized in that fragmented cell walls were studied, and, also, chemically cleared material was constantly compared with fresh material under light and electron microscopes. It is concluded that the cell wall, as a taxonomic character, is of value only in delimiting the Phaeophyta and Rhodophyta.     

Colchicine and microfibril orientation

Summary Investigations on the mechanism of orientation of the cellulose microfibrils of the green algaOocystis solitaria have been carried out. This organism demonstrates easily observable and highly ordered microfibrils in its wall, which are arranged parallel to one another and regularly alternate at 90 from layer to layer of which there are approximately 30. During the entire wall development, and always parallel to one of the microfibril directions, are microtubules lying in the cortical cytoplasm. In the presence of 10^–2 M colchicine, microtubules are no longer detected and the typical cell wall pattern is not developed. The possible role of microtubules in the orientation of cellulose microfibrils is briefly discussed.     

Observations on the Fine Structure of Plant Cell Walls III. The Sieve Tube Wall in Cucurbita

The sieve tube wall in Cucurbita was examined in ultra-thinsections of petioles treated in different ways for the removalof non-cellulosic wall components. The sections were stainedwith permanganate. The microfibrillar components of the wallare arranged in concentric lamellae. The earliest (outermost)part of the wall is similar to that of ordinary parenchyma inhaving its lamellae composed of thinly-distributed microfibrilsreadily separated from one another by certain treatments suchas pectinase extraction. In the characteristically-thickenedinner (nacreous) layer the microfibrils are very densely packedand the lamellae do not separate readily. The microfibrils inthis layer of the wall are very close to transverse and the‘crossed fibrillar’ orientation is not easily discernible.     

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.     

Microfibrillar Structure, Cortical Microtubule Arrangement and the Effect of Amiprophos-methyl on Microfibril Orientation in the Thallus Cells of the Filamentous Green Alga, Chamaedoris orientalis

Microfibrillar structure, cortical microtubule orientation andthe effect of amiprophos-methyl (APM) on the arrangement ofthe most recently deposited cellulose microfibrils were investigatedin the marine filamentous green alga, Chamaedoris orientalis.The thallus cells of Chamaedoris showed typical tip growth.The orientation of microfibrils in the thick cell wall showedorderly change in longitudinal, transverse and oblique directionsin a polar dependent manner. Microtubules run parallel to thelongitudinally arranged microfibrils in the innermost layerof the wall but they are never parallel to either transverseor obliquely arranged microfibrils. The ordered change in microfibrilorientation is altered by the disruption of the microtubuleswith APM. The walls, deposited in the absence of the microtubules,showed typical helicoidal pattern. However, the original crossedpolylamellate pattern was restored by the removal of APM. Thissuggests that cortical microtubules in this alga do not controlthe direction of microfibril orientation but control the orderedchange of microfibril orientation. Amiprophos-methyl, Chamaedoris orientalis, coenocytic green alga, cortical microtubule, microfibrillar structure, tip growth     

Cell wall structure of the marine fungus,Atkinsiella dubia

Summary Cell walls of the marine Oomycete, Atkinsiella dubia were prepared and an analysis of the wall constituents was made. The walls contained approximately 80% polysaccharides and 14% proteins along with small quantities of lipid and ash. The carbohydrate fraction was composed primarily of glucan along with 1.8% glucosamine and a trace of galactosamine. An analysis of the amino acid composition of the protein fraction showed the presence of 18 identified amino acids including a surprisingly high (20% of total amino acids) hydroxyproline content. The polysaccharide fractions of the wall were mostly glucans with solubility properties similar to those reported for other Oomycetes. As anticipated, the glucans of mechanically isolated walls were virtually identical to those prepared from chemically isolated walls. The minor glucan component, cellulose, was found to occur in the form of poorly crystalline cellulose I As expected, electron microscopy of wall specimens showed microfibrillar and amorphous regions. It was stressed that Atkinsiella walls, like those of other Oomycetes, contain large quantities of -13 and -16 linked glucan along with a smaller amount of cellulose.     

