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.     

An examination of the developing cotton fiber: Wall and plasmalemma

Summary The ultrastructure of developing cotton fibers has been examined using novel modifications of the techniques of surface replication, freeze-etching and thin-sectioning. The fiber surface was found to be coated with a lamellar cuticle, which is stretched and thinned as the fiber elongates. It is marked by bars which run parallel with the fiber long axis. Beneath the cuticle, the outermost microfibrils of the primary wall lie parallel with the fiber axis, while those adjacent to the plasma membrane are transverse. Primary wall microfibrils are present in bundles, disposed in left-handed and right-handed helices, which correspond with the fibrils observed optically. Microfibrils within bundles form in-phase waves, with wavelengths and amplitudes in the ranges 0.3–7 m and 0.01–0.1 m in primary and secondary walls respectively. As elongation proceeds bundles become displaced towards the cell axis. Microfibrils of the secondary wall, disposed around the cell as fast helices, are similarly bundled and wavy (though with a reduced amplitude). In surface-replicas, large (20–30 nm) granules are present on the cytoplasmic face of the wall which probably correspond with 20–40 nm low prominences visible on freeze-etch EF plasma membrane fracture faces. It is proposed that these may be microfibril-synthesizing centers. Plasma membranes fracture such that the membrane-associated-particles segregate 6040 between P and E fracture moieties, but the prominence and total number of these particles is reduced at the stage of secondary wall formation as compared with primary wall formation. Beneath the plasmalemma the axes of microtubules parallel secondary wall microfibril orientation. Cross-bridges, which stain heavily after glutaraldehyde/tannic acid fixation, link microtubules to plasma membrane. The use of butyl benzene to cement fragments of cotton fibers, employed in this work, may prove useful in other freeze-etch studies of long fibers which are readily ruptured during preparation.     

Penetration of chemicals into the Malus leaf cuticle

The adaxial leaf cuticle of Malus pumila was examined by electron microscopy to determine possible avenues for transcuticular movement of foliarly applied chemicals. Cutin-embedded polysaccharide microfibrils originated at the outer epidermal cell wall and occasionally extended to the cuticle surface. Lamellae, ca. 4 nm wide, usually were oriented parallel to the cuticle surface. When oriented perpendicular to the surface, they extended nearly to the subjacent wall layer from the surface. Aqueous solutions of uranyl acetate, silver nitrate and phenyl mercuric acetate applied to the cuticle surface of leaf segments floated on solutions of phosphate salts or thiocarbohydrazide (TCH) reacted within the cuticle to form insoluble electron-opaque deposits indicative of their avenues of transcuticular movement. Uranyl phosphate deposits were observed only in the polysaccharide microfibrils of chloroform: methanolextracted leaves. Silver-TCH deposits were observed in the microfibrils of both extracted and nonextracted leaf cuticles. Phenyl mercuric acetate-TCH deposits were randomly dispersed throughout the extracted cuticle and not associated with the polysaccharide microfibrils.Abbreviations TCH thiocarbohydrazide - PMA phenyl mercuric acetate     

A novel microsurgery method for intact plant tissue at the single cell level using ArF excimer laser microprojection

A novel microsurgery technique for the partial removal of rigid cell-walls in intact plant tissue is established. Using a size-variable slit, an ArF excimer laser was microprojected on the surface of the targeted cell, and this method enabled the area- and depth-controllable processing of the cortical structure of plant cells including the cuticle and cell wall layer. In epidermal cells of all tested plants, viabilities of more than 90% were retained 24 h after irradiation. Scanning electron microscope (SEM) observation revealed that the cuticle layer of the irradiated region was completely ablated, and the cellulose microfibrils of the secondary cell wall were partially removed; furthermore, 4 days after laser treatment, the regeneration of cell wall fibrils was observed. As a model experiment, the transient expression of synthetic green fluorescent protein (sGFP) was performed by the microinjection of cauliflower mosaic virus (CMV) 35S promoter-derived sGFP gene through an "aperture" in the treated cell surface. Moreover, micron-sized fluorescent beads were successfully introduced by the same method into the onion cells indicating that this method can be used to introduce foreign materials as large as organelles.     

