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.     

Cell growth pattern and wall microfibrillar arrangement: experiments with nitella

In cylindrical cells growing throughout their length, over-all transverse reinforcement of the wall by microfibrils is believed to be required for cell elongation. The multinet theory states that in such cells microfibrils are deposited at the inner surface of the wall with transverse orientation and are then passively reoriented toward the longitudinal direction by the predominant longitudinal strain (surface expension). In the present study young Nitella cells were physically forced to grow in highly abnormal patterns: in length only, in girth only, or with localized suppression of growth. Subsequent gradients of microfibrillar arrangement within the wall cross-section were measured with polarized light and interference microscopes. The novel wall structures produced were in all cases explainable by passive reorientation, i.e. by the multinet theory. The study also showed that orientation of synthesis remains insensitive to several of the physical manipulations that strongly influence the passive behavior of wall microfibrils. Only the localized complete suppression of surface growth led to the deposition of nontransverse cellulose. These results suggest that the presence of strain is needed for continued oriented synthesis, but that the directional aspect of strain is not an “instructional” agent continuously guiding the orientation of synthesis, once this orientation has been established.     

A mechanics model for the compression of plant and vegetative tissues

The mechanics analysis of plant or vegetable tissue under a compressive stress has been developed based on large deformation elasticity theory. The tissue was treated as a lattice of regular perfect three-dimensional hexagonal cells. The cell walls were assumed to be impermeable under the time-scale of the loading. The cell walls of plants and vegetables are polymeric composite materials, consisting of a relatively amorphous matrix and a highly structured network of microfibrils embedded in the cell wall matrix. The micromechanical features of the individual cells have been related to the macroscopic properties of the whole tissue. The effects of microfibrillar stiffening factors k(1) and k(2), the cell wall matrix property alpha and the initial cell expansion ratio nu(i) on the compressive behaviour of a plant or vegetable tissue have been investigated. The predicted results have also been related to some experimental evidence.     

Two separate zones of helicoidally orientated microfibrils are present in the walls ofNitella internodes during growth

Summary The dynamic changes in microfibril architecture in the internode cell walls of the giant unicellular algaNitella translucens were studied during cell expansion. Thin section electron microscopy in conjunction with mild matrix polysaccharide extraction techniques revealed three distinct architectural zones in the walls of fully grown cells. These zones were related to distinct phases of growth by monitoring changes in cell wall architecture of internodes during active cell expansion. The initial microfibril deposition before the onset of active cell growth is helicoidal. A helicoid is a structurally complex but ordered arrangement of microfibrils that has been detected increasingly often in higher plant cell walls. During active cell elongation microfibrils are deposited transversely to the direction of cell elongation as shown in earlier studies by birefringence measurements in the polarizing microscope. The gradual decline in cell elongation corresponds with a final helicoidal deposition which continues after cell expansion ceases entirely.The continual presence of the initial helicoidal zone in the outer wall region during the whole growth process suggests that these microfibrils do not experience strain reorientation and are continually reorganized, or maintained, in a well ordered helicoidal arrangement.     

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.     

The arrangement of microfibrils in sporangial walls of Allomyces

Summary The microfibrillar component of the walls of zoosporangia and resistant sporangia of the phycomycete Allomyces arbusculus has been studied in the electron microscope, after chemical removal of the amorphous wall materials. In the zoosporangium wall the microfibrils are randomly arranged, as in the outer layer of the hyphal walls, and the sporangial wall is of even thickness. In the resistant sporangia the microfibrillar layer of the wall is perforated by numerous pores 0.25 in diameter. The microfibrils are randomly arranged over much of the wall but tend to be concentrically arranged in the vicinity of the pores. On the inside of the wall the microfibrils form a thickened rim around the pore.     

Effects of gibberellin and colchicine on microfibril arrangement in epidermal cell walls of Vigna angularis Ohwi et Ohashi epicotyls

Gibberellic-acid (GA₃) treatment of azukibean epicotyls resulted in alterations of the direction of newly deposited microfibrils, on the cell walls. Cells having transverse microfibrils on the inner surface of the wall were observed more frequently in GA₃-treated epicotyls than in untreated or water-treated ones. This effect of GA₃ was negated by simultaneously supplied colchicine. A crossed polylamellate structure was observed in the inner portion of the walls of GA₃-treated cells, but not in the inner portion of the walls of colchicine-treated cells. The wall formed under the influence of colchicine consisted of microfibrils running in the same direction.Abbreviations GA gibberellin - GA₃ gibberellic acid (gibberellin A₃)     

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.     

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.     

