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.     

Atomic force microscopy of microfibrils in primary cell walls

Examination of angiosperm primary cell walls by transmission electron microscopy shows that they contain microfibrils that probably consist of cellulose microfibrils surrounded by associated non-cellulosic polysaccharides. Previous studies using solid-state (13)C NMR spectroscopy have shown that the cellulose is all crystalline with crystallites of cross-sectional dimensions of 2-3 nm. However, it is not known if each microfibril contains only one, or more than one crystallite because there is no agreement about the dimensions of the microfibrils. Partially hydrated primary cell walls isolated from onion ( Allium cepa L.) and Arabidopsis thaliana (L.) Heynh. were examined by atomic force microscopy and the microfibril diameters determined. The cell walls of both species contained tightly interwoven microfibrils of uniform diameter: 4.4+/-0.13 nm in the onion and 5.8+/-0.17 nm in A. thaliana. The effect was also examined of extracting the A. thaliana cell walls to remove pectic polysaccharides. The microfibrils in the extracted cell walls of A. thaliana were significantly narrower (3.2+/-0.13 nm) than those in untreated walls. The results are consistent with the microfibrils containing only one cellulose crystallite.     

Lattice images from ultrathin sections of cellulose microfibrils in the cell wall of Valonia macrophysa Kütz.

The crystalline ultrastructure and orientation of cellulose microfibrils in the cell wall of Valonia macrophysa were investigated by means of high-resolution electron microscopy of ultrathin (approx. 28 nm) sections. With careful selection of imaging conditions, ultrastructural aspects of the cell wall that had remained unresolved in previous studies were worked out by direct imaging of crystal lattice of cellulose microfibrils. It was confirmed that each microfibril is a single crystal having a lateral dimension of 20·20 nm², because lattice images of 0.39 nm resolution were clearly recorded with no major disruption in the whole area of the cross section of the microfibril. There was no evidence for the existence of 3.5-nm elementary fibrils which have been considered to be basic crystallographic and morphological units of cellulose in general. It was also confirmed that the axial directions (crystallographic fiber direction) of adjacent microfibrils in each single lamella of the cell wall are opposite to each other.     

Cellulose microfibril assembly inErythrocladia subintegra Rosenv.: An ideal system for understanding the relationship between synthesizing complexes (TCs) and microfibril crystallization

Summary The marine red algaErythrocladia subintegra synthesizes cellulose microfibrils as determined by CBH I-gold labelling, X-ray and electron diffraction analyses. The cellulose microfibrils are quite thin, ribbon-like structures, 1–1.5 nm in thickness (constant), and 10–33 nm in width (variable). Several laterally associated minicrystal components contribute to the variation in microfibrillar width. Electron diffraction analysis suggested a uniplanar orientation of the microfibrils with their (101) lattice planes parallel to the plasma membrane surface of the cell. The linear particle arrays bound in the plasma membrane and associated with microfibril impressions recently demonstrated inErythrocladia have been shown in this study to be the cellulose-synthesizing terminal complexes (TCs). The TCs appear to be organized by a repetition of transverse rows consisting of four TC subunits, rather than by four rows of longitudinallyarranged TC subunits. The number of transverse rows varied between 8–26, corresponding with variation in the length of the TCs and the width of the microfibrils. The spacings between the neighboring transverse rows are almost constant being 10.5–11.5 nm. Based on the knowledge thatAcetobacter, Vaucheria, andErythrocladia synthesize similar thin, ribbon-like cellulose microfibrils, the structural characteristics common to the organization of distinctive TCs occurring in these three organisms has been discussed, so that the mode of cellulose microfibril assembly patterns may be deciphered.     

Cellulose-synthesizing terminal complexes and microfibril structure in the brown alga Sphacelaria rigidula (Sphacelariales,Phaeophyceae)*

The brown alga Sphacelaria rigidula Kützing synthesizes cellulose microfibrils as determined by CBH I-gold labeling. The cellulose microfibrils are thin, ribbon-like structures with a uniform thickness of about 2.6 nm and a variable width in the range of 2.6-30 nm. Some striations appear along the longitudinal axis of the microfibrils. The developed cell wall in Sphacelaria is composed of three to four layers, and cellulose micro-fibrils are deposited in the third layer from the outside of the wall. A freeze fracture investigation of this alga revealed cellulose-synthesizing terminal complexes (TCs), which are associated with the tip of microfibril impressions in the plasmatic fracture face of the plasma membrane. The TCs consist of subunits arranged in a single linear row. The average diameter of the sub-units is about 6 nm, and the intervals between the neighboring subunits, about 9 nm, are relatively constant. The number of subunits constituting the TC varies between 10 and 100, so that the length of the whole TC varies widely. A model that has been proposed for the assembly of thin, ribbon-like microfibrils was applied to microfibril assembly in Sphacelaria.     