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     

Electron diffraction from the primary wall of cotton fibers

Summary Electron diffraction patterns have been obtained from selected areas of disencrusted microfibrils isolated from the primary cell wall of cotton fibers. The resultant fiber diagram has the same meridional repeat distance as a corresponding pattern of secondary wall microfibrils but differs markedly in the equatorial reflections. The primary wall diagram displays only two strong equatorial reflections centered at 0.570 nm and 0.416 nm. The similarity of these spacings with those of cellulose IV suggests that the crystalline structure of the primary wall cellulose is similar to that of cellulose IV_I and is best explained in term of native cellulose I crystals having good longitudinal coherence (i.e., coherence along the length of the microfibrils) but with poor lateral organization of the network of inter chain hydrogen bonds. Similar results were also obtained for other primary wall specimens.     

The pattern of cell wall deterioration in lignocellulose fibers throughout enzymatic cellulose hydrolysis

Cell wall deterioration throughout enzymatic hydrolysis of cellulosic biomass is greatly affected by the chemical composition and the ultrastructure of the fiber cell wall. The resulting pattern of cell wall deterioration will reveal information on cellulose activity throughout enzymatic hydrolysis. This study investigates the progression and morphological changes in lignocellulose fibers throughout enzymatic hydrolysis, using (transmission electron microscopy) TEM and field emission scanning electron microscopy (FE‐SEM). Softwood thermo‐mechanical pulp (STMP) and softwood bleached kraft pulp (SBKP), lignocellulose substrates containing almost all the original fiber composition, and with lignin and some hemicellulose removed, respectively, was compared for morphology changes throughout hydrolysis. The difference of conversion between STMP and SBKP after 48 h of enzymatic hydrolysis is 11 and 88%, respectively. TEM images revealed an even fiber cell wall cross section density, with uneven middle lamella coverage in STMP fibers. SKBP fibers exhibited some spaces between cell wall and lamella layers due to the removal of lignin and some hemicellulose. After 1 h hydrolysis in SBKP fibers, there were more changes in the fiber cross‐sectional area than after 10 h hydrolysis in STMP fibers. Cell wall degradation was uneven, and originated in accessible cellulose throughout the fiber cell wall. FE‐SEM images illustrated more morphology changes in SBKP fibers than STMP fibers. Enzymatic action of STMP fiber resulted in a smoother fiber surface, along with fiber peeling and the formation of ribbon‐disjunction layers. SBKP fibers exhibited structural changes such as fiber erosion, fiber cutting, and fiber splitting throughout enzymatic hydrolysis. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Homofusion of Golgi secretory vesicles in flax phloem fibers during formation of the gelatinous secondary cell wall

The gelatinous type of secondary cell wall is present in tension wood and in phloem fibers of many plants. It is characterized by the absence of xylan and lignin, a high cellulose content and axially orientated microfibrils in the huge S2 layer. In flax phloem fiber, the major non-cellulosic component of such cell walls is tissue-specific galactan, which is tightly bound to cellulose. Ultrastructural analysis of flax fiber revealed that initiation of gelatinous secondary cell wall formation was accompanied by the accumulation of specific Golgi vesicles, which had a characteristic bicolor (dark-light) appearance and were easily distinguishable from vesicles made in different tissues and during the other stages of fiber development. Many of the bicolor vesicles appeared to fuse with each other, forming large vacuoles. The largest observed was 4 mum in diameter. Bicolor vesicles and vacuoles fused with the plasma membrane and spread their content in a characteristic "syringe-like" manner, covering a significant area of periplasm and forming "dark" stripes on the inner wall surface. Both Golgi derivatives and cell wall layers were labeled by LM5 antibody, indicating the presence of tissue- and stage-specific (1-->4)-beta-galactan. We suggest that this specific type of galactan secretion, which allows coverage of a large area of periplasm, is designed to increase the chance of the galactan meeting the cellulose microfibrils while they are still in the process of construction. The membrane fusion machinery of flax fiber must possess special components, which may be crucial for the formation of the gelatinous type cell wall.     