Cell wall synthesis during growth and maturation of Nitella internodal cells

An improved ¹³C-density-labeling method was used to study cell wall synthesis in rapidly expanding, slowly expanding and recently mature internodes of Nitella translucens var axillaris (A.Br.) R.D.W. As cells matured, the rate of wall synthesis slowed and the deposition of cellulose microfibrils changed from a predominantly transverse direction in the primary wall of rapidly expanding internodes to a helicoidal array in the secondary wall of mature internodes. The secondary wall was characterized by relatively higher rates of cellulose synthesis and lower rates of pectin synthesis than the primary wall. The synthesis of xyloglucan also decreased markedly at the transition to secondary wall synthesis, while the synthesis of mannose-rich hemicellulose increased. Even though structural differences were striking between the primary and secondary walls of Nitella, compositional differences between the two types of wall were quantitative rather than qualitative. The authors appreciate the assistance of Martin Yousef with the electron microscopy.     

Fine structure of cell wall surfaces in the giant-cellular xanthophycean alga <Emphasis Type="Italic">Vaucheria terrestris</Emphasis>

The mechanical strength of cell walls in the tip-growing cells of Vaucheria terrestris is weakened by treatment with proteolytic enzymes. To clarify the morphological characteristics of the components maintaining cell wall strength, the fine structures of the cell walls, with and without protease treatment, were observed by transmission electron microscopy (TEM) and atomic force microscopy (AFM). Observations indicated that cellulose microfibrils were arranged in random directions and overlapped each other. Most of the microfibrils observed in the inner surface of the cell wall were embedded in amorphous materials, whereas in the outer surface of the cell wall, microfibrils were partially covered by amorphous materials. The matrix components embedding and covering microfibrils were almost completely removed by protease treatment, revealing layers of naked microfibrils deposited deeply in the cell wall. Topographic data taken from AFM observations provided some additional information that could not be obtained by TEM, including more detailed images of the granular surface textures of the matrix components and the detection of microfibrils in the interior of the cell wall. In addition, quantitative AFM data of local surface heights enabled us to draw three-dimensional renderings and to quantitatively estimate the extent of the exposure of microfibrils by the enzymatic treatment.     

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     

Unusual structures in the cell wall ofNitella

Summary Electron micrographs of the transverse sections of internodal cell walls of the freshwater algaNitella flexilis show spindle-shaped networks of aggregated microfibrils perforated by oval pores embedded in the cylindrical wall.Chargé de Recherches du F. N. R. S.     

Plants control the properties and actuation of their organs through the orientation of cellulose fibrils in their cell walls

Plants use the orientation of cellulose microfibrils to create cell walls with anisotropic properties related to specific functions. This enables organisms to control the shape and size of cells during growth, to adjust the mechanical performance of tissues, and to perform bending movements of organs. We review the key function of cellulose orientation in defining structural-functional relationships in cell walls from a biomechanics perspective, and illustrate this by examples mainly from our own work. First, primary cell-wall expansion largely depends on the organization of cellulose microfibrils in newly deposited tissue and model calculations allow an estimate of how their passive re-orientation may influence the growth of cells. Moreover, mechanical properties of secondary cell walls depend to a large extent on the orientation of cellulose fibrils and we discuss strategies whereby plants utilize this interrelationship for adaptation. Lastly, we address the question of how plants regulate complex organ movements by designing appropriate supramolecular architectures at the level of the cell wall. Several examples, from trees to grasses, show that the cellulose architecture in the cell wall may be used to direct the swelling or shrinking of cell walls and thereby generate internal growth stress or movement of organs.     

Characteristic thickened cell walls of the bracts of the 'eternal flower' Helichrysum bracteatum

Background and Aims

Helichrysum bracteatum is called an ‘eternal flower’ and has large, coloured, scarious bracts. These maintain their aesthetic value without wilting or discoloration for many years. There have been no research studies of cell death or cell morphology of the scarious bract, and hence the aim of this work was to elucidate these characteristics for the bract of H. bracteatum.

Methods

DAPI (4''6-diamidino-2-phenylindol dihydrochloride) staining and fluorescence microscopy were used for observation of cell nuclei. Light microscopy (LM), transmission electron microscopy (TEM) and polarized light microscopy were used for observation of cells, including cell wall morphology.

Key Results

Cell death occurred at the bract tip during the early stage of flower development. The cell wall was the most prominent characteristic of H. bracteatum bract cells. Characteristic thickened secondary cell walls on the inside of the primary cell walls were observed in both epidermal and inner cells. In addition, the walls of all cells exhibited birefringence. Characteristic thickened secondary cell walls have orientated cellulose microfibrils as well as general secondary cell walls of the tracheary elements. For comparison, these characters were not observed in the petal and bract tissues of Chrysanthemum morifolium.

