The Cellulose System in the Cell Wall ofMicrasterias

The cellulose system of the cell wall ofMicrasterias denticulataandMicrasterias rotatawas analyzed by diffraction contrast transmission electron microscopy, electron diffraction, and X-ray analysis. The studies, achieved on disencrusted cell ghosts, confirmed that the cellulose microfibrils occurred in crisscrossed bands consisting of a number of parallel ribbon-like microfibrils. The individual microfibrils had thicknesses of 5 nm for a width of around 20 nm, but in some instances, two or three microfibrils merged into one another to yield larger monocrystalline domains reaching up to 60 nm in lateral size. The orientation of the cellulose ofMicrasteriasis very unusual, as it was found that in the cell wall, the equatorial crystallographic planes of cellulose having ad-spacing of 0.60 nm [(110) in the Iβ cellulose unit cell defined by Sugiyamaet al.,1991,Macromolecules24, 4168–4175] were oriented perpendicular to the cell wall surface. Up to now, such orientation has been found only inSpirogyra,another member of the Zygnemataceae group. The unusual structure of the secondary wall cellulose ofMicrasteriasmay be tentatively correlated with the unique organization of the terminal complexes, which in this alga occur as hexagonal arrays of rosettes.     

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.     

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.     

LIGNIFICATION DURING SECONDARY WALL FORMATION IN COLEUS: AN ELECTRON MICROSCOPIC STUDY

The fine structure of lignin deposition was examined in developing secondary walls of wound vessel members in Coleus. KMnO₄, which was used as the fixative, selectively reacts with the lignin component of the cell wall and thus can be used as a highly sensitive electron stain to follow the course of lignification during secondary wall deposition. Lignin was first detected as conspicuuos electron-opaque granules in the primary wall in the region where the secondary wall thickening arises and as fine granular striations extending into the very young secondary wall. As the secondary wall develops lignification becomes progressively more extensive. In cross sections the lignified secondary wall appears as concentric, fine granular striations; in tangent al or oblique sections it is seen as delicate, beaded fibrils paralleling the long axis of the thickening. High magnification of tangential or oblique sections shows that the fibrillar appearance is due to the presence of alternating light and dark layers each approximately 25-35 A wide. It is assumed that the light layers are the cellulose microfibrils and the dark regions contain lignin which fills the space between the microfibrils. KMnO₄, by selectively reacting with lignin, thus negatively stains the cellulose microfibrils revealing their orientation and dimensions.     

On the polarity of cellulose in the cell wall of Valonia

The orientation of the triclinic phase of cellulose in the cell wall of Valonia ventricosa J. Agardh was investigated by X-ray- and electron-diffraction analysis. In addition to the well-documented uniplanar-axial organization of the cell wall which requires that the a ^* axis should be always perpendicular to the wall surface, the direction of this axis was also found to be pointing outward from the plasma membrane side of the wall. This unidirectionality was persistent throughout the various layers that constitute the cell wall and also for the three microfibrillar orientations that occur in Valonia cell walls. The unidirectionality of the a ^* axis indicates, in particular, that the Valonia cellulose microfibrils are not twisted along their axis. These observations are consistent with a cellulose biosynthetic scheme where a close association exists between terminal-complex orientations and those of the cellulose microfibrils. In this context, the unidirectionality of the a ^* axis of cellulose seems to be related to the restricted mobility of the terminal complexes which are able to slide in the plasma membrane but not to rotate along their long axis.Abbreviations TC terminal complex This work was initiated during a visit of J.F.R at Grenoble in the framework of a France-Québec exchange program. J.S. was recipient of a CNRS fellowship. The diagram in Fig. 8 was kindly drawn for us by Miss Yukie Saito from the Department of Forest Products, the University of Tokyo.     

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.     

A new model for packing of type-I collagen molecules in the native fibril

A specific fibril model is presented consisting of bundles of five-stranded microfibrils, which are usually disordered (except axially) but under lateral compression become ordered. The features are as follows (whereD = 234 residues or 67 nm): (1)D-staggered collagen molecules 4.5D long in the helical microfibril have a left-handed supercoil with a pitch of 400–700 residues, but microfibrils need not have helical symmetry. (2) Straight-tilted 0.5-D overlap regions on a near-hexagonal lattice contribute the discrete x-ray diffraction reflections arising from lateral order, while the gap regions remain disordered. (3) The overlap regions are equivalent, but are crystallographically distinguished by systematic displacements from the near-hexagonal lattice. (4) The unit cell is the same as in a recently proposed three-dimensional crystal model, and calculated intensities in the equatorial region of the x-ray diffraction pattern agree with observed values.     

