The cyclic reorientation of cortical microtubules on walls with a crossed polylamellate structure: effects of plant hormones and an inhibitor of protein kinases on the progression of the cycle

Summary The outer tangential wall (OTW) of epidermal cells of azuki bean epicotyls has a crossed polylamellate structure, in which lamellae of longitudinal cellulose microfibrils alternate with lamellae of transverse cellulose microfibrils. This implies that the cyclic reorientation of cortical microtubules (MTs) from longitudinal to transverse and from transverse to longitudinal occurs on the OTW. Treatment with a solution that contained no auxin caused the accumulation of cells with longitudinal MTs, suggesting that auxin is required for the reorientation of MTs from longitudinal to transverse during the reorientation cycle. Treatment with 6-dimethylaminopurine (DMAP), an inhibitor of protein kinases that promoted the reorientation of MTs from transverse to longitudinal, resulted in the accumulation of cells with longitudinal MTs. Subsequent treatment with auxin caused a marked increase in the percentage of cells with transverse MTs and then a decrease in the percentage, indicating that the reorientation of MTs from longitudinal to transverse and then from transverse to longitudinal occurred during treatment with auxin. The percentage of cells with transverse MTs decreased more slowly in segments that had been pretreated with gibberellin A₃ (GA) than in segments that had been pretreated without GA, suggesting that GA, in cooperation with auxin, caused the suppression of the reorientation of MTs from transverse to longitudinal.Abbreviations BL brassinolide - BSA bovine serum albumin - GA gibberellin A₃ - DMAP 6-dimethylaminopurine - DMSO dimethylsulfoxide - FITC fluorescein isothiocyanate - IAA indoleacetic acid - MT microtubule - OTW outer tangential wall - PBS phosphate-buffered saline Dedicated to Professor Eldon H. Newcomb in recognition of his contributions to cell biology     

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.     

Relationship between microtubule orientation and patterns of cell expansion in developing cortical cells of Lemna minor roots

In actively growing cortical cells in the elongation zone of Lemna minor L. roots, both longitudinal (radial and tangential) and transverse walls expand in both length and width. The longitudinal walls of the three types of cortical cells in the root (i.e. outer, middle and inner) showed the largest expansion in the longitudinal axis. In contrast, the inner cortical cells exhibited the least expansion in width, whereas the middle cortical cells displayed the largest expansion in width. Thus, the profiles of the expansion of longitudinal walls were characteristic for the three types of cortical cells. In this study, both the orientation of cortical microtubule (MT) arrays and their dynamic reorientation, and the density of cortical MTs, were documented and correlated to the patterns of cell wall expansion. Significantly, transverse arrays of cortical MTs were most prominent in the radial walls of the inner cortical cells, and least so in those of the middle cortical cells. Toward the base of roots, beyond the elongation zone, the orientation of cortical MTs shifted continuously from transverse to oblique and then to longitudinal. In this case, the rate of shift in the orientation of cortical MTs along the root axis was appreciably faster in the middle cortical cells than in the other two types of cortical cells. Interestingly, the continuous change in cortical MT orientation was not confirmed in the transverse walls which showed much smaller two-dimensional expansion than the radial walls. Additionally, the presence of fragmented or shortened cortical MTs rapidly increased concomitantly with the decrease of transversely oriented cortical MTs. This relationship was especially prominent in the transverse walls of the inner cortical cells, which displayed the least expansion among the three types of cortical cells investigated. In the root elongation zone, the density of cortical MTs in the inner cortical cells was about three times higher than that in the other two cortical cell types. These results indicate that in the early stage of cell expansion, the orientation of cortical MTs determines a preferential direction of cell expansion and both the shifting orientation and density of cortical MTs affect the magnitude of expansion in width of the cell wall.     

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.     

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.     

