Changes in microfibril arrangement on the inner surface of the epidermal cell walls in the epicotyl of Vigna angularis ohwi et ohashi during cell growth

Throughout the entire period of cell growth, the microfibrils on the inner surface of the outer tangential walls of the epidermal cells of Vigna angularis epicotyls are running parallel to one another and their orientation differs from cell to cell. Although transverse, oblique and longitudinal microfibrils can be observed irrespective of cell age, the frequency distribution of microfibril orientation changes with age. In young cells, transversely oriented microfibrils predominate. In cells of medium age, which are still undergoing elongation, transverse, oblique and longitudinal microfibrils are present in quite similar frequencies. In old, non-growing cells, longitudinally oriented microfibrils are predominent. A decrease in the relative frequency of transversely oriented microfibrils with cell age was also observed in the radial epidermal walls.     

Wall architecture in the cellulose-deficientrsw1 mutant ofArabidopsis thaliana: Microfibrils but not microtubules lose their transverse alignment before microfibrils become unrecognizable in the mitotic and elongation zones of roots

Summary Thersw1 mutant ofArabidopsis thaliana has a single amino acid substitution in a putative glycosyl transferase that causes a temperature-dependent reduction in cellulose production. We used recently described methods to examine root growth by surface marker particles, cell wall structure by field emission scanning electron microscopy and microtubule alignment by immunofluorescence after the mutant is transferred to its restrictive temperature. We find that raising the temperature quickly accelerates root elongation in both wild type and mutant, presumably as a result of general metabolic stimulation, but that in the mutant, the rate declines within 7–8 h and elongation almost ceases after 24 h. Radial swelling begins at about 6 h in the mutant and root diameter continues to increase until about 24 h. The normal transverse alignment of microfibrils is severely impaired in the mutant after 8 h, and chemical inhibition of cellulose synthesis by 2,6-dichlorobenzonitrile causes a similar loss of orientation. After 24 h, microfibrils are not clearly visible in the walls of cells that would have been in the mitotic and early-elongation zone of wild-type roots. Changes in older cells are less marked; loss of transverse microfibril orientation occurs without disruption to the transverse orientation of cortical microtubules. The wild type shows none of the changes except for acceleration of elongation, which in its case is sustained. We conclude that microfibril alignment requires the normal functioning of RSW1 and that, in view of the effects of dichlorobenzonitrile, there may be a more general linkage between the rate of cellulose production and its proper alignment.Abbreviations DCB 2,6-dichlorobenzonitrile - REGR relative elemental growth rate Dedicated to Professor Brian E. S. Gunning on the occasion of his 65th birthday     

Effects of hypergravity on the elongation growth in radish and cucumber hypocotyls

The elongation growth of the hypocotyls of radish and cucumber seedlings was examined under hypergravity in a newly developed centrifuge (Kasaharaet al. 1995). The effects of hypergravity on elongation growth differed between the two species. The rate of elongation of radish hypocotyls was reduced under basipetal hypergravity (H+20g) but not under acropetal hypergravity (H-13g), as compared to growth under the control conditions (C+1g and C-1g). In cucumber hypocotyls, elongation growth was inhibited not only by basipetal but also by acropetal hypergravity. Under these conditions, the reduction in the elongation growth of both radish and cucumber hypocotyls was accompanied by an increase in their thickness. Although no distinct differences in relative composition of neutral sugars were found, the amounts of cell-wall components (pectic substances, hemicelluloses and cellulose) per unit length of hypocotyls were increased by exposure to hypergravity.     

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.     

