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     

New techniques enable comparative analysis of microtubule orientation, wall texture, and growth rate in intact roots of Arabidopsis

This article explores root epidermal cell elongation and its dependence on two structural elements of cells, cortical microtubules and cellulose microfibrils. The recent identification of Arabidopsis morphology mutants with putative cell wall or cytoskeletal defects demands a procedure for examining and comparing wall architecture and microtubule organization patterns in this species. We developed methods to examine cellulose microfibrils by field emission scanning electron microscopy and microtubules by immunofluorescence in essentially intact roots. We were able to compare cellulose microfibril and microtubule alignment patterns at equivalent stages of cell expansion. Field emission scanning electron microscopy revealed that Arabidopsis root epidermal cells have typical dicot primary cell wall structure with prominent transverse cellulose microfibrils embedded in pectic substances. Our analysis showed that microtubules and microfibrils have similar orientation only during the initial phase of elongation growth. Microtubule patterns deviate from a predominantly transverse orientation while cells are still expanding, whereas cellulose microfibrils remain transverse until well after expansion finishes. We also observed microtubule-microfibril alignment discord before cells enter their elongation phase. This study and the new technology it presents provide a starting point for further investigations on the physical properties of cell walls and their mechanisms of assembly.     

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.     

Changes in the structural properties and rate of hydrolysis of cotton fibers during extended enzymatic hydrolysis

An extended enzymatic hydrolysis of cotton fibers by crude cellulase from Trichoderma pseudokoningii S-38 is described with characterization of both the enzyme changes of activities and cellulose structure. The hydrolysis rates declined drastically during the early stage and then slowly and steadily throughout the whole hydrolysis process the same trend could be seen during the following re-hydrolysis process. Morphological and structural changes to the fibers, such as swelling, frequent surface erosion, and variation in the packing and orientation of microfibrils, were investigated by scanning electron microscopy (SEM) and atomic force microscopy (AFM). Observation of X-ray diffraction and IR spectra suggests that the hydrolysis process results in a gradual increase in the relative intensity of the hydrogen bond network, and a gradual decrease in the apparent crystal size of cellulose. The I(alpha) crystal phase was hydrolyzed more easily than was the I(beta) crystal phase. Apart from the inactivation of CBHs activity, changes in the packing and arrangement of microfibrils and the structural heterogeneity of cellulose during hydrolysis could be responsible for the reduction in the rate of reaction, especially in its later stages. The results indicate that the enzymatic hydrolysis of cellulose occurs on the outer layer of the fiber surface and that, following this, the process continues in a sub-layer manner.     

Cellulose microfibril alignment recovers from DCB-induced disruption despite microtubule disorganization

Cellulose microfibril deposition patterns define the direction of plant cell expansion. To better understand how microfibril alignment is controlled, we examined microfibril orientation during cortical microtubule disruption using the temperature-sensitive mutant of Arabidopsis thaliana, mor1-1. In a previous study, it was shown that at restrictive temperature for mor1-1, cortical microtubules lose transverse orientation and cells lose growth anisotropy without any change in the parallel arrangement of cellulose microfibrils. In this study, we investigated whether a pre-existing template of well-ordered microfibrils or the presence of well-organized cortical microtubules was essential for the cell to resume deposition of parallel microfibrils. We first transiently disrupted the parallel order of microfibrils in mor1-1 using a brief treatment with the cellulose synthesis inhibitor 2,6-dichlorobenzonitrile (DCB). We then analysed the alignment of recently deposited cellulose microfibrils (by field emission scanning electron microscopy) as cellulose synthesis recovered and microtubules remained disrupted at the mor1-1 mutant's non-permissive culture temperature. Despite the disordered cortical microtubules and an initially randomized wall texture, new cellulose microfibrils were deposited with parallel, transverse orientation. These results show that transverse cellulose microfibril deposition requires neither accurately transverse cortical microtubules nor a pre-existing template of well-ordered microfibrils. We also demonstrated that DCB treatments reduced the ability of cortical microtubules to form transverse arrays, supporting a role for cellulose microfibrils in influencing cortical microtubule organization.     