Nucleus-associated microtubules help determine the division plane of plant epidermal cells: avoidance of four-way junctions and the role of cell geometry

To investigate the spatial relationship between the nucleus and the cortical division site, epidermal cells were selected in which the separation between these two areas is large. Avoiding enzyme treatment and air drying, Datura stramonium cells were labeled with antitubulin antibodies and the three-dimensional aspect of the cytoskeletons was reconstructed using computer-aided optical sectioning. In vacuolated cells preparing for division, the nucleus migrates into the center of the cell, suspended by transvacuolar strands. These strands are now shown to contain continuous bundles of microtubules which bridge the nucleus to the cortex. These nucleus-radiating microtubules adopt different configurations in cells of different shape. In elongated cells with more or less parallel side walls, oblique strands radiating from the nucleus to the long side walls are presumably unstable, for they are progressively realigned into a transverse disc (the phragmosome) as broad, cortical, preprophase bands (PPBs) become tighter. The phragmosome and the PPB are both known predictors of the division plane and our observations indicate that they align simultaneously in elongated epidermal cells. These observations suggest another hypothesis: that the PPB may contain microtubules polymerized from the nuclear surface. In elongated cells, the majority of the radiating microtubules, therefore, come to anchor the nucleus in the transverse plane, consistent with the observed tendency of such cells to divide perpendicular to the long axis. In nonrectangular isodiametric epidermal cells, which approximate regular hexagons in section, the radial microtubular strands emanating from the nucleus tend to remain associated with the middle of each subtending cell wall. The strands are not reorganized into a single dominant transverse bar, but remain as a starlike array until mitosis. PPBs in these cells are not as tight; they may only be a sparse accumulation of microtubules, even forming along non-diametrical radii. This arrangement is consistent with the irregular division patterns observed in epidermal mosaics of isodiametric D. stramonium cells. The various conformations of the radial strands can be modeled by springs held in two-dimensional hexagonal frames, and by soap bubbles in three-dimensional hexagonal frames, suggesting that the division plane may, by analogy, be selected by minimal path criteria. Such behavior offers a cytoplasmic explanation of long-standing empirically derived "rules" which state that the new cell wall tends to meet the maternal wall at right angles. The radial premitotic strands and their analogues avoid taking the longer path to the vertex of an angle where a cross wall is already present between neighboring cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Transient gibberellin application promotes Arabidopsis thaliana hypocotyl cell elongation without maintaining transverse orientation of microtubules on the outer tangential wall of epidermal cells

The phytohormone gibberellin (GA) promotes plant growth by stimulating cellular expansion. Whilst it is known that GA acts by opposing the growth-repressing effects of DELLA proteins, it is not known how these events promote cellular expansion. Here we present a time-lapse analysis of the effects of a single pulse of GA on the growth of Arabidopsis hypocotyls. Our analyses permit kinetic resolution of the transient growth effects of GA on expanding cells. We show that pulsed application of GA to the relatively slowly growing cells of the unexpanded light-grown Arabidopsis hypocotyl results in a transient burst of anisotropic cellular growth. This burst, and the subsequent restoration of initial cellular elongation rates, occurred respectively following the degradation and subsequent reappearance of a GFP-tagged DELLA (GFP-RGA). In addition, we used a GFP-tagged α-tubulin 6 (GFP-TUA6) to visualise the behaviour of microtubules (MTs) on the outer tangential wall (OTW) of epidermal cells. In contrast to some current hypotheses concerning the effect of GA on MTs, we show that the GA-induced boost of hypocotyl cell elongation rate is not dependent upon the maintenance of transverse orientation of the OTW MTs. This confirms that transverse alignment of outer face MTs is not necessary to maintain rapid elongation rates of light-grown hypocotyls. Together with future studies on MT dynamics in other faces of epidermal cells and in cells deeper within the hypocotyl, our observations advance understanding of the mechanisms by which GA promotes plant cell and organ growth.     

Rearrangements of F-actin during Stomatogenesis Visualised by Confocal Microscopy in Fixed and Permeabilised Tradescantia Leaf Epidermis

