CYTOPLASMIC MICROTUBULE AND WALL MICROFIBRIL ORIENTATION IN ROOT HAIRS OF RADISH

The fine structure of young root hairs of radish was studied, with special attention to cytoplasm-wall relationships. Hairs up to 130 µ in length were examined after fixation of root tips in glutaraldehyde followed by osmium tetroxide. Microtubules occur axially aligned in the cytoplasm just beneath the plasmalemma, and extend from the base of the hair to within 2 to 3 µ of the tip. Poststaining with uranyl acetate and lead citrate clearly reveals in thin sections the presence of the two layers of cellulose microfibrils known from studies on shadowed wall preparations: an outer layer of randomly arranged microfibrils arising at the tip, and a layer of axially oriented microfibrils deposited on the inside of this layer along the sides. The youngest microfibrils of the inner, oriented layer first appear at a distance of about 25 µ from the tip. Although the microfibrils of the inner layer and the adjacent microtubules are similarly oriented, the oriented microtubules also extend through the 20- to 25-µ zone near the tip where the wall structure consists of random microfibrils. This suggests that the role of microtubules in wall deposition or orientation may be indirect.     

Cellulose microfibril orientation and cell shaping in developing guard cells of Allium: The role of microtubules and ion accumulation

Summary The role of microtubules and ions in cell shaping was investigated in differentiating guard cells of Allium using light and electron microscopy and cytochemistry. Microtubules appear soon after cytokinesis in a discrete zone close to the plasmalemma adjacent to the common wall between guard cells. The microtubules fan out from this zone, which corresponds to the future pore site, towards the other sides of the cell. Soon new cellulose microfibrils are deposited on the wall adjacent to the microtubules and oriented parallel to them. As the wall thickens, the shape of the cell shifts from cylindrical to kidney-like. Studies with polarized light show that guard cells gradually assume a birefringence pattern during development characteristic of wall microfibrils radiating away from the pore site. Retardation increases from 10 Å when cells just begin to take shape, to 80–100 Å at maturity. Both microfibril and microtubule orientation remain constant during development. Observations on aberrant cells including those produced under the influence of drugs such as colchicine, which leads to loss of microtubules, abnormal wall thickenings and disruption of wall birefringence, further support the role of microtubules in cell shaping through their function in the localization of wall deposition and the orientation of cellulose microfibrils in the new wall layer. Potassium first appears in guard mother cells before division and rapidly accumulates afterwards during cell shaping, as judged by the cobaltinitrite reaction. Some chloride and perhaps organic acid anions also accumulate. Thus, these ions, which are known to play a role in the function of mature guard cells, also seem to be important in the early growth and shaping of these cells.Abbreviations IPC isopropyl-N-phenylcarbamate - CB cytochalasin B - GMC guard mother cell - MTOC microtubule organizing center     

Progress in understanding the role of microtubules in plant cells

Microtubules have long been known to play a key role in plant cell morphogenesis, but just how they fulfill this function is unclear. Transverse microtubules have been thought to constrain the movement of cellulose synthase complexes in order to generate transverse microfibrils that are essential for elongation growth. Surprisingly, some recent studies demonstrate that organized cortical microtubules are not essential for maintaining or re-establishing transversely oriented cellulose microfibrils in expanding cells. At the same time, however, there is strong evidence that microtubules are intimately associated with cellulose synthesis activity, especially during secondary wall deposition. These apparently conflicting results provide important clues as to what microtubules do at the interface between the cell and its wall. I hypothesize that cellulose microfibril length is an important parameter of wall mechanics and suggest ways in which microtubule organization may influence microfibril length. This concept is in line with current evidence that links cellulose synthesis levels and microfibril orientation. Furthermore, in light of new evidence showing that a wide variety of proteins bind to microtubules, I raise the broader question of whether a major function of plant microtubules is in modulating signaling pathways as plants respond to sensory inputs from the environment.     