Homogeneous suspensions of individualized microfibrils from TEMPO-catalyzed oxidation of native cellulose

Never-dried native celluloses (bleached sulfite wood pulp, cotton, tunicin, and bacterial cellulose) were disintegrated into individual microfibrils after oxidation mediated by the 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) radical followed by a homogenizing mechanical treatment. When oxidized with 3.6 mmol of NaClO per gram of cellulose, almost the totality of sulfite wood pulp and cotton were readily disintegrated into long individual microfibrils by a treatment with a Waring Blendor, yielding transparent and highly viscous suspensions. When observed by transmission electron microscopy, the wood pulp and cotton microfibrils exhibited a regular width of 3-5 nm. Tunicin and bacterial cellulose could be disintegrated by sonication. A bulk degree of oxidation of about 0.2 per one anhydroglucose unit of cellulose was necessary for a smooth disintegration of sulfite wood pulp, whereas only small amounts of independent microfibrils were obtained at lower oxidation levels. This limiting degree of oxidation decreased in the following order: sulfite wood pulp > cotton > bacterial cellulose, tunicin.     

Disruption of cellulose synthesis by 2,6‐dichlorobenzonitrile affects the structure of the cytoskeleton and cell wall construction in Arabidopsis

Cellulose is the major component of plant cell walls and is an important source of industrial raw material. Although cellulose biosynthesis is one of the most important biochemical processes in plant biology, the regulatory mechanisms of cellulose synthesis are still unclear. Here, we report that 2,6‐dichlorobenzonitrile (DCB), an inhibitor of cellulose synthesis, inhibits Arabidopsis root development in a dose‐ and time‐dependent manner. When treated with DCB, the plant cell wall showed altered cellulose distribution and intensity, as shown by calcofluor white and S4B staining. Moreover, pectin deposition was reduced in the presence of DCB when immunostained with the monoclonal antibody JIM5, which was raised against pectin epitopes. This result was confirmed using Fourier transform infrared (FTIR) analysis. Confocal microscopy revealed that the organisation of the microtubule cytoskeleton was significantly disrupted in the presence of low concentrations of DCB, whereas the actin cytoskeleton only showed changes with the application of high DCB concentrations. In addition, the subcellular dynamics of Golgi bodies labelled with N‐ST‐YFP and TGN labelled with VHA‐a1‐GFP were both partially blocked by DCB. Transmission electron microscopy indicated that the cell wall structure was affected by DCB, as were the Golgi bodies. Scanning electron microscopy showed changes in the organisation of cellulose microfibrils. These results suggest that the inhibition of cellulose synthesis by DCB not only induced changes in the chemical composition of the root cell wall and cytoskeleton structure, but also changed the distribution of cellulose microfibrils, implying that cellulose plays an important role in root development in Arabidopsis.     

Changes in the Arrangement of Microtubules and Microfibrils in Differentiating Conifer Tracheids During the Expansion of Cells

The arrangements of microtubules and the cellulose microfibrilsof radial walls in tracheids of Abies sachalinensis Mastersduring the expansion of cells were examined by immunofluorescenceand field-emission scanning electron microscopy. The radialdiameter of tracheids increased to three to four times thatof cambial initial cells. Microfibrils on the innermost surfaceof primary walls of conifer tracheids at early stages were notwell ordered and most of the microfibrils were oriented longitudinally.As each cell expanded, microfibrils in the process of depositionwere still not well ordered but their orientation changed fromlongitudinal to transverse. When cell expansion ceased, microfibrilswere well ordered and oriented transversely. Cortical microtubulesshowed a change in orientation similar to that of the microfibrils.These results indicate that the orientation of cortical microtubulesis correlated with that of microfibrils as they are being laiddown and with cell morphogenesis in conifer tracheids.Copyright1995, 1999 Academic Press Microfibril, microtubule, tracheid, cell expansion, Abies sachalinensis Masters, field-emission scanning electron microscopy, immunofluorescence microscopy     

Structure of Acetobacter cellulose composites in the hydrated state

The structure of composites produced by the bacterium Acetobacter xylinus have been studied in their natural, hydrated, state. Small-angle X-ray diffraction and environmental scanning electron microscopy has shown that the ribbons have a width of 500 A and contain smaller semi-crystalline cellulose microfibrils with an essentially rectangular cross-section of approximately 10 x 160 A(2). Incubation of Acetobacter in xyloglucan or pectin results in no changes in the size of either the microfibrils or the ribbons. Changes in the cellulose crystals are seen upon dehydration of the material, resulting in either a reduction in crystal size or an increase in crystal disorder.     