Further evidence from sectioned material in support of the existence of a linear terminal complex in cellulose synthesis

Summary Transmembrane linear terminal complexes considered to be involved in the synthesis of cellulose microfibrils have been described in the plasma membrane ofBoergesenia forbesii. Evidence for the existence of these structures has been obtained almost exlusively using the freeze etching technique. In the present study an attempt has been made to complete these studies using conventional fixation, staining, and sectioning procedures. In developing cells ofBoergesenia forbesii, strongly stained structures traversing the plasma membrane and averaging 598.9 nm ± 171.3 nm in length, 28.7 nm ± 4.2 nm in width, and 35.2 nm ± 6.6 nm in depth have been demonstrated. These structures are considered to be linear terminal complexes. At their distal (cell wall) surface, they appear to be closely associated with cellulose microfibrils. At the proximal (cytoplasmic) surface, they are associated with microtubules and polysomes. A model of the possible interrelation of the terminal complexes and microtubules leading to the generation of cell wall microfibrils is proposed.     

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.     

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.     

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.     

Cellulose microfibrils from banana rachis: Effect of alkaline treatments on structural and morphological features

Four different alkaline treatments for isolation of cellulose microfibrils from vascular bundles of banana rachis were comparatively studied. Isolated cellulose microfibrils were characterized using high performance anion exchange chromatography for neutral sugar composition, as well as attenuated total reflection Fourier transform infrared spectroscopy, transmission electron microscopy, X-ray and electron diffraction and solid-state ¹³C NMR. The cellulose microfibrils treated with peroxide alkaline, peroxide alkaline–hydrochloric acid or 5 wt% potassium hydroxide had average diameters of 3–5 nm, estimated lengths of several micrometers. Although the interpretation of their structure is difficult because of the low cristallinity, X-ray diffraction, ¹³C NMR and ATR-FTIR results suggested that cellulose microfibrils from banana rachis could be either interpreted as cellulose IV₁ or cellulose Iβ. The specimens treated with a more concentrated KOH solution (18 wt%) were still microfibrillated but their structure was converted to cellulose II.     

Almost Pure Iα Cellulose in the Cell Wall of Glaucocystis

Crystalline features of cellulose microfibrils in the cell walls of Glaucocystis (Glaucophyta) were studied by combined spectroscopy and diffraction techniques, and the results were compared with those of Oocystis (Chlorophyta). Although these algae are grouped into two different classes, by the composition of their chloroplasts for instance, their cell walls are quite similar in size and morphology. The most striking features of their cellulose crystallites are that they have the highest cellulose I_α contents reported to date. In particular, the I_α fraction of cellulose from Glaucocystis was found to be as high as 90% from ¹³C NMR analysis. The mode of preferential orientation of cellulose crystallites in their cell walls is also interesting; equatorial 0.53-nm lattice planes were oriented parallel to the cell surface in the case of Glaucocystis, while the 0.62-nm planes were parallel to the Oocystis cell surface. Such a structural variation provides another link to the evolution of cellulose structure, biosynthesis, and its biocrystallization mechanism.     

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.     

Three AtCesA6‐like members enhance biomass production by distinctively promoting cell growth in Arabidopsis

Cellulose is an abundant biopolymer and a prominent constituent of plant cell walls. Cellulose is also a central component to plant morphogenesis and contributes the bulk of a plant's biomass. While cellulose synthase (CesA) genes were identified over two decades ago, genetic manipulation of this family to enhance cellulose production has remained difficult. In this study, we show that increasing the expression levels of the three primary cell wall AtCesA6‐like genes (AtCesA2, AtCesA5, AtCesA6), but not AtCesA3, AtCesA9 or secondary cell wall AtCesA7, can promote the expression of major primary wall CesA genes to accelerate primary wall CesA complex (cellulose synthase complexes, CSCs) particle movement for acquiring long microfibrils and consequently increasing cellulose production in Arabidopsis transgenic lines, as compared with wild‐type. The overexpression transgenic lines displayed changes in expression of genes related to cell growth and proliferation, perhaps explaining the enhanced growth of the transgenic seedlings. Notably, overexpression of the three AtCesA6‐like genes also enhanced secondary cell wall deposition that led to improved mechanical strength and higher biomass production in transgenic mature plants. Hence, we propose that overexpression of certain AtCesA genes can provide a biotechnological approach to increase cellulose synthesis and biomass accumulation in transgenic plants.     