Feinstruktur von Zellwand und Plasmamembran beiMicrasterias denticulata Bréb. nach Gefrierätzung

Zusammenfassung Gefrierätz-Untersuchungen an rasch eingefrorenen sonst aber unvorbehandelten Zellen vonMicrasterias denticulata ergaben, daß die Sekundärwand aus einer fibrillenfreien Außenschicht, die möglicherweise Lipid-Charakter aufweist, und einer Fibrillenschicht aufgebaut ist.Jede der zu Bändern zusammengefügten Mikrofibrillen (mit Durchmessern bis zu 300 Å) der Fibrillenschicht besteht aus zahlreichen Elementarfibrillen, die einen ungefähren Durchmesser von 40 bis 50 Å aufweisen. In tieferen Lagen der Fibrillenschicht laufen die Mikrofibrillen um den Porus herum, während die äußerste Lage der Fibrillenschicht vom Porus durchbrochen wird. Nur diese Lage wird sekundär perforiert; der übrige Porenkanal entsteht gleichzeitig während des Sekundärwandwachstums.Die Bruchfläche der Plasmamembran ist mit zahlreichen, statistisch verteilten Partikeln bedeckt und weist Eindellungen unter jedem Porus auf. Am Rande dieser Eindellungen sind Membranlöcher festzustellen, die als Fusionsorte von schleimenthaltenden Vesikeln und der Plasmamembran gedeutet werden.

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Fine-structure of the cell wall and plasma membrane inMicrasterias denticulata bréb. after freeze-etching

Summary Cells of the algaMicrasterias denticulata, frozen in their growth medium without further pretreatment, have been examined by means of freeze-etching. The outer surface of the secondary wall as well as the side walls of the slime pores are covered with a continuous thin, fibril free layer with cleaving properties resembling those of multiple lipid bilayers. Elementary fibrils (40 to 50 Å in diameter) are shown to make up the microfibrils (200 to 300 Å in diameter), 2 to 15 of which may aggregate laterally to form the characteristic bands of secondary wall fibrils. While the microfibrils in the inner layers of the secondary wall appear to circumvent the pores those belonging to the outermost layer seem to be frequently disrupted by the pore apparatus. These observations suggest that the main part of the pore channel is formed during secondary cell wall formation. Only the outermost fibrillar layer of the secondary wall seems to be perforated after its deposition.The cleaved face of the plasma membrane carries numerous randomly distributed particles and is marked by large depressions underlying each of the pores. In the rim areas of these depressions the membrane is frequently interrupted by small holes, which are interpreted as points of fusion between the slime secreting vesicles and the plasma membrane.

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Die elektronenmikroskopischen Untersuchungen wurden in den Biological Laboratories der Harvard University, Cambridge, U.S.A., durchgeführt. Die Arbeit wurde durch die Deutsche Forschungsgemeinschaft und durch einen Health Science Advancement Award Grant 5-SO 4-FRO 6084 an die University of Colorado unterstützt.     

Observations on the Fine Structure of Plant Cell Walls II. The Microfibrillar Framework of the Parenchymatous Cell Wall in Cucurbita

The microfibrillar framework of parenchymatous walls in Cucurbitawas observed in petioles treated so as to remove various non-cellulosiccell wall components. Such extraction typically results in separationof the microfibrillar components into concentric lamellae. Thenumber and thickness of these lamellae vary according to theage and type of cell wall. The microfibrils appear to be orientatedwithin the plane of their lamellae but the orientation may varyin successive lamellae, and in many walls the crossed polylamellatecondition was detected. The collenchyma—and the outerepidermal cell walls show an alternation of lamellae with almostvertical microfibrils with those with a practically transverseorientation. In ordinary parenchymatous walls the alternationis not so extreme and is revealed only by the occasional presenceof the ‘herring bone pattern’ in non-radial sections.As a rule the lamellae are continuous around the circumferenceof a cell though individual lamellae may vary in thickness andsometimes appear to ‘fade out’. The present observationssuggest that growth of the primary wall occurs by depositionof microfibrils in successive lamellae thus confirming the basicpremise of the multinet theory of growth.     