Alteration of oriented deposition of cellulose microfibrils by mutation of a katanin-like microtubule-severing protein

It has long been hypothesized that cortical microtubules (MTs) control the orientation of cellulose microfibril deposition, but no mutants with alterations of MT orientation have been shown to affect this process. We have shown previously that in Arabidopsis, the fra2 mutation causes aberrant cortical MT orientation and reduced cell elongation, and the gene responsible for the fra2 mutation encodes a katanin-like protein. In this study, using field emission scanning electron microscopy, we found that the fra2 mutation altered the normal orientation of cellulose microfibrils in walls of expanding cells. Although cellulose microfibrils in walls of wild-type cells were oriented transversely along the elongation axis, cellulose microfibrils in walls of fra2 cells often formed bands and ran in different directions. The fra2 mutation also caused aberrant deposition of cellulose microfibrils in secondary walls of fiber cells. The aberrant orientation of cellulose microfibrils was shown to be correlated with disorganized cortical MTs in several cell types examined. In addition, the thickness of both primary and secondary cell walls was reduced significantly in the fra2 mutant. These results indicate that the katanin-like protein is essential for oriented cellulose microfibril deposition and normal cell wall biosynthesis. We further demonstrated that the Arabidopsis katanin-like protein possessed MT-severing activity in vitro; thus, it is an ortholog of animal katanin. We propose that the aberrant MT orientation caused by the mutation of katanin results in the distorted deposition of cellulose microfibrils, which in turn leads to a defect in cell elongation. These findings strongly support the hypothesis that cortical MTs regulate the oriented deposition of cellulose microfibrils that determines the direction of cell elongation.     

Formation of root hairs by the wheat Triticum Vulgare Host. Trichoblasts.

The process of root hair formation has been studied by light-, transmission-and scanning electron microscopy. In the course of root hair development a break of the outer cell wall is observed by electron microscopy. It apparently occurs after the break of the fibrillar layer. The break of the outer layer of the cell wall is assumed to represent the break of the outer mucilage, the cuticle and the adjoining amorphous matrix with irregularly oriented cellulose microfibrils. The scheme of successive ultrastructural changes in the outer cell wall pattern during root hair formation is presented.     

STRUCTURE OF THE EUGLENOID STORAGE CARBOHYDRATE,PARAMYLON

Paramylon, the euglenoid storage carbohydrate, was studied by transmission electron microscopy (thin sectioning, negative staining, and freeze-etching) and scanning electron microscopy in order to develop a three-dimensional model of paramylon structure. The paramylon granule is a membrane-bound crystal composed of two types of segments, rectangular solids and wedges. The segments meet in the central region of the crystal. Both the segments and the granule as a whole are composed of several layers. Fibers traverse the paramylon granule in an overall concentric pattern. These fibers appear to be composed of bundles of microfibrils, which measure 4.0 nm in diameter and are similar to cellulose elementary microfibrils.     

A survey of cellulose microfibril patterns in dividing,expanding, and differentiating cells of Arabidopsis thaliana

Cellulose microfibrils are critical for plant cell specialization and function. Recent advances in live cell imaging of fluorescently tagged cellulose synthases to track cellulose synthesis have greatly advanced our understanding of cellulose biosynthesis. Nevertheless, cellulose deposition patterns remain poorly described in many cell types, including those in the process of division or differentiation. In this study, we used field emission scanning electron microscopy analysis of cryo-planed tissues to determine the arrangement of cellulose microfibrils in various faces of cells undergoing cytokinesis or specialized development, including cell types in which cellulose cannot be imaged by conventional approaches. In dividing cells, we detected microfibrillar meshworks in the cell plates, consistent with the concentration at the cell plate of cellulose synthase complexes, as detected by fluorescently tagged CesA6. We also observed a loss of parallel cellulose microfibril orientation in walls of the mother cell during cytokinesis, which corresponded with the loss of fluorescently tagged cellulose synthase complexes from these surfaces. In recently formed guard cells, microfibrils were randomly organized and only formed a highly ordered circumferential pattern after pore formation. In pit fields, cellulose microfibrils were arranged in circular patterns around plasmodesmata. Microfibrils were random in most cotyledon cells except the epidermis and were parallel to the growth axis in trichomes. Deposition of cellulose microfibrils was spatially delineated in metaxylem and protoxylem cells of the inflorescence stem, supporting recent studies on microtubule exclusion mechanisms.     