Differential regulation of cellulose orientation at the inner and outer face of epidermal cells in the Arabidopsis hypocotyl

It is generally believed that cell elongation is regulated by cortical microtubules, which guide the movement of cellulose synthase complexes as they secrete cellulose microfibrils into the periplasmic space. Transversely oriented microtubules are predicted to direct the deposition of a parallel array of microfibrils, thus generating a mechanically anisotropic cell wall that will favor elongation and prevent radial swelling. Thus far, support for this model has been most convincingly demonstrated in filamentous algae. We found that in etiolated Arabidopsis thaliana hypocotyls, microtubules and cellulose synthase trajectories are transversely oriented on the outer surface of the epidermis for only a short period during growth and that anisotropic growth continues after this transverse organization is lost. Our data support previous findings that the outer epidermal wall is polylamellate in structure, with little or no anisotropy. By contrast, we observed perfectly transverse microtubules and microfibrils at the inner face of the epidermis during all stages of cell expansion. Experimental perturbation of cortical microtubule organization preferentially at the inner face led to increased radial swelling. Our study highlights the previously underestimated complexity of cortical microtubule organization in the shoot epidermis and underscores a role for the inner tissues in the regulation of growth anisotropy.     

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.     

Fine structural observations on the epidermis

Summary The organization of the wall of epidermal cells in the petiole of species of Apium, Eryngium, Rumex, and Abutilon as well as that of the epidermis of Avena coleoptile has been investigated. The outer and inner tangential walls consist of layers in which the cellulose microfibrils are oriented alternately parallel or transverse to the longitudinal cell axis. This organization resembles that previously described for collenchyma cell walls (Wardrop, 1969; Chafe, 1970). On the radial (anticlinal) walls the orientation of the microfibrils is transverse and these appear continuous with the layers of transverse orientation of the outer and inner tangential walls. Variation in thickness of the outer tangential, and radial, and inner tangential walls appears to result from the variation in thickness of those layers in which the microfibrils have a longitudinal orientation. The extent to which these observations can interpreted in terms of some type of modified multi-net growth is discussed.     

Interaction between wall deposition and cell elongation in dark-grown hypocotyl cells in Arabidopsis

A central problem in plant biology is how cell expansion is coordinated with wall synthesis. We have studied growth and wall deposition in epidermal cells of dark-grown Arabidopsis hypocotyls. Cells elongated in a biphasic pattern, slowly first and rapidly thereafter. The growth acceleration was initiated at the hypocotyl base and propagated acropetally. Using transmission and scanning electron microscopy, we analyzed walls in slowly and rapidly growing cells in 4-d-old dark-grown seedlings. We observed thick walls in slowly growing cells and thin walls in rapidly growing cells, which indicates that the rate of cell wall synthesis was not coupled to the cell elongation rate. The thick walls showed a polylamellated architecture, whereas polysaccharides in thin walls were axially oriented. Interestingly, innermost cellulose microfibrils were transversely oriented in both slowly and rapidly growing cells. This suggested that transversely deposited microfibrils reoriented in deeper layers of the expanding wall. No growth acceleration, only slow growth, was observed in the cellulose synthase mutant cesA6(prc1-1) or in seedlings, which had been treated with the cellulose synthesis inhibitor isoxaben. In these seedlings, innermost microfibrils were transversely oriented and not randomized as has been reported for other cellulose-deficient mutants or following treatment with dichlorobenzonitrile. Interestingly, isoxaben treatment after the initiation of the growth acceleration in the hypocotyl did not affect subsequent cell elongation. Together, these results show that rapid cell elongation, which involves extensive remodeling of the cell wall polymer network, depends on normal cellulose deposition during the slow growth phase.     

Light-induced inhibition of elongation growth in sunflower hypocotyls

Summary The long-term effects of white light (WL) on epidermal cell elongation and the mechanical properties and ultrastructure of cell walls were investigated in the subapical regions of hypocotyls of sunflower seedlings (Helianthus annuus L.) that were grown in darkness. Upon transition to WL a drastic inhibition of epidermal cell elongation was observed. However, the mechanical properties of the inner tissues (cortex, vascular bundles, and pith) were unaffected by WL. Thus, the light-induced decrease in cell wall plasticity measured on entire stems occurs exclusively in the peripheral tissues (epidermis and 2 to 3 subepidermal cell layers).An electronmicroscopic investigation of the epidermal cell walls showed that they are of the helicoidal type with the direction of microfibrils monotonously changing during deposition. This cell wall type was identified by the appearance of arced patterns of microfibrils in cell walls sectioned oblique to the plane of their synthesis. WL irradiation did not change the periodicity of this pattern nor the thickness of the lamellae. Thus, the inhibition of cell elongation was not caused or accompanied by a shift in the direction of microfibril deposition in the growth-limiting outer tissues. However, cell wall thickness, the number of lamellae and hence the amount of cellulose oriented parallel and transverse to the longitudinal cell axis increased in WL. This may account for the effect of WL on the reduction of cell wall plasticity and growth.Abbreviations D darkness - PATAg periodic acid-thiocarbohydracide-silver protein - WL white light     