STRUCTURE AND COMPOSITION OF THE RESISTANT SPORANGIAL WALL IN THE FUNGUS ALLOMYCES

The fine structure and chemical composition of the wall of resistant sporangia of Allomyces neo-moniliformis were investigated. Studies with the electron microscope showed that the wall is approximately 1.3 μ in thickness and is of complex construction. It consists essentially of three parts: a five-layered outer wall, two layers of “cementing substances,” and a single-layered inner wall. The presence of a highly convoluted cell membrane was also demonstrated. Six structural components were found to make up the walls of the resistant sporangia: glucose, glucosamine, chitin, melanin, protein, and lipids. Comparison of the structure and composition of the walls of resistant sporangia with the walls of hyphae and zoospores of Allomyces as reported by other investigators showed that, while the structure is very different, the composition is quite similar with only melanin and lipids apparently being absent from the zoospore and hyphal walls.     

Fine structural observations on the epidermis

Summary The organization of the wall of epidermal cells in the petiole of species of Apium, Eryngium, Rumex, and Abutilon as well as that of the epidermis of Avena coleoptile has been investigated. The outer and inner tangential walls consist of layers in which the cellulose microfibrils are oriented alternately parallel or transverse to the longitudinal cell axis. This organization resembles that previously described for collenchyma cell walls (Wardrop, 1969; Chafe, 1970). On the radial (anticlinal) walls the orientation of the microfibrils is transverse and these appear continuous with the layers of transverse orientation of the outer and inner tangential walls. Variation in thickness of the outer tangential, and radial, and inner tangential walls appears to result from the variation in thickness of those layers in which the microfibrils have a longitudinal orientation. The extent to which these observations can interpreted in terms of some type of modified multi-net growth is discussed.     

Ultrastructural aspects of cell wall synthesis inSpirogyra

Summary Filaments ofSpirogyra were fixed in 2% osmium tetroxide dehydrated in alcohol and embedded in Araldite. The fine structure of cells with regard to wall synthesis was studied. The cell wall was shown to have four layers. The inner one contains microfibrils and is considered to be the cell wall proper. The outer three layers are components of the slime layer. The innermost of these, the second layer of the wall, was shown to be between 1m to 3m and the third 0.3m to 1m. The fourth layer appears as no more than a dark black line measuring 10 nm across. In the cytoplasm two types of vesicles were seen. The largest of these has contents similar in appearance to the slime layer of the wall. This same material was also seen in the large vesicles attached to the Golgi bodies. It is suggested that the smaller vesicles are derived from the larger vesicles and later fuse with the cell membrane. The Golgi bodies were found to be fairly large measuring up to 5m across. Small electron opaque blobs and flecks on the outside of the plasmalemma and in between the microfibrils of the cell wall proper are considered to be mucilage droplets travelling to the slime layer. It cannot be excluded that some of the material of the large vesicles is released directly into the cytoplasm and is transferred without vesicles through the plasma membrane. The negative contrast appearance of the microfibrils seen in the cell wall is thought to be due to the spaces between them being filled with this electron opaque mucilage.Intercisternal rodlets measuring 2.5 nm across were seen in the Golgi bodies.Transverse microtubules were found to occur near the plasmalemma having the same orientation as some of the microfibrils.Lomasome-like structures sometimes with many 5 nm fibrils in their vicinity were seen.     

Wall Structure and spore Germination in Fusarium culmorum

The conidial and germ-tube walls of Fusarium culmorum (W. G.Smith) Sacc. have been examined by various chemical and electron-microscopetechniques. On the basis of these results and hypothesis isproposed for the organization of these walls. Microchemicaltests indicate the presence of chitin in the walls and suggestthat the mucilaginous layer around the conidium is mainly composedof xylan. Chemical analyses of isolated wall material confirmthe presence of chitin constituents in the wall, and a rylanlayer around the conidium. Furthermore, the wall contains apolypeptide moiety which has a different amino acid compositionfrom the rest of the protein of the cell. Electron microscopestudies of replicas and sections of conidia, germ tubes, andhyphae reveal a layered structure for the wall. The centrallayer is non-microfibrillar and is overlaid on both sides witha layer of randomly orientated microfibrils. The mucilaginouslayer of the conidium obscures the microfibrillar structurebeneath it unless the mucilage is removed by hydrolysis. Theproblem of hyphal growth is discussed on the basis of the structureof germ-tube tips and mature hyphae observed.     