Making parallel lines meet: Transferring information from microtubules to extracellular matrix

The extracellular matrix is constructed beyond the plasma membrane, challenging mechanisms for its control by the cell. In plants, the cell wall is highly ordered, with cellulose microfibrils aligned coherently over a scale spanning hundreds of cells. To a considerable extent, deploying aligned microfibrils determines mechanical properties of the cell wall, including strength and compliance. Cellulose microfibrils have long been seen to be aligned in parallel with an array of microtubules in the cell cortex. How do these cortical microtubules affect the cellulose synthase complex? This question has stood for as many years as the parallelism between the elements has been observed, but now an answer is emerging. Here, we review recent work establishing that the link between microtubules and microfibrils is mediated by a protein named cellulose synthase-interacting protein 1 (CSI1). The protein binds both microtubules and components of the cellulose synthase complex. In the absence of CSI1, microfibrils are synthesized but their alignment becomes uncoupled from the microtubules, an effect that is phenocopied in the wild type by depolymerizing the microtubules. The characterization of CSI1 significantly enhances knowledge of how cellulose is aligned, a process that serves as a paradigmatic example of how cells dictate the construction of their extracellular environment.     

Diurnal difference in the amount of immunogold-labeled glucomannans detected with field emission scanning electron microscopy at the innermost surface of developing secondary walls of differentiating conifer tracheids

The differences between cell wall formation at night, when the tangential strain used as an index of the volumetric changes in differentiating cells is high, and in the day, when the tangential strain is low, were investigated in Cryptomeria japonica D. Don. Samples containing differentiating xylem were collected at 0500 hours and 1400 hours. The innermost surface of developing secondary walls in differentiating tracheids was observed by field emission scanning electron microscopy. In the specimens collected at 0500 hours, an amorphous material was observed covering the cellulose microfibrils. The cell wall surface was immunogold-labeled with an anti-glucomannan antiserum. After chlorite treatment, the amorphous material disappeared, and immunogold labeling was rarely observed. In the specimens collected at 1400 hours, cellulose microfibrils were clearly evident, and amorphous material and immunogold labeling were rarely observed. We thus confirmed that much amorphous material containing glucomannans is observed at night, when differentiating tracheids are turgid due to the increase in their volume, while the amorphous material was rarely observed during the day when cellulose microfibrils are clearly observed.     

Investigation into the structural, morphological, mechanical and thermal behaviour of bacterial cellulose after a two-step purification process

Bacterial cellulose (BC) is a natural hydrogel, which is produced by Acetobacter xylinum (recently renamed Gluconacetobacter xylinum) in culture and constitutes of a three-dimensional network of ribbon-shaped bundles of cellulose microfibrils. Here, a two-step purification process is presented that significantly improves the structural, mechanical, thermal and morphological behaviour of BC sheet processed from these hydrogels produced in static culture. Alkalisation of BC using a single-step treatment of 2.5 wt.% NaOH solution produced a twofold increase in Young's modulus of processed BC sheet over untreated BC sheet. Further enhancements are achieved after a second treatment with 2.5 wt.% NaOCl (bleaching). These treatments were carefully designed in order to prevent any polymorphic crystal transformation from cellulose I to cellulose II, which can be detrimental for the mechanical properties. Scanning electron microscopy and thermogravimetric analysis reveals that with increasing chemical treatment, morphological and thermal stability of the processed films are also improved.     