Abstract: New details of F-actin organisation in leaf epidermal and stomatal cells were revealed by rhodamine — and fluorescein — phalloidin staining of fixed epidermal peels of Tradescantia virginiana and visualisation by confocal microscopy. Non-specialised epidermal cells contain highly organised arrays of fine cortical actin filaments aligned in transverse or oblique orientations. In interphase guard mother cells (GMCs), the arrangement of cortical F-actin changes on the periclinal and anticlinal cell walls at different times during differentiation. Initially, cortical F-actin on the periclinal surfaces is oriented transversely and F-actin is evenly distributed around the anticlinal walls. Following polarisation of the adjacent subsidiary mother cells (SMCs), actin in GMCs concentrates on the lateral anticlinal walls, but not on the transverse walls. Subsequently, F-actin on the periclinal walls reorients to radial and then longitudinal. Organisation of F-actin in SMCs appears to be influenced by the adjacent GMCs and co-ordination in F-actin arrangements in cells of the stomatal complex continues through to the formation of the guard cell pair. Our studies indicate that actin bands marking the division site in prophase cells, and detected in microinjected living material, are a particularly labile subset of F-actin. Actin bands were difficult to preserve, even when aldehyde fixation was avoided, in contrast to all interphase and mitotic F-actin.     

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

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

Root contraction in hyacinth III. Orientation of cortical microtubules visualized by immunofluorescence microscopy

Summary Cortical microtubules (MTs) were visualized in root cortex cells ofHyacinthus orientalis L. using immunofluorescence techniques. Cellular MT orientation was determined adjacent to radial longitudinal and transverse walls of root tip, uncontracted, contracting, and fully contracted regions. As seen in longitudinal views, MTs formed parallel, apparently helical arrays which were oriented transversely, axially or obliquely depending upon the region. Transverse sectional views showed that MTs adjacent to transverse cell walls formed a variety of patterns which varied with developmental stage and cell location. Microtubules were oriented in crisscross or parallel arrays. The parallel arrays were oriented either parallel, perpendicular or oblique to the radius of the root. There was an apparent temporal progression in MT reorientation from outer cortical to inner cortical cell layers. A resultant progression of reoriented cell growth could account for root contraction. These findings corroborate earlier electron microscopic observations of changing MT orientation accompanying root contraction, and provide cytological evidence to test mathematical and biophysical models of the mechanics of cell expansion.Abbreviations MT microtubule - MF microfibril - MTSB microtubule stabilizing buffer - PBS phosphate buffered saline     

Developmental reorientation of transverse cortical microtubules to longitudinal directions: a role for actomyosin-based streaming and partial microtubule-membrane detachment

Transversely oriented cortical microtubules in elongating cells typically reorient themselves towards longitudinal directions at the end of cell elongation. We have investigated the reorientation mechanism along the outer epidermal wall in maturing leek (Allium porrum L.) leaves using a GFP-MBD microtubule reporter gene and fluorescence microscopy. Incubating leaf segments for 14-18 h with the anti-actin or anti-actomyosin agents, 20 microm cytochalasin D or 20 mM 2,3-butanedione monoxime, inhibited the normal developmental reorientation of microtubules to the longitudinal direction. Observation of living cells revealed a small subpopulation of microtubules with their free ends swinging into oblique or longitudinal directions, before continuing to assemble in the new direction. Electron microscopy confirmed that longitudinal microtubules are partly detached from the plasma membrane. Incubating leaf segments with 0.2% 1 degree-butanol, an activator of phospholipase D, which has been implicated in plasma membrane-microtubule anchoring, promoted the reorientation, presumably by promoting microtubule detachment from the membrane. Stabilizing microtubules with 10 microm taxol also promoted longitudinal orientation, even in the absence of cytoplasmic streaming. These results were consistent with confocal microscopy of live cells before and after drug treatments, which also revealed that the slow (days) global microtubule reorientation is superimposed over short-term (hours) regional cycling in a clockwise and an anti-clockwise direction. We propose that partial detachment of transverse microtubules from the plasma membrane in maturing cells exposes them to hydrodynamic forces of actomyosin-driven cytoplasmic streaming, which bends or shifts pivoting microtubules into longitudinal directions, and thus provides an impetus to push microtubule dynamics in the new direction.     