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.     

Composition of the cell wall of Chlorella fusca

Isolated cell walls of mature Chlorella fusca consisted of about 80% carbohydrate, 7% protein, and 13% unidentified material. Mannose and glucose were present in a ratio of about 2.7:1 and accounted for most of the carbohydrate. Minor components were glucuronic acid, rhamnose, and traces of other sugars; galactose was absent. After treatment with 2 M trifluoroacetic acid or with 80% acetic acid/HNO₃ (10/1, v/v), a residue with a mannose/glucose ratio of 0.3:1 was obtained, probably representing a structural polysaccharide. An X-ray diffraction diagram of the walls showed one diffuse reflection at 0.44 nm and no reflections characteristic of cellulose. Walls from young cells contained about 51% carbohydrate, 12% protein, and 37% unidentified material. Mannose and glucose were also the main sugars; their absolute amounts per wall increased 6–7 fold during cell growth. Walls isolated with omission of a dodecylsulphate/mercaptoethanol/urea extraction step had a higher protein content and, with young walls, a significantly higher glucose and fucose content. These data and other published cell wall analyses show a wide variability in cell wall composition of the members of the genus Chlorella.Abbreviations GLC gas liquid chromatography - TFA trifluoroacetic acid     

Ethylene is a modulator of gibberellic acid-induced antheridiogenesis in <Emphasis Type="Italic">Anemia phyllitidis</Emphasis> gametophytes

In fern (Anemia phyllitidis) gametophytes cellulose in the walls of the antheridial zone cells which was organized in clusters and spots was transformed via dispersed form to fibrillar arrangement (layered in oblique and perpendicular array in relation to the transverse direction of cell expansion) during antheridiogenesis induced by gibberellic acid (GA₃) and/or enhanced by 1-aminocyclopropane-1-carboxylic acid (ACC). In the ACC-treated gametophytes, where antheridia were not induced, the cellulose was arranged in the same manner. Aminooxyacetic acid (AOA), which inhibits antheridiogenesis and development of fern gametophytes, produced in the cell walls both random and longitudinal type of organization of cellulose microfibrils, however, in the GA₃/AOA-treated plants the oblique type was also observed. The total numbers of cells with perpendicular and/or oblique type of cellulose microfibrils in the GA₃-, GA₃/ACC-and GA₃/AOA-treated gametophytes corresponded to the average number of antheridia formed. Moreover, it was found that the extracts from the gametophytes treated with GA₃ or with the mixture of GA₃ and ACC contained significantly less soluble sugars but more α-amylase-and endoglucanase-released sugars than the extracts from the gametophytes of the other series. Thin layer chromatography of the samples from the cell wall extracts hydrolyzed by endoglucanase contained xylose and cellobiose which suggested that these sugars built the xyloglucans, hemicellulose polymers responsible for tethering of walls of fern gametophyte cells like in higher plants.     