On the alignment of cellulose microfibrils by cortical microtubules: A review and a model

Summary The hypothesis that microtubules align microfibrils, termed the alignment hypothesis, states that there is a causal link between the orientation of cortical microtubules and the orientation of nascent microfibrils. I have assessed the generality of this hypothesis by reviewing what is known about the relation between microtubules and microfibrils in a wide group of examples: in algae of the family Characeae,Closterium acerosum, Oocystis solitaria, and certain genera of green coenocytes and in land plant tip-growing cells, xylem, diffusely growing cells, and protoplasts. The salient features about microfibril alignment to emerge are as follows. Cellulose microfibrils can be aligned by cortical microtubules, thus supporting the alignment hypothesis. Alignment of microfibrils can occur independently of microtubules, showing that an alternative to the alignment hypothesis must exist. Microfibril organization is often random, suggesting that self-assembly is insufficient. Microfibril organization differs on different faces of the same cell, suggesting that microfibrils are aligned locally, not with respect to the entire cell. Nascent microfibrils appear to associate tightly with the plasma membrane. To account for these observations, I present a model that posits alignment to be mediated through binding the nascent microfibril. The model, termed templated incorporation, postulates that the nascent microfibril is incorporated into the cell wall by binding to a scaffold that is oriented; further, the scaffold is built and oriented around either already incorporated microfibrils or plasma membrane proteins, or both. The role of cortical microtubules is to bind and orient components of the scaffold at the plasma membrane. In this way, spatial information to align the microfibrils may come from either the cell wall or the cell interior, and microfibril alignment with and without microtubules are subsets of a single mechanism.Dedicated to Professor Brian E. S. Gunning on the occasion of his 65th birthday     

AN ULTRASTRUCTURAL STUDY OF THE RELATIONSHIP BETWEEN MICROTUBULES AND MICROFIBRILS IN COTTON (GOSSYPIUM HIRSUTUM L.) CELL WALL REVERSALS

The arrangement of cellulosic fibrils in the cell walls of cotton fibers is very unusual; rather than exhibiting a continuous spiraling in one direction, they intermittantly reverse their gyre. Microtubules that line the periphery of the protoplasm, subjacent to the plasmalemma, tend to parallel the deployment of the cell wall microfibrils. It was not known whether this parallelism persisted through the reversal. By studying tangential sections of the cell wall/protoplasmic interfaces at the reversals, we show that congruity continues even through the reversals. Colchicine treatment did not appear to inhibit cellulose synthesis but it did abolish microtubules in the cotton fiber cells and deranged normal cell wall microfibrillar orientation. Previously, cotton fibers have been shown to possess most of the familiar organelles, but we found two new features not reported heretofore. They are microfilaments and peculiar “polygonal structures” that appear to be associated with the plasma membrane.     

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.     

Fine structure of the plasmalemma surface of poplar parenchyma cells observed by the freeze etching technique

The plasmalemma surface of poplar parenchyma cells observed by the freeze etching technique is characterized by 11 nm particles, primary pit fields, lomasome-like structures and fibrillar structures. The most remarkable feature is the occurrence of fibrillar structures, which are considered to be the imprint of underlying microtubules on the plasmalemma surface. The previously reported observation on the possible appearance of microtubules at the cytoplasmic surface inside the plasmalemma is questionable. Although the fibrillar structures run almost perpendicular to the main cell axis, longitudinally oriented ones are also found, the occurrence of which is discussed in relation to the orientation of the cellulose microfibrils.     

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

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

Microfibril orientation in plant cell walls

Summary The distribution of particles on the surface of the plasmalemma in the collenchyma of Apium graveolens was studied by the freeze-etching technique. The aim was to determine whether the distribution of particles was related to the known longitudinal or transverse orientation of cellulose microfibrils in different layers of the walls of these cells. Preliminary statistical studies have shown no obvious correlation between particle distribution and microfibril orientation although the distribution appeared uniform rather than random. Qualitatively, the particle distribution on the plasmalemma of differentiating xylem fibres of Eucalyptus maculata and of the cortical parenchyma of Avena sativa coleoptiles appeared to be similar to that observed on the plasmalemma of Apium. No correlation between the particle distribution and the microfibril orientation known to exist in the walls of these cells could be discerned.The orientation of microtubules in the cytoplasm of collenchyma cells of Apium graveolens was parallel to the microfibril orientation in many instances, but exceptions were noted. A possible interpretation for this variation is discussed. It is concluded that the microtubules are the structures which are most likely to be involved in determining microfibril orientation in the cell wall.     

Spatial relationship between microtubules and plasma-membrane rosettes during the deposition of primary wall microfibrils in Closterium sp.