Conclusions

Bracts at anthesis are composed of dead cells. Helichrysum bracteatum bracts have characteristic thickened secondary cell walls that have not been observed in the parenchyma of any other flowers or leaves. The cells of the H. bracteatum bract differ from other tissues with secondary cell walls, suggesting that they may be a new cell type.Key words: Helichrysum bracteatum, scarious bract, secondary cell wall, primary cell wall, cell morphology, birefringence, orientated cellulose microfibrils, cell death, DAPI, transmission electron microscopy, polarized light microscopy     

Real-Time Imaging of Cellulose Reorientation during Cell Wall Expansion in Arabidopsis Roots

Cellulose forms the major load-bearing network of the plant cell wall, which simultaneously protects the cell and directs its growth. Although the process of cellulose synthesis has been observed, little is known about the behavior of cellulose in the wall after synthesis. Using Pontamine Fast Scarlet 4B, a dye that fluoresces preferentially in the presence of cellulose and has excitation and emission wavelengths suitable for confocal microscopy, we imaged the architecture and dynamics of cellulose in the cell walls of expanding root cells. We found that cellulose exists in Arabidopsis (Arabidopsis thaliana) cell walls in large fibrillar bundles that vary in orientation. During anisotropic wall expansion in wild-type plants, we observed that these cellulose bundles rotate in a transverse to longitudinal direction. We also found that cellulose organization is significantly altered in mutants lacking either a cellulose synthase subunit or two xyloglucan xylosyltransferase isoforms. Our results support a model in which cellulose is deposited transversely to accommodate longitudinal cell expansion and reoriented during expansion to generate a cell wall that is fortified against strain from any direction.The walls of growing plant cells must fulfill two simultaneous and seemingly contradictory requirements. First, they must expand to accommodate cell growth, which is anisotropic in many tissues and determines organ morphology. Second, they must maintain their structural integrity, both to constrain the turgor pressure that drives cell growth and to provide structural rigidity to the plant. These requirements are met by constructing primary cell walls that can expand along with growing cells, whereas secondary cell walls are deposited after cell growth has ceased and serve the latter function.One of the major constituents of both types of cell walls is cellulose, which exists as microfibrils composed of parallel β-1,4-linked glucan chains that are held together laterally by hydrogen bonds (Somerville, 2006). Microfibrils are 2 to 5 nm in diameter, can extend to several micrometers in length, and exhibit high tensile strength that allows cell walls to withstand turgor pressures of up to 1 MPa (Franks, 2003). In vascular plants, cellulose is synthesized by a multimeric cellulose synthase (CESA) complex composed of at least three types of glycosyl transferases arranged into a hexameric rosette (Somerville, 2006). After delivery to the plasma membrane, CESA initially moves in alignment with cortical microtubules (Paredez et al., 2006), but its trajectory can be maintained independently of microtubule orientation. For example, in older epidermal cells of the root elongation zone in Arabidopsis (Arabidopsis thaliana), cellulose microfibrils at the inner wall face are oriented transversely despite the fact that microtubules reorient from transverse to longitudinal along the elongation zone (Sugimoto et al., 2000), suggesting that microtubule orientation and cellulose deposition are independent in at least some cases.Depending on species, cell type, and developmental stage, cellulose microfibrils may be surrounded by additional networks of polymers, including hemicelluloses, pectins, lignin, and arabinogalactan proteins (Somerville et al., 2004). Hemicelluloses are composed of β-1,4-linked carbohydrate backbones with side branches and include xyloglucans, mannans, and arabinoxylans. Xyloglucan is thought to interact with the surface of cellulose and form cross-links between adjacent microfibrils (Vissenberg et al., 2005). In some cell types, pectin or lignin may also participate in cross-linking or entrapment of other cell wall polymers. It is unclear how the associations between networks of different cell wall components are relaxed to allow for cell wall expansion during growth.Several models have been proposed for the behavior of cell wall components during wall expansion. The passive reorientation hypothesis (also called the multinet growth hypothesis; Preston, 1982) postulates that in longitudinally expanding cells, cellulose microfibrils are synthesized in a transverse pattern and are then reoriented toward the longitudinal axis due to the strain generated by turgor pressure (Green, 1960). This phenomenon has been observed in the multicellular alga Nitella (Taiz, 1984). In higher plants, there is less direct evidence for passive reorientation, and another hypothesis holds that wall expansion involves active, local, and controlled remodeling of cellulose microfibrils along a diversity of orientations (Baskin, 2005). Such remodeling could be achieved by proteins such as xyloglucan endotransglycosylases (XETs), which break and rejoin xyloglucan chains, and expansins, which loosen cell walls in vitro in a pH-dependent manner (Cosgrove, 2005). Marga et al. measured cellulose microfibril orientation at the innermost layer of the cell wall before and after in vitro extension and did not observe reorientation (Marga et al., 2005). This suggests that processes other than microfibril reorientation might be involved in wall expansion, at least under certain circumstances or in some wall layers. Thus, the degree to which cellulose microfibrils are reoriented after their synthesis during wall expansion has remained unclear.One difficulty in resolving this problem has been the inability to directly image cellulose microfibrils in the growing cell wall. Existing methods to assess cellulose structure and orientation in plant cell walls are limited by the low contrast of cellulose in transmission electron microscopy, the ability to image only the surface of the wall using field emission scanning electron microscopy, and the use of polarized light microscopy in combination with dyes such as Congo red to measure only the bulk orientation of cellulose microfibrils (Baskin et al., 1999; Sugimoto et al., 2000; Verbelen and Kerstens, 2000; MacKinnon et al., 2006). In addition, the sample manipulation required for the former two methods has the potential to introduce artifacts (Marga et al., 2005). Although cellulose microfibril orientation differs at the inner and outer surfaces of the cell wall (Sugimoto et al., 2000) and presumably changes over time, the dynamics of cellulose reorientation during cell wall expansion have not been observed to date.In this study, we tested fluorescent dyes for their potential to allow imaging of cellulose distribution in the walls of Arabidopsis seedlings by confocal microscopy. We used one of these dyes to characterize the distribution of cellulose in wild-type root cells and in mutants with reduced cellulose or xyloglucan. By directly observing the fine structure of cellulose over time in growing wild-type root cells, we concluded that cellulose microfibrils in these cells reorient in a transverse to longitudinal direction as predicted by the passive reorientation hypothesis.     