Establishing and maintaining axial growth: wall mechanical properties and the cytoskeleton

Organ morphology depends on cell placement and directional cell expansion. Microtubules are involved in both of these processes so genetic approaches to understand the role microtubules play in organ expansion are not straightforward. Our use of the temperature-sensitive mor1-1 mutants led to the surprising discovery that Arabidopsis thaliana (L.) Heynh. root cells can establish and maintain transverse cellulose texture without well organized microtubule arrays. This work also demonstrated that cells can lose the ability to expand anisotropically without losing transversely oriented cellulose microfibrils. We suggest that microtubule disruption affects the cells ability to generate long cellulose microfibrils, which may be essential for achieving growth anisotropy. Thus organ shape may depend not only on the orientation but also on the relative length of cellulose microfibrils during axis establishment and growth. More recent work has shown an important correlation between microtubule organization and the deposition patterns of the glycosylphosphatidylinositol (GPI)-anchored wall protein COBRA. Loss of microtubule organization is associated with the dissipation of transverse banding patterns of COBRA, suggesting that COBRAs function in maintaining anisotropic expansion may be microtubule-dependent.     

Cell-wall synthesis and elongation growth in hypocotyls of Helianthus annuus L.

The relationship between growth and increase in cell-wall material (wall synthesis) was investigated in hypocotyls of sunflower seedlings (Helianthus annuus L.) that were either grown in the dark or irradiated with continuous white light (WL). The peripheral three to four cell layers comprised 30–50% of the entire wall material of the hypocotyl. The increase in wall material during growth in the dark and WL, respectively, was larger in the inner tissues than in the peripheral cell layers. The wall mass per length decreased continuously, indicating that wall thinning occurs during growth of the hypocotyl. When dark-grown seedlings were transfered to WL, a 70% inhibition of growth was observed, but the increase in wall mass was unaffected. Likewise, the composition of the cell walls (cellulose, hemicellulose, pectic substances) was not affected by WL irradiation. Upon transfer of dark-grown seedlings into WL a drastic increase in wall thickness and a concomitant decrease in cell-wall plasticity was measured. The results indicate that cell-wall synthesis and cell elongation are independent processes and that, as a result, WL irradiation of etiolated hypocotyls leads to a thickening and mechanical stiffening of the cell walls.     

FT-IR study of the Chara corallina cell wall under deformation

Fourier-transform infrared (FT-IR) microspectroscopy was used to investigate both the chemical composition of, and the effects of an applied strain on, the structure of the Chara corallina cell wall. The inner layers of the cell wall are known to have a transverse cellulose orientation with a gradient through the thickness to longitudinal orientation in the older layers. In both the native state and following the removal of various biopolymers by a sequential extraction infrared dichroism was used to examine the orientation of different biopolymers in cell-wall samples subjected to longitudinal strain. In the Chara system, cellulose microfibrils were found to be aligned predominantly transverse to the long axis of the cell and became orientated increasingly transversely as longitudinal strain increased. Simultaneously, the pectic polysaccharide matrix underwent molecular orientation parallel to the direction of strain. Following extraction in CDTA, microfibrils were orientated transversely to the strain direction, and again the degree of transverse orientation increased with increasing strain. However, the pectic polysaccharides of the matrix were not detected in the dichroic difference spectra. After a full sequential extraction, the cellulose microfibrils, now with greatly reduced crystallinity, were detected in a longitudinal direction and they became orientated increasingly parallel to the direction of strain as it increased.     