The mechanism by which cortical microtubules (MTs) control the orientation of cellulose microfibril deposition in elongating plant cells was investigated in cells of the green alga, Closterium sp., preserved by ultrarapid freezing. Cellulose microfibrils deposited during formation of the primary cell wall are oriented circumferentially, parallel to cortical MTs underlying the plasma membrane. Some of the microfibrils curve away from the prevailing circumferential orientation but then return to it. Freeze-fracture electron microscopy shows short rows of particle rosettes on the P-face of the plasma membrane, also oriented perpendicular to the long axis of the cell. Previous studies of algae and higher plants have provided evidence that such rosettes are involved in the deposition of cellulose microfibrils. The position of the rosettes relative to the underlying MTs was visualized by deep etching, which caused much of the plasma membrane to collapse. Membrane supported by the MTs and small areas around the rosettes resisted collapse. The rosettes were found between, or adjacent to, MTs, not directly on top of them. Rows of rosettes were often at a slight angle to the MTs. Some evidence of a periodic structure connecting the MTs to the plasma membrane was apparent in freeze-etch micrographs. We propose that rosettes are not actively or directly guided by MTs, but instead move within membrane channels delimited by cortical MTs attached to the plasma membrane, propelled by forces derived from the polymerization and crystallization of cellulose microfibrils. More widely spaced MTs presumably allow greater lateral freedom of movement of the rosette complexes and result in a more meandering pattern of deposition of the cellulose fibrils in the cell wall.Abbreviations E-face exoplasmic fracture face - MT microtubule - P-face protoplasmic fracture-face     

The relationship of echinate inclusions and coated vesicles on wound healing inNitella flexilis (Characeae)

Summary Wound healing in internodal cells of the freshwater algaNitella flexilis (Characeae) was studied in the light and electron microscope. Immediately after punctation of the cell wall a wound plug is formed which stops outflow of cytoplasm. The plug consists of echinate inclusions which are normally located in the central vacuole. A wound wall consisting of pectin and cellulose microfibrils is formed beneath the plug within one to several hours. During that time the wound shows intensive fluorescence when treated with chlorotetracycline indicating transmembrane Ca²⁺ fluxes. Numerous coated pits and vesicles are found at the plasmalemma. The glycosomes undergo pronounced structural changes. Neither plug nor wound wall formation depend on actin filaments or microtubules as shown by inhibitor experiments with cytochalasin and amiprophos-methyl. The function of the coated vesicles and their interrelationship with other cell organelles is discussed.     

Colchicine and microfibril orientation

Summary Investigations on the mechanism of orientation of the cellulose microfibrils of the green algaOocystis solitaria have been carried out. This organism demonstrates easily observable and highly ordered microfibrils in its wall, which are arranged parallel to one another and regularly alternate at 90 from layer to layer of which there are approximately 30. During the entire wall development, and always parallel to one of the microfibril directions, are microtubules lying in the cortical cytoplasm. In the presence of 10^–2 M colchicine, microtubules are no longer detected and the typical cell wall pattern is not developed. The possible role of microtubules in the orientation of cellulose microfibrils is briefly discussed.     

The orientation of cell wall microtubules in wheat coleoptile segments subjected to phytohormone treatment

The effect of plant hormones was studied on the growth of excised coleoptile segments of wheat plantlets grown under daylight conditions. In addition to the change in growth, that in the orientation of microtubules and cellulose microfibrils was investigated in parenchyma cells. Following a 6-h treatment gibberellin, and still more kinetin, stímulated the thickening of segments, which became evident also in an altered orientation of microtubules. Whereas in the control the microtubules and wall microfibrils were oriented randomly, following gibberellin treatment they were all parallel and formed an acute angle with the longitudinal cell axis. A still more pronounced difference resulted after kinetin treatment, when microtubules were localized parallel with the longitudinal cell axis. Auxin had the opposite effect: it stimulated the elongation of the segments, which became evident in a transverse orientation of both wall microtubules and microfibrils.     