Ultrastructure and localization of wall polymers during regeneration and reversion of protoplasts ofSchizophyllum commune

Summary Cell-wall regeneration and reversion of protoplasts ofSchizophyllum commune were investigated using electron microscopic methods and X-ray diffraction.After 3 hours of regeneration protoplasts have formed a loosely organized wall which does not react with Thiéry's stain for periodic acid sensitive carbohydrates. This wall largely consists of chitin microfibrils which are adpressed to the plasmalemma and which are covered by loose aggregates of alkali-soluble S-glucan (-1,3-glucan). Both components are microcrystalline, at least partly. Walls formed in the presence of polyoxin D only consist of thick loose fibers of S-glucan.From 3 hours onward the inner chitin microfibrils of the wall of the primary cells become embedded in alkali-insoluble material that stains heavily with the Thiéry reagent and probably is similar to the R-glucan of the mature wall (i.e., -1,3--1,6-glucan). The outer chitin microfibrils remain free of this matrix and are covered by S-glucan only.Bud-like structures that arise have the same wall architecture as the primary cells,i.e., only the inner chitin microfibrils are embedded in R-glucan and the S-glucan forms a fluffy coat. The walls of hyphal tubes that arise are distinct, however, in that all chitin microfibrils are embedded in R-glucan and the S-glucan forms a compact coat.Cytoplasmic vesicles are sparse in primary cells except at the sites of emergence of budlike structures and hyphae. They continue to be present in the apex of growing hyphae.     

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.     

Cell expansion patterns and directionality of wall mechanical properties in nitella

As a means of assessing the extent to which deformation of isolated walls relates to in vivo cell expansion, the directionality of wall mechanical properties was examined in Nitella. Measurements were made of plastic and elastic deformation and creep under both uniaxial and multiaxial stress conditions. Walls of different structural characteristics were obtained from control, isopropyl N-phenylcarbamate (IPC)-treated and IPC recovery cells. Although microfibrils in the inner portion of the wall were transverse for control and recovery cells but random for IPC cells, all walls had similar over-all microfibrillar orientations. Consequently, differences in wall mechanical properties should reflect structural differences in the inner wall. It is the action of the prevailing stress pattern on the inner, not overall, wall microfibrillar organization which dictates the directionality of growth in Nitella. The results indicate that the directional character of expansion is preserved to a large extent in the mechanical properties of isolated walls, and that most, but not all, of the deformation is determined by the inner wall. In addition, directional differences in the threshold for acid-induced extension varied in accord with the pattern of inner wall microfibrils.     

Cell wall structure in the xylem parenchyma ofCryptomeria

Summary Cell wall structure in ray and axial parenchyma cells in the wood ofCryptomeria was shown to be typically crossed polylamellate and dissimilar to the characteristically layered wall of fibers and tracheids. Ray cells differed from axial cells in terms of form and also in the relative inclination of crossed microfibrillar helices in the cell wall. This feature was reflected by positive birefringence in ray cells and negative birefringence in axial cells. Localized wall thickenings,viz. transverse bars in ray cells and longitudinal ribs in axial cells, also displayed crossed polylamellate structure. This observation contrasts with the exclusively longitudinal microfibrillar orientation previously reported for longitudinal ribs in elongated parenchyma cells of primary tissue. On the basis of similar microfibrillar orientations between outer and inner wall lamellae, the cell walls ofCryptomeria parenchyma were judged to be predominantly secondary.Lignin was heterogeneously distributed in lamellate fashion and a high concentration characterized the thin middle lamella. Both types of parenchyma suggested a higher lignin content than adjacent longitudinal tracheids.