Stem Elongation and Cell Wall Proteins in Flowering Plants

Abstract: The growth of stems (hypocotyls, epicotyls) and stem-like organs (coleoptiles) in developing seedlings is largely due to the elongation of cells in the sub-apical region of the corresponding organ. According to the organismal concept of plant development, the thick outer epidermal wall, which can be traced back to the peripheral cell wall of the zygote, creates a sturdy organ sheath that determines the rate of stem elongation. The cells of the inner tissues are the products of secondary partitioning of one large protoplast; these turgid, thin-walled cells provide the driving force for organ growth. The structural differences between these types of cell walls are described (outer walls: thick, sturdy, helicoidal cellulose architecture; inner walls: thin, extensible, transversely-oriented cellulose microfibrils). On the basis of these facts, current models of cell wall loosening (and wall stiffening) are discussed with special reference to the expansin, enzymatic polymer remodelling and osmiophilic particle hypothesis. It is concluded that the exact biochemical mechanism(s) responsible for the coordinated yielding of the growth-controlling peripheral organ wall(s) have not yet been identified.     

Regulation of cell division in pea root tips after wounding: A possible role for ethylene

Summary Calcofluor White ST is a fluorescent brightener that has previously been shown to alter cellulose ribbon assembly in the bacteriumAcetobacter xylinum. In this report, we demonstrate that Calcofluor also disrupts cell wall assembly in the eukaryotic algaOocystis apiculata. When observed with polarization microscopy, walls altered by Calcofluor show reduced birefringence relative to controls. Electron microscopy has shown that these altered walls contain regions which consist primarily of amorphous material and which generally lack organized microfibrils. We propose that wall alteration occurs because Calcofluor binds with the glucan chains polymerized by the cellulose synthesizing enzymes as they are produced. As a consequence, the glucan chains are prevented from co-crystallizing to form microfibrils. Synthesis of normal walls resumes when Calcofluor is removed, which is consistent with our proposal that Calcofluor acts by direct physical interaction with newly synthesized wall components.Several types of fluorescent patterns at the cell wall/plasmalemma interface have also been observed following Calcofluor treatment. Fluorescent spots, striations; helical bands, and lens-shaped thickenings have been documented. Each of these patterns may be the result of the interaction of Calcofluor with cellulose at different spatial or temporal levels or from varying concentrations of the brightener itself. Helical bands and lens-shaped thickenings also have been examined with the electron microscope. Like other regions of wall alteration, they are found to contain primarily amorphous material. Finally, we note that cells with severely disrupted walls are unable to complete their normal life cycle.     

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

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

A principal role for AtXTH18 in Arabidopsis thaliana root growth: a functional analysis using RNAi plants

Rearrangement of cellulose microfibrils within cell-wall matrices is considered one of the most critical steps in the regulation of both the orientation and extent of cell expansion in plants. Xyloglucan endotransglucosylase/hydrolases (XTHs) are a family of enzymes that mediate the construction and restructuring of load-bearing cross links among cellulose microfibrils. The Arabidopsis thaliana XTH genes AtXTH17, 18, 19, and 20 are phylogenetically closely related to one another and are preferentially expressed in the roots. However, they exhibit different expression profiles within the root and respond to hormonal signals differently. To investigate their functions in root growth, we examined phenotypes of loss-of-function mutants for these genes using T-DNA insertion lines and RNAi plants. These functional analyses disclosed a principal role for the AtXTH18 gene in primary root elongation. Of the four XTH genes, AtXTH18 exhibits the highest level of mRNA expression. We also determined auxin-signaling pathways for these genes using a mutant with a defect in the AXR2/IAA7 gene and found that the expression of AtXTH19 in the elongation/maturation region of the root is under the control of the AXR2/IAA7 signaling pathway.     