THE STRUCTURE OF THE PRIMARY EPIDERMAL CELL WALL OF AVENA COLEOPTILES

The primary walls of epidermal cells in Avena coleoptiles ranging in length from 2 to 40 mm. have been studied in the electron and polarizing microscopes and by the low-angle scattering of x-rays. The outer walls of these cells are composed of multiple layers of cellulose microfibrils oriented longitudinally; initially the number of layers is between 10 and 15 but this increases to about 25 in older tissue. Where epidermal cells touch, these multiple layers fuse gradually into a primary wall of the normal type between cells. In these radial walls, the microfibrils are oriented transversely. Possible mechanisms for the growth of the multilayered outer wall during cell elongation are discussed.     

Arrangement of Cellulose Microfibrils in Walls of Elongating Parenchyma Cells

The arrangement of cellulose microfibrils in walls of elongating parenchyma cells of Avena coleoptiles, onion roots, and celery petioles was studied in polarizing and electron microscopes by examining whole cell walls and sections. Walls of these cells consist firstly of regions containing the primary pit fields and composed of microfibrils oriented predominantly transversely. The transverse microfibrils show a progressive disorientation from the inside to the outside of the wall which is consistent with the multinet model of wall growth. Between the pit-field regions and running the length of the cells are ribs composed of longitudinally oriented microfibrils. Two types of rib have been found at all stages of cell elongation. In some regions, the wall appears to consist entirely of longitudinal microfibrils so that the rib forms an integral part of the wall. At the edges of such ribs the microfibrils can be seen to change direction from longitudinal in the rib to transverse in the pit-field region. Often, however, the rib appears to consist of an extra separate layer of longitudinal microfibrils outside a continuous wall of transverse microfibrils. These ribs are quite distinct from secondary wall, which consists of longitudinal microfibrils deposited within the primary wall after elongation has ceased. It is evident that the arrangement of cellulose microfibrils in a primary wall can be complex and is probably an expression of specific cellular differentiation.     

Tensile strength of cell walls of living cells

A gas decompression technique was used to determine the breaking strength of cell walls of single cells. Breaking strengths of the bacterium Salmonella typhimurium and the unicellular green alga Chlamydomonas eugametos were 100 and 95 atmospheres, respectively, while those of sporophytes of the water mold Blastocladiella emersonii were 65 atmospheres, and those of suspension cultured cells of carrot were only 30 atmospheres. Estimation of wall tensile stress based on breaking pressures, cell radii, and estimation of wall thickness, indicates that microfibrillar walls are not necessarily stronger than walls of primitive organisms. Hence, alternative hypotheses for their evolution must be considered.