Strain rate does not affect cortical microtubule orientation in the isolated epidermis of sunflower hypocotyls

A hypothesis exists that external and internal factors affect the orientation of cortical microtubules in as much as these lead to changes in cell elongation rate. Factors that stimulate elongation are proposed to lead to transverse microtubule orientation, whereas factors that inhibit elongation lead to longitudinal orientation. The elongation rate is equal to the rate of longitudinal irreversible strain in cell walls. Incubated epidermis peeled from sunflower hypocotyls does not extend unless it is stretched by loading and the pH of the incubation medium is appropriately low. Thus, peels provide a convenient model to investigate the relationship between longitudinal strain rate and cortical microtubule orientation. In the present study, it was found that peeling affects microtubule orientation. Peels were incubated for several hours in Murashige & Skoog medium (both unbuffered and buffered) to attain a steady state of microtubule orientation before loading. The effects of loading and pH on strain rate and orientation of microtubules under the outer epidermal walls were examined in three portions of peels positioned with respect to the cotyledonary node. Appropriate loading caused longitudinal strain of peels at pH 4.5 but not at pH 6.5. However, no clear effect of strain rate on microtubule orientation in the peels was observed. Independent of applied load and pH of the incubation medium, the microtubule orientation remained unchanged, i.e. orientation was mainly oblique. Our results show that strain rate does not affect cortical microtubule orientation in isolated epidermis of the sunflower hypocotyl model system, although orientation could be changed by white light.     

Cortical microtubules optimize cell-wall crystallinity to drive unidirectional growth in Arabidopsis

The shape of plants depends on cellulose, a biopolymer that self-assembles into crystalline, inextensible microfibrils (CMFs) upon synthesis at the plasma membrane by multi-enzyme cellulose synthase complexes (CSCs). CSCs are displaced in directions predicted by underlying parallel arrays of cortical microtubules, but CMFs remain transverse in cells that have lost the ability to expand unidirectionally as a result of disrupted microtubules. These conflicting findings suggest that microtubules are important for some physico-chemical property of cellulose that maintains wall integrity. Using X-ray diffraction, we demonstrate that abundant microtubules enable a decrease in the degree of wall crystallinity during rapid growth at high temperatures. Reduced microtubule polymer mass in the mor1-1 mutant at high temperatures is associated with failure of crystallinity to decrease and a loss of unidirectional expansion. Promotion of microtubule bundling by over-expressing the RIC1 microtubule-associated protein reduced the degree of crystallinity. Using live-cell imaging, we detected an increase in the proportion of CSCs that track in microtubule-free domains in mor1-1, and an increase in the CSC velocity. These results suggest that microtubule domains affect glucan chain crystallization during unidirectional cell expansion. Microtubule disruption had no obvious effect on the orientation of CMFs in dark-grown hypocotyl cells. CMFs at the outer face of the hypocotyl epidermal cells had highly variable orientation, in contrast to the transverse CMFs on the radial and inner periclinal walls. This suggests that the outer epidermal mechanical properties are relatively isotropic, and that axial expansion is largely dependent on the inner tissue layers.     

Disruption of cellulose synthesis by isoxaben causes tip swelling and disorganizes cortical microtubules in elongating conifer pollen tubes

Summary.  In elongating pollen tubes of the conifer Picea abies (Norway spruce), microtubules form a radial array beneath the plasma membrane only at the elongating tip and an array parallel with elongation throughout the tube. Tips specifically swell following microtubule disruption. Here we test whether these radial microtubules coordinate cell wall deposition and maintain tip integrity as tubes elongate. Control pollen tubes contain cellulose throughout the walls, including the tip. Pollen tubes grown in the presence of isoxaben, which disrupts cellulose synthesis, are significantly shorter with a decrease in cellulose throughout the walls. Isoxaben also significantly increases the frequency of tip swelling, with no effect on tube width outside of the swollen tip. The decrease in cellulose is more pronounced in pollen tubes with swollen tips. The effects of isoxaben are reversible. Following isoxaben treatment, the radial array of microtubules persists beneath the plasma membrane of nonswollen tips, while this array is specifically disrupted in swollen tips. Microtubules instead form a random network throughout the tip. Growth in these pollen tubes is turgor driven, but the morphological changes due to isoxaben are not just the result of weakened cell walls since pollen tubes grown in hypoosmotic media are not significantly shorter but do have swollen tips and tubes are wider along their entire length. We conclude that the radial microtubules in the tip do maintain tip integrity and that the specific inhibition of cellulose microfibril deposition leads to the disorganization of these microtubules. This supports the emerging model that there is bidirectional communication across the plasma membrane between cortical microtubules and cellulose microfibrils. Received January 15, 2002; accepted August 3, 2002; published online March 11, 2003     