Winding threads around plant cells Applications of the geometrical model for microfibril deposition

Summary Based on precise information about the orientations of cellulose microfibrils (CMFs) in the secondary cell wall of theEquisetum hyemale root hair, a geometrical model was recently put forward to account for the deposition orientation of CMFs. The model supposes that synthases spin out the CMFs and that geometrical laws dictate their movement. Taking space-limiting conditions into account, CMF orientation is dependent on cell morphology, the amount of other wall molecules adhering to the CMFs, and the number and distribution pattern of synthases. In the present paper this geometrical model for CMF deposition is further applied to nontip-growing angular cells with varying diameters, cells with tapering morphology, various distribution patterns of synthases, various matrix/fibril ratios, and intercalarily elongating cells. The model can accurately predict the actual wall textures in a great variety of cell walls. In the proposed model for CMF orientation, microtubules are not required as cellular guiding structures for the CMFs, not even in elongating walls. They are supposed to be involved in cell elongation, possibly by delivering wall material including CMF synthases.Abbreviation CMF cellulose microfibril     

Anisotropic cell growth and cell wall structure in lenticular cell of Valonia utricularis (Ulvophyceae)

During cell division of the giant-celled green alga, Valonia utricularis, a lenticular cell is newly formed, which grows from disc-shaped to globular to obovoid. During the early developmental stages of growth, the cell surface shows a remarkable outward protrusion. In the present study, the anisotropy of cell growth, i.e. the difference between cell surface extension in meridional and radial orientation, was investigated by analyzing the movement of the surface markers in a living cell. Growth was isotropic around the cell zenith but of two different kinds of anisotropic growth in other regions; radial extension was dominant in cell periphery and meridional extension in intermediate regions between zenith and periphery. Moreover, local orientation of cellulose microfibrils was observed on the inner surface of the cell wall during different stages of early development in lenticular cell using an atomic force microscope. Cellulose microfibrils showed meridional orientation overall and this phenomenon was most remarkable in the periphery of the cell, suggesting the possibility of cellulose microfibrils promoting radial extension of cells by suppressing meridional extension of cell wall.     

Solid-state 13C-NMR spectroscopy shows that the xyloglucans in the primary cell walls of mung bean (Vigna radiata L.) occur in different domains: a new model for xyloglucan-cellulose interactions in the cell wall

Xyloglucans (XG) with different mobilities were identified in the primary cell walls of mung beans (Vigna radiata L.) by solid-state 13C-NMR spectroscopy. To improve the signal:noise ratios compared with unlabelled controls, Glc labelled at either C-1 or C-4 with 13C-isotope was incorporated into the cell-wall polysaccharides of mung bean hypocotyls. Using cell walls from seedlings labelled with d-[1-13C]glucose and, by exploiting the differences in rotating-frame and spin-spin proton relaxation, a small signal was detected which was assigned to Xyl of XGs with rigid glucan backbones. After labelling seedlings with d-[4-13C]glucose and using a novel combination of spin-echo spectroscopy with proton spin relaxation-editing, signals were detected that had 13C-spin relaxations and chemical shifts which were assigned to partly-rigid XGs surrounded by mobile non-cellulosic polysaccharides. Although quantification of these two mobility types of XG was difficult, the results indicated that the partly-rigid XGs were predominant in the cell walls. The results lend support to the postulated new cell-wall models in which only a small proportion of the total surface area of the cellulose microfibrils has XG adsorbed on to it. In these new models, the partly-rigid XGs form cross-links between adjacent cellulose microfibrils and/or between cellulose microfibrils and other non-cellulosic polysaccharides, such as pectic polysaccharides.     

A xyloglucan endotransglucosylase/hydrolase involves in growth of primary root and alters the deposition of cellulose in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Xyloglucan endotransglucosylase/hydrolases (XTHs) are a class of enzymes that mediate the construction and restructure of the cellulose/xyloglucan framework by splitting and reconnecting xyloglucan molecule cross-linking among cellulose microfibrils. Remodification of cellulose microfibrils within cell-wall matrices is realized to be one of the most critical steps in the regulation of cells expansion in plants. Thirty-three XTH genes have been found in Arabidopsis thaliana but their roles remain unclear. AtXTH21 (At2g18800), an Arabidopsis XTH gene that mainly expresses in root and flower, exhibits different expression profiles from other XTH members under hormone treatment. We examined loss-of-function mutants using T-DNA insertion lines and overexpression lines and found that the AtXTH21 gene played a principal role in the growth of the primary roots by altering the deposition of cellulose and the elongation of cell wall.