Reorientation of Microfibrils and Microtubules at the Outer Epidermal Wall of Maize Coleoptiles During Auxin-Mediated Growth

Auxin-mediated elongation growth of isolated subapical coleoptile segments of maize (Zea mays L.) is controlled by the extensibility of the outer cell wall of the outer epidermis (Kutschera et al., 1987). Here we investigate the hypothesis that auxin controls the extensibility of this wall by changing the orientation of newly deposited microfibrils through a corresponding change in the orientation of cortical microtubules. On the basis of electron micrographs it is shown that cessation of growth after removal of the endogenous source of auxin is correlated with a relative increase of longitudinally orientated microfibrils and microtubules at the inner wall surface. Conversely, reinduction of growth by exogenous auxin is correlated with a relative increase of transversely orientated microfibrils and microtubules at the inner wall surface. These changes can be detected 30–60 min after the removal and addition of auxin, respectively. The functional significance of directional changes of newly desposited wall microfibrils for the control of elongation growth is discussed.     

Chemical genetic screening identifies a novel inhibitor of parallel alignment of cortical microtubules and cellulose microfibrils

It is a well-known hypothesis that cortical microtubules control the direction of cellulose microfibril deposition, and that the parallel cellulose microfibrils determine anisotropic cell expansion and plant cell morphogenesis. However, the molecular mechanism by which cortical microtubules regulate the orientation of cellulose microfibrils is still unclear. To investigate this mechanism, chemical genetic screening was performed. From this screening, 'SS compounds' were identified that induced a spherical swelling phenotype in tobacco BY-2 cells. The SS compounds could be categorized into three classes: those that disrupted the cortical microtubules; those that reduced cellulose microfibril content; and thirdly those that had neither of these effects. In the last class, a chemical designated 'cobtorin' was found to induce the spherical swelling phenotype at the lowest concentration, suggesting strong binding activity to the putative target. Examining cellulose microfibril regeneration using taxol-treated protoplasts revealed that the cobtorin compound perturbed the parallel alignment of pre-existing cortical microtubules and nascent cellulose microfibrils. Thus, cobtorin could be a novel inhibitor and an attractive tool for further investigation of the mechanism that enables cortical microtubules to guide the parallel deposition of cellulose microfibrils.     

Microtubules in statocytes from roots of cress (Lepidium sativum L.)

Summary Statocytes in root caps ofLepidium sativum L. were examined by means of ultrathin serial sections to evaluate the amount and distribution of cortical microtubules. The microtubules encircle the cell, oriented normal to the root length axis. In the distal cell edges, microtubules form a network, separating the distal complex of endoplasmic reticulum from the plasmalemma. Preprophase bands in meristem cells are observable rarely, structures which can be regarded as nucleating sites for microtubules are lacking. During ageing of the root cap cells, the number of microtubules increases in combination with a decrease of microtubule length. Development of the roots on a horizontal clinostat preserves a younger developmental stage of the microtubule system regarding amount and length of the individual microtubules. Evidence for an involvement of microtubules in graviperception is low, whereas their role in orienting cellulose microfibrils cannot be ruled out. Compression of the distal network of microtubules after centrifugation of the roots indicates that microtubules in statocytes ofLepidium sativum L. roots might function in stabilizing the distal complex of endoplasmic reticulum.     

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.     

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.     

On the ultrastructure of differentiating secondary xylem in willow

Summary Studies of differentiating xylem inSalix fragilis L. show the immediate cambial derivatives to be ultrastructurally similar. The Golgi apparatus is important at all stages of wall synthesis, possibly producing (amongst other substances) hemicellulose material which is carried to the wall in vesicles or multivesicular bodies. The endoplasmic reticulum also contributes one or more components to the developing wall: at some stages during differentiation the endoplasmic reticulum produces electron opaque bodies which appear to be guided towards the wall by microtubules. Compact structures formed from concentric membranes (myelin-like bodies) have been found joined to rough endoplasmic reticulum, but their presence is not explained.Two types of plasmalemma elaboration occur: invagination of the plasmalemma itself to form vesicles which may contain cytoplasmic material; and vesicles between the plasmalemma and cell wall which are the result of single vesicles or multivesicular bodies traversing the plasmalemma. Both systems provide a means for transporting cytoplasmic material across the plasmalemma.Microtubules have been seen associated with all vesicles derived from the cytoplasm which appear to be moving towards the wall. The presence of microtubules may generally be explained in terms of orientation of vesicles, even if they also happen coincidentally to parallel the supposed orientation of microfibrils in the wall itself. It is possible to resolve connections between the microtubules and the plasmalemma.