A kinesin-like protein is essential for oriented deposition of cellulose microfibrils and cell wall strength

Cortical microtubules have long been hypothesized to regulate the oriented deposition of cellulose microfibrils. However, the molecular mechanisms of how microtubules direct the orientation of cellulose microfibril deposition are not known. We have used fibers in the inflorescence stems of Arabidopsis to study secondary wall deposition and cell wall strength and found a fragile fiber (fra1) mutant with a dramatic reduction in the mechanical strength of fibers. The fra1 mutation did not cause any defects in cell wall composition, secondary wall thickening, or cortical microtubule organization in fiber cells. An apparent alteration was found in the orientation of cellulose microfibrils in fra1 fiber walls, indicating that the reduced mechanical strength of fra1 fibers probably was attributable to altered cellulose microfibril deposition. The FRA1 gene was cloned and found to encode a kinesin-like protein with an N-terminal microtubule binding motor domain. The FRA1 protein was shown to be concentrated around the periphery of the cytoplasm but absent in the nucleus. Based on these findings, we propose that the FRA1 kinesin-like protein is involved in the microtubule control of cellulose microfibril order.     

Xylan deposition on secondary wall of Fagus crenata fiber

Summary. Delignified and/or xylanase-treated secondary walls of Fagus crenata fibers were examined by field emission scanning electron microscopy. Microfibrils with a smooth surface were visible in the innermost surface of the differentiating fiber secondary wall. There was no ultrastructural difference between control and delignified sections, indicating that lignin deposition had not started in the innermost surface of the cell wall. There was no ultrastructural difference between control and xylanase-treated sections. Microfibrils on the outer part of the differentiating secondary wall surface had globular substances in delignified sections. These globular substances disappeared following xylanase treatment, indicating that these globules are xylan. The globular substances were not visible near the inner part of the differentiating secondary wall but gradually increased toward the outer part of the secondary wall, indicating that xylan penetrated into the cell wall and continuously accumulated on the microfibrils. Mature-fiber secondary walls were also examined by field emission scanning electron microscopy. Microfibrils were not apparent in the secondary wall in control specimens. Microfibrils with many globular substances were observed in the delignified specimens. Following xylanase treatment, the microfibrils had a smooth surface without any globules, indicating that the globular substance is xylan. These results suggest that cellulose microfibrils synthesized on the plasma membrane are released into the innermost surface of the secondary wall and coated with a thin layer of xylan. Successive deposition of xylan onto the cell wall increases the microfibril diameter. The large amounts of xylan that accumulated on microfibrils appear globular but are covered with lignin after they are deposited. Received February 20, 2001/Accepted September 1, 2001     

Immunolocalization of hemicelluloses in Arabidopsis thaliana stem. Part II: Mannan deposition is regulated by phase of development and its patterns of temporal and spatial distribution differ between cell types