An NMR relaxation and spin diffusion study of cellulose structure during water adsorption

The goal of this paper is a systematic investigation of changes in the supramolecular structure of cellulose during its water uptake. The main attention is concentrated on the analysis of the mechanism of dispersion of microfibrils by proton NMR relaxation techniques. Spin diffusion NMR experiments made it possible to estimate the linear dimensions of the surface thickness of cellulose crystallites and the average depth of micropores that are formed between elementary fibrils, as well as the character of the filling of micropores during adsorption. It has been shown that when the relative water content gradually increases to 7–8%, water molecules occupy the space between cellulose microfibrils, which is accompanied by an increase in the pore sizes and their specific surface area and a simultaneous decrease in the degree of crystallinity. Upon acquiring a free induction decay signal, a magic sandwich echo sequence was used, due to which the accuracy and information value of the results were considerably improved.     

Crystallographic aspects of sub-elementary cellulose fibrils occurring in the wall of rose cells culturedin vitro

Summary Quantities of disencrusted sub-elementary cellulose fibrils from the cell wall of rose cells culturedin vitro were prepared. Following an X-ray and electron diffraction analysis, these fibrils gave a cellulose diffraction pattern which presented only two strong equatorial diffraction spacings at 0.409 and 0.572 nm indicating that the fibrils have a crystalline structure resembling that of cellulose IV_I. This observation is best explained in terms of a lateral disorganization of the cellulose chains within the fibrils. This disorganization cannot be eliminated and is connected with the small width of the fibrils which contain from 12 to 25 cellulose chains only. In these fibrils, most of the cellulose chains are superficial and not locked with neighboring chains in a tight hydrogen bond system as in thicker cellulose microfibrils.     

THE MICROCRYSTALLINE STRUCTURE OF CELLULOSE IN CELL WALLS OF COTTON, RAMIE, AND JUTE FIBERS AS REVEALED BY NEGATIVE STAINING OF SECTIONS

With a new technique of negative staining of sections, it has been possible to observe directly, in ultrathin sections under the electron microscope, the original microcrystalline and microfibrillar structure of cellulose as it occurs in living cells. This method has advantages over the study of isolated fibers used so far by others, in that the original arrangement of microfibrils is better preserved, and their collapse into larger fibrillar units is prevented. With this method, the cell walls of ramie, jute, and cotton fibers have been studied. The size (diameter, 25 to 40 A) and the longitudinal periodicity observed in the single microfibrils and the orientation and spatial arrangement of the microcrystallite within the microfibrils are found to correspond with the latest models derived by others from data obtained by indirect methods such as X-ray diffraction. The microfibril size of about 35 A, found by measuring these structures in sections, agrees with the latest conclusions reached by others in recent work with isolated fibrils.     

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.     

Dermal collagen fibrils are hybrids of type I and type III collagen molecules

It has been suggested that dermal collagen fibrils with 67-nm periodicity consist of hybrids of type I and type III collagens. This is based on the assumption that all these banded fibrils are coated with type III collagen regardless of their diameter. However, conclusive evidence for this form of hybridization is lacking. In order to clarify this problem dermal collagen fibrils were disrupted into microfibrils using 8 M urea. Single and double indirect immunoelectron microscopy showed type III collagen at the periphery of intact collagen fibrils but no labeling with type I collagen antibodies, suggesting that the epitopes for this collagen were masked. Disrupted collagen fibrils revealed type I collagen throughout the fibril except for the periphery which was coated with type III collagen. Almost no type III collagen was noted in the interior of the collagen fibrils. Since type III collagen is present only at the periphery it suggests that this collagen has a different role than type I collagen and may have a regulatory function in fibrillogenesis.     

SYNTHESIS OF CELLULOSE BY ACETOBACTER XYLINUM : V. Ultrastructure of Polymer

Appearance of cellulose microfibrils in the medium of a suspension of cells of Acetobacter xylinum in buffered glucose solution was preceded by a stage during which the cellulose in the medium was amorphous within the available resolution. The size of the vertical axis of the microfibrils of the bacterial cellulose was found on the basis of measurement of shadow length to be only about 16 A. In good agreement with findings of earlier workers, the size of the lateral axis ("width") of the image of the metal-shadowed cellulose microfibrils was found to be 11 mµ. After correcting for a large part probably contributed by deposited metal in the observed width of the microfibrils, the real width is estimated roughly to be in the neighborhood of 3 mµ. To account for the occurrence of diverse morphological elements in the fields and for the fact that the cellulose fibrils are free entities rather than physical appendages of the cell, it is suggested that individual cellulose molecules are released at the cell surface and diffuse into the medium, wherein they finally enter into crystal-line patterns.     