Evidence for the role of the glomerulocyte in cellulose synthesis in the tunicate,Metandrocarpa uedai

Summary The tunicate,Metandrocarpa uedai, contains a large quantity of cellulose; however, it is not known how and where the cellulose is synthesized. Based on evidence from electron diffraction and conventional thin-sectioning for electron microscopy, this study shows that the glomerulocyte is involved in the synthesis of cellulose. The bundles of microfibrils in the glomerulocyte as well as the tunic were identified as cellulose I using selected area electron diffraction analysis. The diffraction pattern of cellulose in the glomerulocyte was similar to that from the tunic, suggesting that the crystallization of cellulose already is initiated in the glomerulocyte. The diameter of cellulose microfibrils, both in the glomerulocyte and the tunic was the same, about 16 nm. These results suggest that the glomerulocyte is the most probable site for the synthesis of cellulose in the tunic ofM. uedai. Using thin-sectioning techniques, a series of observations showed that individual microfibrils are primarily assembled in structures tentatively identified as vacuole-like structures, then they are bundled by a tapering region within the vacuole-like structures. These bundles of microfibrils are deposited in a continuously circular arrangement. The microtubules are oriented parallel to the bundles of microfibrils at the tapering vacuole-like structure, and they may be involved in the tapering of these structures (perhaps controlling the shape). This study also provides the first account for the involvement of a vacuole-like structure in the synthesis of cellulose microfibrils among living organisms.     

TEMPO-mediated oxidation of native cellulose: Microscopic analysis of fibrous fractions in the oxidized products

The 2,2,6,6-tetramethylpiperidine-1-oxy radial (TEMPO)-mediated oxidation was applied to aqueous suspensions of cotton linters, ramie and spruce holocellulose at pH 10.5, and water-insoluble fractions of the TEMPO-oxidized celluloses collected by filtration with water were analyzed by optical and transmission electron microscopy and others. The results showed that both fibrous forms and microfibrillar nature of the original native celluloses were maintained after the TEMPO-mediated oxidation, even though carboxylate and aldehyde groups of 0.67–1.16 and 0.09–0.21 mmol/g, respectively, were introduced into the water-insoluble fractions. Neither crystallinity nor crystal size of cellulose I of the original native celluloses was changed under the conditions adopted in this study. Carboxylate groups in the TEMPO-oxidized ramie were mapped by labeling with lead ions as their counter ions. The transmission electron micrographs indicated that some heterogeneous distribution of carboxylate groups along each cellulose microfibril or each bundle of cellulose microfibrils seemed to be present in the TEMPO-oxidized celluloses.     

Distribution of carboxylate groups introduced into cotton linters by the TEMPO-mediated oxidation

The 2,2,6,6-tetramethylpiperidine-1-oxy radial (TEMPO)-mediated oxidation was applied to aqueous slurries of cotton linters. The water-insoluble fibrous fractions thus obtained in the yields of more than 78% were characterized by solid-state ¹³C-NMR, X-ray diffraction and scanning electron microscopic analyses for evaluation of distribution of carboxylate groups formed in the TEMPO-oxidized celluloses. The patterns of solid-state ¹³C-NMR spectra revealed that the oxidation occurred at the C6 primary hydroxyl groups of cellulose. X-ray diffraction and scanning electron microscopic analyses showed that such C6 oxidation took place at the surfaces of cellulose I crystallites without any oxidation at the C6 of inside cellulose I crystallites. Thus, carboxylate and aldehyde groups introduced into the TEMPO-oxidized celluloses are densely present on the surfaces of cellulose I crystallites. In addition, the obtained results revealed that the shoulder signal due to non-crystalline C6 carbons at about 63 ppm in solid-state ¹³C-NMR spectra of native celluloses is ascribed to those of surfaces of cellulose I crystallites or those of cellulose microfibrils.     

Assessment of in vitro binding of isolated pectic domains to cellulose by adsorption isotherms, electron microscopy, and X-ray diffraction methods

Isolated pectic domains representative of the pectic backbone and the neutral sugar side chains were tested for their ability to interact with cellulose in comparison to the well-known binding of xyloglucan. Pectic side chains displayed a significant in vitro binding capacity to cellulose, whereas pectic backbone domains exhibited only slight adsorption to cellulose microfibrils. To support the binding results, electron microscopy and X-ray diffraction were applied. Celluloses from bacteria and sugar beet cell walls were used as substrates for the precipitation of isolated pectic domains or xyloglucan by acetone vapor diffusion. Pectic side chains grew attached to the cellulose surfaces, whereas pectic backbone domains were observed separately from cellulose microfibrils. Xyloglucan seeded with cellulose provoked a decrease of microfibrils entanglement, but no clear cross-links between neighboring microfibrils were observed. These results led to the elucidation of the pectic domains responsible for binding with cellulose microfibrils.