Microdistribution of mannans in Arabidopsis stem was examined using immunolocalization with mannan-specific monoclonal antibodies (LM21 and LM22). Mannan labeling in secondary xylem cells (except for protoxylem vessels) was initially detected in the cell wall during S₂ formation and increased gradually during development. Labeling in metaxylem vessels (vessels) was detected earlier than that in xylary fibers (fibers), but was much weaker than fibers. The S₁ layer of vessels and fibers showed much less labeling than the S₂ layer. Some strong labeling was also detected in pit membranes of vessel pits. Interfascicular fibers (If-fibers) showed more heterogeneous labeling patterns than fibers by LM21. Unlike fibers, If-fibers also revealed some strong labeling in the cell corner of the S₁ layer, indicating different mannan labeling patterns between If-fibers and fibers. Interestingly, protoxylem vessels (proto-vessels) showed strong labeling at the early stage of secondary xylem formation with more intense labeling in the outer- than inner cell wall even though fibers and vessels showed no or very low labeling at this stage. Labeling intensity of proto-vessels was also much stronger than vessels and stronger or slightly weaker than fibers by LM21 and LM22, respectively. Using pectinase and mild alkali treatment, the presence of mannans in parenchymatous cells was also confirmed. Together our observations indicate that there are temporal and spatial variations in mannan labeling between cell types in the secondary xylem of Arabidopsis stems. Some similar features of mannan labeling between Arabidopsis and poplar are also discussed.     

The legume Rhizosphere

Summary Examination of the root surfaces of Medicago tribuloides Desr. with phase contrast microscopy or electron microscopy using thin sections revealed the presence of a layer of material outside the root surface. In thin sections of KMnO₄ fixed roots this layer was composed of a thin electron dense layer, an electron dense granular matrix of varying width and an enclosing electron dense membrane. After inoculation with an effective Rhizobium strain, rhizobia were found aggregated in a definite zone adjacent to the root surface when either living roots were examined by phase microscopy or thin sections by electron microscopy. This layer was also found in inoculated and uninoculated roots of Trifolium fragiferum and T. pratense. The bacteria were packed with inclusion granules and lay enclosed by a membrane layer adjacent to the granular matrix seen in uninoculated roots. The ultrastructural organisation of root hairs is essentially similar to that of other differentiated root cells. The replicated surface of the uninoculated root hair wall is largely amorphous with a few sculptured portions resembling a cuticle layer. The inoculated root hair wall often shows areas of exposed, open microfibrillar meshwork with rhizobia sitting on the microfibrils. The rhizobia resemble a flagellated, coccoid swarmer form of Rhizobium which is found in the barrel medic rhizosphere.     

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.     

Reciprocal influence of ethylene and gibberellins on response-gene expression in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

A cortical band of fiber cells originate de novo in tendrils of redvine [Brunnichia ovata (Walt.) Shiners] when these convert from straight, supple young filaments to stiffened coiled structures in response to touch stimulation. We have analyzed the cell walls of these fibers by in situ localization techniques to determine their composition and possible role(s) in the coiling process. The fiber cell wall consists of a primary cell wall and two lignified secondary wall layers (S₁ and S₂) and a less lignified gelatinous (G) layer proximal to the plasmalemma. Compositionally, the fibers are sharply distinct from surrounding parenchyma as determined by antibody and affinity probes. The fiber cell walls are highly enriched in cellulose, callose and xylan but contain no homogalacturonan, either esterified or de-esterified. Rhamnogalacturonan-I (RG-I) epitopes are not detected in the S layers, although they are in both the gelatinous layer and primary wall, indicating a further restriction of RG-I in the fiber cells. Lignin is concentrated in the secondary wall layers of the fiber and the compound middle lamellae/primary cell wall but is absent from the gelatinous layer. Our observations indicate that these fibers play a central role in tendril function, not only in stabilizing its final shape after coiling but also generating the tensile strength responsible for the coiling. This theory is further substantiated by the absence of gelatinous layers in the fibers of the rare tendrils that fail to coil. These data indicate that gelatinous-type fibers are responsible for the coiling of redvine tendrils and a number of other tendrils and vines.     