Structure of Cellulose Microfibrils in Primary Cell Walls from Collenchyma

In the primary walls of growing plant cells, the glucose polymer cellulose is assembled into long microfibrils a few nanometers in diameter. The rigidity and orientation of these microfibrils control cell expansion; therefore, cellulose synthesis is a key factor in the growth and morphogenesis of plants. Celery (Apium graveolens) collenchyma is a useful model system for the study of primary wall microfibril structure because its microfibrils are oriented with unusual uniformity, facilitating spectroscopic and diffraction experiments. Using a combination of x-ray and neutron scattering methods with vibrational and nuclear magnetic resonance spectroscopy, we show that celery collenchyma microfibrils were 2.9 to 3.0 nm in mean diameter, with a most probable structure containing 24 chains in cross section, arranged in eight hydrogen-bonded sheets of three chains, with extensive disorder in lateral packing, conformation, and hydrogen bonding. A similar 18-chain structure, and 24-chain structures of different shape, fitted the data less well. Conformational disorder was largely restricted to the surface chains, but disorder in chain packing was not. That is, in position and orientation, the surface chains conformed to the disordered lattice constituting the core of each microfibril. There was evidence that adjacent microfibrils were noncovalently aggregated together over part of their length, suggesting that the need to disrupt these aggregates might be a constraining factor in growth and in the hydrolysis of cellulose for biofuel production.Growth and form in plants are controlled by the precisely oriented expansion of the walls of individual cells. The driving force for cell expansion is osmotic, but the rate and direction of expansion are controlled by the mechanical properties of the cell wall (Szymanski and Cosgrove, 2009). Expanding, primary cell walls are nanocomposite materials in which long microfibrils of cellulose, a few nanometers in diameter, run through a hydrated matrix of xyloglucans, pectins, and other polymers (Knox, 2008; Mohnen, 2008; Szymanski and Cosgrove, 2009; Scheller and Ulvskov, 2010). Native cellulose microfibrils are partially crystalline (Nishiyama, 2009; Fernandes et al., 2011). Formerly, primary wall cellulose was thought to have a unique crystal structure called cellulose IV₁ (Dinand et al., 1996), but NMR evidence suggests the presence of forms similar to the better characterized cellulose Iα and Iβ crystalline forms together with large quantities of less ordered cellulose (Wickholm et al., 1998; Sturcová et al., 2004; Wada et al., 2004). Nevertheless, cellulose is much more ordered than any other component of the primary cell wall (Bootten et al., 2004), in keeping with its key role of providing strength and controlling growth.The stiffness of the cell wall is greatest in the direction of the cellulose microfibrils, where growth is directional and the predominant microfibril orientation is usually transverse to the growth direction (Green, 1999; MacKinnon et al., 2006; Szymanski and Cosgrove, 2009). Expansion of the cell wall then requires either widening of the spacing between microfibrils (Marga et al., 2005) or slippage between them (Cosgrove, 2005), or both, and the microfibrils reorient toward the direction of growth (Anderson et al., 2010). Polymer cross bridges between microfibrils (McCann et al., 1990) are thought to resist these deformations of the cell wall nanostructure and, thus, to control the rate of growth. Until recently, most attention was focused on bridging xyloglucans, hydrogen bonded to microfibril surfaces (Scheller and Ulvskov, 2010). However, there is evidence that not all xyloglucans are appropriately positioned (Fujino et al., 2000; Park and Cosgrove, 2012a) and that other bridging polymers may be involved (Zykwinska et al., 2007). It has also been suggested that bundles of aggregated microfibrils, not single microfibrils, might be the key structural units in primary cell walls (Anderson et al., 2010), as in wood (Fahlén and Salmén, 2005; Fernandes et al., 2011). If so, single microfibrils could bridge between microfibril bundles. In summary, the growth of plant cells is not well understood, and we need more information on how cellulose orientation is controlled and on the nature of the bridging polymers, the cellulose surfaces to which these polymers bind, and the cohesion between microfibril surfaces that might mediate aggregation.Cellulose microfibrils are synthesized at the cell surface by large enzyme complexes having hexagonal symmetry, sometimes called “rosettes” (Somerville, 2006). Each complex contains multiple cellulose synthases that differ between primary cell walls and wood, although the appearance of the complexes is similar (Somerville, 2006; Atanassov et al., 2009). The simultaneous synthesis, from the same end, of all the chains in a native cellulose microfibril is why they are parallel (Nishiyama et al., 2002, 2003), in contrast to the entropically favored antiparallel structure found in man-made celluloses like rayon (Langan et al., 2001). The number of chains in a microfibril and the number of cellulose synthases in the synthetic complex are evidently related. It is commonly assumed that the number of chains is divisible by six, matching the hexagonal rosette symmetry, and 36-chain models (Himmel et al., 2007) bounded by the hydrophilic [110] and [1-10] crystal faces, as in algal celluloses (Bergenstråhle et al., 2008), have been widely adopted. The assembly and orientation of cellulose are connected, as several cellulose synthase mutants have phenotypes defective in cellulose orientation and plant form as well as depleted in cellulose content (Paredez et al., 2008). In certain other mutant lines, the crystallinity of the microfibrils appears to be affected (Fujita et al., 2011; Harris et al., 2012; Sánchez-Rodríguez et al., 2012).Therefore, a detailed understanding of the structure of primary wall cellulose microfibrils would help us to understand cellulose synthesis as well as the growth and structural mechanics of living plants (Burgert and Fratzl, 2009). Primary cell walls and their cellulose skeletons also affect food quality characteristics like the crispness of salad vegetables and apples (Malus domestica; Jarvis, 2011). When biofuels are produced from lignocellulosic biomass, lignification leads to recalcitrance (Himmel et al., 2007), but some of the cell types in Miscanthus spp., switchgrass (Panicum virgatum), and arable crop residues have only primary walls with no lignin, and recalcitrance then depends on the nature of the cellulose microfibrils (Beckham et al., 2011).A relatively detailed structure has recently been proposed for the microfibrils of spruce (Picea spp.) wood (Fernandes et al., 2011), which are 3.0 nm in diameter, allowing space for only about 24 cellulose chains. Evidence from x-ray diffraction supported a “rectangular” shape (Matthews et al., 2006) bounded by the [010] and [200] faces. There was considerable disorder increasing toward the surface, and the microfibrils were aggregated into bundles about 15 to 20 nm across, with some, but not all, of the lateral interfaces being resistant to water (Fernandes et al., 2011). Disordered domains are a feature of other strong biological materials such as spider silk (van Beek et al., 2002).Therefore, it is of interest whether any of these features of wood cellulose might also be found in the cellulose microfibrils of primary (growing) cell walls. It would be particularly useful to characterize the disorder known to be present in primary wall microfibrils, that is, to define how cellulose that is not measured as “crystalline” differs from crystalline cellulose. Many of the experiments leading toward a structure for wood cellulose were dependent on exceptionally uniform orientation of the cellulose microfibrils (Sturcová et al., 2004; Fernandes et al., 2011). However, in growing cell walls, the microfibrils are not uniformly oriented. When microfibrils are first laid down at the inner face of the primary cell wall, their orientation is normally transverse to the direction of growth, but as the cell wall expands, the microfibrils reorient so that the orientation distribution, integrated across the thickness of the expanded cell wall, becomes progressively closer to random (Cosgrove, 2005; MacKinnon et al., 2006).This technical problem does not apply to the cell walls of celery (Apium graveolens) collenchyma, which are similar in composition to other primary cell walls but have their microfibrils oriented relatively uniformly along the cell axis (Sturcová et al., 2004; Kennedy et al., 2007a, 2007b). Some structural information on celery collenchyma cellulose has already been derived from spectroscopic and scattering experiments (Sturcová et al., 2004; Kennedy et al., 2007a, 2007b), confirming the disorder expected in a primary wall cellulose. Some of these experiments were analogous to what has been done on spruce cellulose (Fernandes et al., 2011), but insufficient data are available to specify the number of chains in each primary wall microfibril, the nature and location of the disorder, and the presence or absence of direct contact between microfibrils. Here, we report x-ray and neutron scattering and spectroscopic experiments addressing these questions and leading to a proposed structure for primary wall cellulose microfibrils. Characterizing a structure containing so much disorder presented unusual challenges, but disorder appears to be central to the enigmatic capacity of primary wall cellulose to provide high strength and yet to permit and control growth.     