A correlated histochemical and ultrastructural study of the epidermis and hypodermis of onion roots

Summary Cell walls of mature epidermal and hypodermal cells are autofluorescent when viewed under ultraviolet or blue light. This autofluorescence develops in a centripetal direction, beginning in the outer tangential wall of the epidermis and ending in the inner tangential wall of the hypodermis. The intercellular regions between the epidermis and hypodermis and between the hypodermis and the cortex are dense and also become autofluorescent. Although the walls of the hypodermis provide a barrier to the movement of a high molecular weight fluorescent dye, the walls of the epidermis are permeable. Histochemical studies indicate that lipids and polyphenolics are components of the epidermal and hypodermal cell walls. Both layers are resistant to the wall-degrading enzyme Driselase and to concentrated sulphuric acid, whereas the cortex is digested with both treatments. Observations with the transmission electron microscope show that a complex suberin lamella encases each hypodermal cell but is absent from the epidermis. However, the outer tangential wall and radial walls of the epidermal cells are complex in that layers of different densities are present. Some of these layers, as well as the intercellular regions and the radial walls of the hypodermal cells, bind ferric ions when tissue is fixed in ferric chloride-glutaraldehyde indicating the presence of poly-phenolics in these regions. An extracellular layer covering the outer tangential wall of the epidermis stained positively with a number of histochemical tests for polyphenolics.     

Effects of ethylene on the orientation of microtubules and cellulose microfibrils of pea epicotyl cells with polylamellate cell walls

Summary Following a 5 hours ethylene treatment, cortical cells of Pea (Pisum sativum L. var Alaska) epicotyl third internode showed a change in the orientation of both microtubules near the plasma membrane and recently deposited cellulose microfibrils. Control cortical cells had mostly transverse microtubules. The ratio of the average frequency of transverse to longitudinal microtubules was 6.0. After 5 hours of ethylene treatment, cortical cells had mostly longitudinal microtubules, with the ratio of transverse to longitudinal microtubules equal to 0.1. Epidermal cells were more variable than cortical cells with regard to the frequency of longitudinal and transverse microtubules. Observation of cortical cell walls in conventionally stained thin sections revealed that recent deposition of microfibrils had been primarily transverse in almost all of the control cortical cells sampled. In contrast, more than half of the ethylene-treated cortical cells had recent deposition oriented primarily longitudinally. This change in microtubule and microfibril orientation may be early enough to constitute the primary effect of ethylene leading to radial cell expansion.Research supported by NSF grant PCM 78-03244, A1, 2 to PBG and by a Research Corporation grant to WRE.     

Zostera capensis setchell: III. Some aspects of wall ingrowth development in leaf blade epidermal cells

Summary The development of wall ingrowths in leaf blade epidermal cells of the marine angiospermZostera capensis was studied by electron microscopy. Prior to the appearance of ingrowths long profiles of endoplasmic reticulum cisternae become arranged peripherally closely following the contours of the walls. The plasmalemma assumes a wavy appearance and in regions where wall ingrowths first start forming (i.e., along the radial, inner tangential and transverse walls) the plasmalemma becomes separated from the walls by an undulating extracytoplasmic space. Small, irregular projections of secondary wall material make their appearance here. Paramural bodies, dictyosomes, endoplasmic reticulum (ER) and possibly also microtubules seem to be closely associated with the initiation and subsequent development of wall projections. As the cells mature, new ingrowths arise in a centrifugal direction along the radial and transverse walls. When wall ingrowths reach a certain stage of their development, mitochondria become strongly polarized towards them and become closely associated with the plasmalemma which ensheaths the ingrowths. There is often also a close association between ER cisternae and the involuted plasmalemma of the wall projections. Initially ingrowths are slender, curved structures, but become more complex as the cells mature. Ingrowths are most extensively developed along the inner tangential and transverse walls. As epidermal cells age there is a loss of wall material from the ingrowths. The probable significance of the formation of wall ingrowths in the epidermal cells is also discussed.     