Tracing cellulose microfibril orientation in inner primary cell walls

Summary By quantitative analysis of cellulose microfibril orientation at different levels in the primary cell wall of a number of cell types, the development of wall texture was studied. Meristematic, isodiametric and cylindrical parenchyma cells and cells of a suspension culture were used. Within the newly deposited microfibril population, various orientations were recognized on the micrographs. Within subpopulations the orientation of undercrossing and overcrossing microfibrils were measured. These measurements showed a gradual shift in cellulose microfibril orientation in the different levels. Microfibrils showed predominant orientations at particular levels but microfibrils of intermediate orientation also occurred, although at a much lower density. As cellulose microfibrils of intermediate orientation were not closely packed, lamellae were not formed. Interwoven microfibrils were occasionally present, indicating that differently orientated microfibrils are occasionally deposited simultaneously. Also gradual changes in orientation over the entire inner cell wall surface were observed. From these observations it was inferred that microfibril deposition occurs with a small but regular and progressive change in orientation, the rotational motion, related to that of a helicoidal system.Dedicated to Professor Dr. M. M. A. Sassen on the occasion of his 65th birthday     

Microfibril-associated MAGP-2 stimulates elastic fiber assembly

Elastic fibers are complex structures composed of a tropoelastin inner core and microfibril outer mantle guiding tropoelastin deposition. Microfibrillar proteins mainly include fibrillins and microfibril-associated glycoproteins (MAGPs). MAGP-2 exhibits developmental expression peaking at elastic fiber onset, suggesting that MAGP-2 mediates elastic fiber assembly. To determine whether MAGP-2 regulates elastic fiber assembly, we used an in vitro model featuring doxycycline-regulated cells conditionally overexpressing exogenous MAGP-2 and constitutively expressing enhanced green fluorescent protein-tagged tropoelastin. Analysis by immunofluorescent staining showed that MAGP-2 overexpression dramatically increased elastic fibers levels, independently of extracellular levels of soluble tropoelastin, indicating that MAGP-2 stimulates elastic fiber assembly. This was associated with increased levels of matrix-associated MAGP-2. Electron microscopy showed that MAGP-2 specifically associates with microfibrils and that elastin globules primarily colocalize with MAGP-2-associated microfibrils, suggesting that microfibril-associated MAGP-2 facilitates elastic fiber assembly. MAGP-2 overexpression did not change levels of matrix-associated fibrillin-1, MAGP-1, fibulin-2, fibulin-5, or emilin-1, suggesting that microfibrils and other elastic fiber-associated proteins known to regulate elastogenesis do not mediate MAGP-2-induced elastic fiber assembly. Moreover, mutation analysis showed that MAGP-2 does not stimulate elastic fiber assembly through its RGD motif, suggesting that integrin receptor binding does not mediate MAGP-2-induced elastic fiber assembly. Because MAGP-2 interacts with Jagged-1 that controls cell-matrix interaction and cell motility, two key factors in elastic fiber macroassembly, microfibril-associated MAGP-2 may stimulate elastic fiber macroassembly by targeting the release of elastin globules from the cell membrane onto developing elastic fibers.     

The elastic fiber. I. The separation and partial characterization of its macromolecular components

The two morphologically different constituents of the mature elastic fiber, the central amorphous and the peripheral microfibrillar components, have been separated and partially characterized. A pure preparation of elastic fibers was obtained from fetal bovine ligamentum nuchae by extraction of the homogenized ligament with 5 M guanidine followed by digestion with collagenase. The resultant preparation consisted of elastic fibers which were morphologically identical with those seen in vivo. The microfibrillar components of these elastic fibers were removed either by proteolytic enzymes or by reduction of disulfide bonds with dithioerythritol in 5 M guanidine. The microfibrils solubilized by both methods were rich in polar, hydroxy, and sulfur-containing amino acids and contained less glycine, valine, and proline than the amorphous component of the elastic fiber. In contrast, the amino acid composition of the amorphous component was identical with that previously described for elastin. This component demonstrated selective susceptibility to elastase digestion, but was relatively resistant to the action of other proteolytic enzymes and to reduction. These observations establish that the microfibrils consist of a different connective tissue protein (or proteins) that is neither collagen nor elastin. During embryologic development the microfibrils form an aggregate structure before the amorphous component is secreted. These microfibrils may therefore play a primary role in the morphogenesis of the elastic fiber.