Deposition of glucuronoxylans on the secondary cell wall of Japanese beech as observed by immuno-scanning electron microscopy

Summary Glucuronoxylans (GXs), the main hemicellulosic component of hardwoods, are localized exclusively in the secondary wall of Japanese beech and gradually increase during the course of fiber differentiation. To reveal where GXs deposit within secondary wall and how they affect cell wall ultrastructure, immuno-scanning electron microscopy using anti-GXs antiserum was applied in this study. In fibers forming the outer layer of the secondary wall (S₁), cellulose fibrils were small in diameter and deposited sparsely on the inner surface of the cell wall. Fine fibrils with approximately 5 nm width aggregated and formed thick fibrils with 12 nm width. Some of these thick fibrils further aggregated to form bundles which labelled positively for GXs. In fibers forming the middle layer of the secondary wall (S₂), fibrils were thicker than those found in S₁ forming fibers and were densely deposited. The S₂ layer labelled intensely for GXs with no preferential distribution recognized. Compared with newly formed secondary walls, previously formed secondary walls were composed of thick and highly packed microfibrils. Labels against GXs were much more prevalent on mature secondary walls than on newly deposited secondary walls. This result implies that the deposition of GXs into the cell wall may occur continuously after cellulose microfibril deposition and may be responsible for the increase in diameter of the microfibrils.Abbreviations GXs glucuronoxylans - PBS phosphate-buffered saline - RFDE rapid-freeze and deep-etching technique - FE-SEM field emission scanning electron microscope - TEM transmission electron microscope     

Illustration of the development of bacterial cellulose bundles/ribbons by Gluconacetobacter xylinus via atomic force microscopy

The development of bacterial cellulose (BC) fibrils biosynthesized by Gluconacetobacter xylinus was investigated using atomic force microscopy (AFM). After various incubation times at 30 °C, both the length of BC fibrils and their average diameters increased significantly. After the first 2-h incubation, not only single BC microfibrils with an average diameter of 5.8?±?0.7 nm were biosynthesized but single microfibrils also began to bind with each other forming bundles. After longer incubation times of 6 h, 16 h, and 48 h, only BC bundles and ribbons or even only ribbons were detectable. The development of BC fibrils and the formation of BC bundles/ribbons along with the biosynthesis time were illustrated using AFM. Furthermore, single BC fibrils were twisted in a right-handed manner. The twisting of BC fibrils possibly promoted the formation of bigger ribbons.     

Assembly of microfibrils in vivo and in vitro from (1»3)-β-d-glucan synthesized by protoplasts of Saccharomyces cerevisiae

Polymer chains of (13)--d-glucan were dissolved with 1 M NaOH at 4° C from native microfibrillar protoplast nets. The chains associated into microfibrils during NaOH neutralization or dialysis. In contrast to the native microfibrils which are of uniform width individually (10 to 20 nm) and arranged in flat bundles, the microfibrils formed in vitro showed no band formation and consisted of fibrous spindle-shaped subunits of variable width or loose elementary fibrils about 1.7 nm wide. X-ray diagrams of native nets indicated a fairly high crystallinity and were different for wet and dry specimens. They corresponded to those of paramylon. Precipitated glucans produced diagrams different from the former and revealing a lower crystallinity especially with the dry samples.The X-ray pattern, combined with other data, allowed the precipitated microfibrils to be identified as aggregates of molecular strands composed each of three intertwined helical glucan chains. Since these triple helical chains are about 1.7 nm wide the elementary fibrils of this width can represent only single triple-helical strands. These helices have 7 glucose residues per turn and therefore a low symmetry which explains the poor crystallizing properties. The 7 membered helix represents a basic difference with the well crystallized native glucan which is built of highly symmetrical triple helices with 6 glucose residues per turn. Since 6₁ helical conformation is not formed in vitro at normal temperatures its generation in vivo must be due to the action of synthesizing enzymes at the protoplast membrane. The intertwining of these helices and crystallization of the strands are determined by their symmetry and physical properties of the chains. This characterizes the native microfibrils as products of self-assembly of enzymegenerated 6₁ helices.     