Microtubules and morphogenesis in ordinary epidermal cells ofVigna sinensis leaves

Summary Undifferentiated ordinary epidermal cells (ECs) ofVigna sinensis leaves possess straight anticlinal walls and cortical microtubules (Mts) scattered along them. At an early stage of EC differentiation cortical Mts adjacent to the above walls form bundles normal to the leaf plane, loosely interconnected through the cortical cytoplasm of the internal periclinal wall. At the upper ends of the Mt bundles, Mts fan out towards the external periclinal wall and form radial arrays. Mt bundles and radial arrays exhibit strict alternate disposition between neighbouring ECs. An identical reticulum of cellulose microfibril (CM) bundles is deposited outside the Mt bundles. Local wall pads rise at the junctions of anticlinal walls with the external periclinal one, where the CM bundles terminate. They display radial CMs fanning towards the external periclinal wall. The CM bundles and radial CM systems prevent local cell bulging, but allow it in the intervening wall areas. In particular, the radial CM systems dictate the pattern of EC waviness by favouring local tangential expansion of external periclinal wall. As a result, ECs obtain an undulate appearance. Constrictions in one EC correspond with protrusions of adjacent ECs. ECs affected by colchicine entirely lose their Mts and do not develop wavy walls, an observation substantiating the role of cortical Mts in EC morphogenesis.Abbreviations CM cellulose microfibril - DTT dithiothreitol - EC epidermal cell - MSB microtubule stabilizing buffer - Mt microtubule - PBS phosphate buffered saline - PMSF phenylmethylsulfonyl fluoride     

Shifts in plant cell axiality: histogenetic influences on cellulose orientation in the succulent, Graptopetalum

The elongation of typical plant meristems can be explained, biophysically, by the cellulosic hoop reinforcement in the longitudinal walls of most of the cells. A cortical microtubule array is believed to govern the reinforcement alignment. The orientation of the reinforcement is studied here with polarized light. Transverse orientation appears to be invariant, regardless of division direction, in interior cells of at least some established meristems. Exceptional longitudinal reinforcement (i.e., in the direction of growth) is occasionally seen, however, in the outer epidermal wall of established Graptopetalum roots, leaves, and stems. It is found in pairs of elongate cells that arise from a longitudinal division. This behavior is prominent when the epidermis is shifting reinforcement to initiate new organs. Usually the division direction and the new cell long axis coincide; reinforcement parallels both. When these two factors are in marked opposition, as in certain longitudinal divisions of very broad cells, the reinforcement can follow the new cell's long axis even though this orients alignment perpendicular to the new wall. The normal prevailing effect of the division direction may relate to the formation of the cortical microtubule array parallel to the orientation of the preprophase band. The normally synergistic effect of the new cell's long axis may stem from the fact that when the band-like array occupies the smaller pair of anticlinal faces of the cuboidal cell (and hence parallels the cell's long dimension in surface view), it will have the densest and presumably most stable structure. Reinforcement orientation is seen as a function of at least two factors which bear on the stability of the newly forming array.     

Microtubule organization in the differentiating transfer cells of the placenta inLilium spp.

Summary Placental cells in the ovarian transmitting tissue ofLilium spp. are organized as transfer cells with inbuddings facing the ovarian locule. A detailed analysis of microtubule (MT) organization during development of these polarized cells is reported here. Formation of wall projections occurs at the apical part of the cell starting on the day of anthesis, and a fully mature secretion zone is found four days after anthesis. MTs are organized into distinct cortical and central arrays. The cortical array undergoes a unique transition at anthesis. MTs in the basal half of the cell remain in longitudinal bundles while in the apical half of the cell their longitudinal orientation is replaced by a transverse alignment. One day after anthesis, these transverse bundles become a meshwork of short, randomly organized MTs, while MTs in the basal half of the cell retain their longitudinal alignment. The realignment of MTs in the apical half of the cell coincides with the deposition of the secondary cell wall. The central array is composed of short, randomly arranged strands of MTs in the cytoplasm between the nucleus and the apical and basal periclinal walls of the cell. This array first appears as solitary strands in the apical part of the cell one day before anthesis. The central array extends during development and is eventually seen in the basal half of the cell. We propose that MTs in the cortical region near the apical wall act as templates for the deposition of cellulose microfibrils in the secondary cell wall. MTs in the central array in these transfer cells may be involved in the trafficking of vesicles and/or positioning of organelles near the secretion zone.Abbreviations MT microtubule - daa day after anthesis - dba day before anthesis