Interference of cell wall regeneration ofBoergesenia forbesii protoplasts by Tinopal LPW,a fluorescent brightening agent

Summary Wounding cells ofBoergesenia forbesii (Harvey) Feldmann induces the synchronous formation of numerous protoplasts which synthesize large cellulose microfibrils within 2–3 hours after wounding. The microfibrils appear to be assembled by linear terminal synthesizing complexes (TCs). TC subunits appear on both E- and P-faces of the plasma membrane, thus suggesting the occurrence of a transmembrane complex. The direction of microfibril synthesis is random during primary wall assembly and becomes ordered during secondary wall assembly. The average density of TCs during secondary wall deposition is 1.7/m², and the average length of the TC is 510 nm. TC organization is similar to that ofValonia macrophysa; however, the larger TCs ofBoergesenia (510 nm vs. 350 nm) produce correspondingly larger microfibrils (30 nm vs. 20 nm).The effects of a fluorescent brightening agent (FBA), Tinopal LPW, on cell wall regeneration ofBoergesenia protoplasts was investigated. The threshold level of Tinopal LPW for interfering with microfibril assembly is 1.5 M. At 95 M Tinopal (for short periods up to 15 minutes), microfibril impressions have atypical spherical impressions at their termini. At longer incubations (24 hours), TCs and microfibril impressions are absent. When washed free of Tinopal, the protoplasts eventually resume normal wall assembly; however, TCs do not reappear until at least 30 minutes after the removal of Tinopal. In consideration of the presence of ordered TCs before FBA treatment, their random distribution upon recovery implies an intermediate stage of assembly or possiblyde novo synthesis.