Formation of macromolecular lignin in ginkgo xylem cell walls as observed by field emission scanning electron microscopy

Formation of macromolecular lignin in ginkgo cell walls. In the lignifying process of xylem cell walls, macromolecular lignin is formed by polymerization of monolignols on the pectic substances, hemicellulose and cellulose microfibrils that have deposited prior to the start of lignification. Observation of lignifying secondary cell walls of ginkgo tracheids by field emission scanning electron microscopy suggested that lignin-hemicellulose complexes are formed as tubular bead-like modules surrounding the cellulose microfibrils (CMFs), and that the complexes finally fill up the space between CMFs. The size of one tubular bead-like module in the middle layer of the secondary wall (S2) was tentatively estimated to be about 16+/-2 nm in length, about 25+/-1 nm in outer diameter, with a wall thickness of 4+/-2 nm; the size of the modules in the outer layer of the secondary wall (S1) was larger and they were thicker-walled than that in the middle layer (S2). Aggregates of large globular modules were observed in the cell corner and compound middle lamella. It was suggested that the structure of non-cellulosic polysaccharides and mode of their association with CMFs may be important factors controlling the module formation and lignin concentration in the different morphological regions of the cell wall.     

Spatial organization of cellulose microfibrils and matrix polysaccharides in primary plant cell walls as imaged by multichannel atomic force microscopy

We used atomic force microscopy (AFM), complemented with electron microscopy, to characterize the nanoscale and mesoscale structure of the outer (periclinal) cell wall of onion scale epidermis – a model system for relating wall structure to cell wall mechanics. The epidermal wall contains ~100 lamellae, each ~40 nm thick, containing 3.5‐nm wide cellulose microfibrils oriented in a common direction within a lamella but varying by ~30 to 90° between adjacent lamellae. The wall thus has a crossed polylamellate, not helicoidal, wall structure. Montages of high‐resolution AFM images of the newly deposited wall surface showed that single microfibrils merge into and out of short regions of microfibril bundles, thereby forming a reticulated network. Microfibril direction within a lamella did not change gradually or abruptly across the whole face of the cell, indicating continuity of the lamella across the outer wall. A layer of pectin at the wall surface obscured the underlying cellulose microfibrils when imaged by FESEM, but not by AFM. The AFM thus preferentially detects cellulose microfibrils by probing through the soft matrix in these hydrated walls. AFM‐based nanomechanical maps revealed significant heterogeneity in cell wall stiffness and adhesiveness at the nm scale. By color coding and merging these maps, the spatial distribution of soft and rigid matrix polymers could be visualized in the context of the stiffer microfibrils. Without chemical extraction and dehydration, our results provide multiscale structural details of the primary cell wall in its near‐native state, with implications for microfibrils motions in different lamellae during uniaxial and biaxial extensions.     

Arrangement of connective tissue components in the walls of seminiferous tubules of man and monkey.

In primates the membrane separating the seminiferous epithelium from the interstitial space is composed of one to three (monkey) or two to six layers (man) of myoid cells associated with one to two layers of fibrocyte-like adventitial cells. All these cells are separated from each other by irregular spaces filled with various connective tissue intercellular components. Subjacent to the elements of the seminiferous epithelium is a continuous, often redundant, basement membrane. A similar basement membrane-like material forms a layer next to and over small areas of the plasma membrane of myoid cells. Collagen fibrils grouped in bundles of various sizes are seen in all connective tissue layers but are particularly abundant in the space between the seminiferous epithelium and the innermost layer of myoid cells. Elastic fibrils demonstrated by the Verhoeff iron hematoxylin technique are also present. Composed of a homogeneous material, the elastic fibrils are short, irregular, branching entities with a diameter comparable to or smaller than that of collagen fibrils. In addition, an abundance of microfibrils with a diameter of 12-15 nm is present in the various connective tissue layers. These microfibrils have a densely stained cortex and a lightly stained core. When seen close to the myoid cells, bundles of micro fibrils appear to insert on well defined areas next to the plasma membrane. These areas commonly face the patches of electron-dense material observed on the inner aspect of the plasma membrane of the myoid cells and in which the actin filaments are inserted. Bundles of microfibrils often span the gap between myoid cells of the same layer as well as those of adjacent layers. Microfibrils are also closely related to the surface of elastic fibrils and are seen intertwining with collagen fibrils. Thus microfibrils appear to bridge and bind together adjacent myoid cells and anchor the surface of these cells to the bundles of elastic and collagen fibrils present in the intercellular spaces of the limiting membrane.     

Structure and association of wall fibrils produced by regenerating tobacco protoplasts

A study has been made of the wall fibrils produced by tobacco protoplasts, using scanning electron microscopy in conjunction with negative staining. It has been shown that the fibres seen in scanning electron microscopy correspond to aggregates of microfibrils. These aggregates are only visible where they are lifted clear of the protoplast surface. Negative staining of fixed protoplasts shows that the aggregation of microfibrils into the fibres visible in scanning electron microscopy is probably produced by air-drying. Gentle disruption of microfibrils produces both random broken fragments and bundles of short pieces of fibrillar material about 60 nm in length. This material is present in undisrupted young walls, but not in undisrupted older walls. The microfibrils in young walls seem much more fragile and liable to breakage than those in older walls. These results are discussed in terms of the interpretation of scanning electron microscope images and the mechanism of cellulose microfibril formation by higher plants.Abbreviations SEM Scanning electron microscopy     

THE SITES OF CELLULOSE SYNTHESIS IN ALGAE: DIVERSITY AND EVOLUTION OF CELLULOSE-SYNTHESIZING ENZYME COMPLEXES

Information on the sites of cellulose synthesis and the diversity and evolution of cellulose-synthesizing enzyme complexes (terminal complexes) in algae is reviewed. There is now ample evidence that cellulose synthesis occurs at the plasma membrane-bound cellulose synthase, with the exception of some algae that produce cellulosic scales in the Golgi apparatus. Freeze-fracture studies of the supramolecular organization of the plasma membrane support the view that the rosettes (a six-subunit complex) in higher plants and both the rosettes and the linear terminal complexes (TCs) in algae are the structures that synthesize cellulose and secrete cellulose microfibrils. In the Zygnemataceae, each single rosette forms a 5-nm or 3-nm single “elementary” microfibril (primary wall), whereas rosettes arranged in rows of hexagonal arrays synthesize criss-crossed bands of parallel cellulose microfibrils (secondary wall). In Spirogyra, it is proposed that each of the six subunits of a rosette might synthesize six β-1,4-glucan chains that cocrystallize into a 36-glucan chain “elementary” microfibril, as is the case in higher plants. One typical feature of the linear terminal complexes in red algae is the periodic arrangement of the particle rows transverse to the longitudinal axis of the TCs. In bangiophyte red algae and in Vaucheria hamata, cellulose microfibrils are thin, ribbon-shaped structures, 1–1.5 nm thick and 5–70 nm wide; details of their synthesis are reviewed. Terminal complexes appear to be made in the endoplasmic reticulum and are transferred to Golgi cisternae, where the cellulose synthases are activated and may be transported to the plasma membrane. In algae with linear TCs, deposition follows a precise pattern directed by the movement and the orientation of the TCs (membrane flow). A principal underlying theme is that the architecture of cellulose microfibrils (size, shape, crystallinity, and intramicrofibrillar associations) is directly related to the geometry of TCs. The effects of inhibitors on the structure of cellulose-synthetizing complexes and the relationship between the deposition of the cellulose microfibrils with cortical microtubules and with the membrane-embedded TCs is reviewed In Porphyra yezoensis, the frequency and distribution of TCs reflect polar tip growth in the apical shoot cell.The evolution of TCs in algae is reviewed. The evidence gathered to date illustrates the utility of terminal complex organization in addressing plant phylogenetic relationships.     

THE CELL WALL OF PYROCYSTIS SPP. (DINOCOCCALES)1,2

The cell of Pyrocystis spp. is covered by an outer layer of material resistant to strong acids and bases. Internal to this layer much of the cell wall is composed of cellulose fibrils. The presence of cellulose fibrils was established by staining raw and ultra-violet–peroxide-cleaned cell walls and by combining X-ray diffraction spectroscopy with electron microscope observation. Carbon replicas of freeze-etched preparations and thin sections of P. lunula walls show outer layers, inside them ca. 24 layers of crossed parallel cellulose fibrils (4–5 nm thick, ca. 12 nm wide), then a region of smaller (ca. 6–12 nm diameter) fibrils in a disperse texture, and then the plasma membrane. Cellulose fibrils in the parallel texture are constructed of 3–5 elementary fibrils ca. 3 nm in diameter. Walls of P. fusiformis and P. pseudonctiluca also have cellulose fibrils in a crossed parallel texture similar to those of P. lunula. The Gymnodinium-type swarmer from lunate P. lunula appears to have a cell wall ultrastructure typical of other “naked” dinoflagellates.