Orientation Of Cortical Microtubules Research Articles

A cellulose synthesis inhibitor affects cellulose synthase complex secretion and cortical microtubule dynamics.

P4B (2-phenyl-1-[4-(6-(piperidin-1-yl) pyridazin-3-yl) piperazin-1-yl] butan-1-one) is a novel cellulose biosynthesis inhibitor (CBI) discovered in a screen for molecules to identify inhibitors of Arabidopsis (Arabidopsis thaliana) seedling growth. Growth and cellulose synthesis inhibition by P4B were greatly reduced in a novel mutant for the cellulose synthase catalytic subunit gene CESA3 (cesa3pbr1). Cross-tolerance to P4B was also observed for isoxaben-resistant (ixr) cesa3 mutants ixr1-1 and ixr1-2. P4B has an original mode of action as compared with most other CBIs. Indeed, short-term treatments with P4B did not affect the velocity of cellulose synthase complexes (CSCs) but led to a decrease in CSC density in the plasma membrane without affecting their accumulation in microtubule-associated compartments. This was observed in the wild type but not in a cesa3pbr1 background. This reduced density correlated with a reduced delivery rate of CSCs to the plasma membrane but also with changes in cortical microtubule dynamics and orientation. At longer timescales, however, the responses to P4B treatments resembled those to other CBIs, including the inhibition of CSC motility, reduced growth anisotropy, interference with the assembly of an extensible wall, pectin demethylesterification, and ectopic lignin and callose accumulation. Together, the data suggest that P4B either directly targets CESA3 or affects another cellular function related to CSC plasma membrane delivery and/or microtubule dynamics that is bypassed specifically by mutations in CESA3.

The cotton protein GhIQD21 interacts with GhCaM7 and modulates organ morphogenesis in Arabidopsis by influencing microtubule stability.

GhIQD21, a cotton IQ67-domain protein, interacts with GhCaM7 and alters organ shape in Arabidopsis by modulating microtubule stability. Calcium ion (Ca2+) and the calcium sensor calmodulin play crucial roles in the growth and development of plants. GhCaM7, a calmodulin in upland cotton (Gossypium hirsutum L.), is highly expressed in cotton fiber cells during the rapid elongation period and plays an important role in fiber cell development. In this study, we screened for GhCaM7-interacting proteins and identified GhIQD21, which contains a typical IQ67-domain. GhIQD21 was preferentially expressed at the fiber rapid elongation stage, and the protein localized to microtubules (MTs). Ectopic expression of GhIQD21 in Arabidopsis resulted in shorter leaves, petals, siliques, and plant height, thicker inflorescences, and more trichomes when compared with wild type (WT). Further investigation indicated that the morphogenesis of leaf epidermal cells and silique cells was altered. There was less consistency in the orientation of cortical microtubules in cotyledon and hypocotyl epidermal cells. Furthermore, compared with WT, transgenic seedling hypocotyls were more sensitive to oryzalin, a MT depolymerization drug. These results indicated that GhIQD21 is a GhCaM7-interacting protein located in MTs and that it plays a role in plant growth and potentially cotton fiber development. This study provides a foundation for further studies of the function and regulatory mechanism of GhIQD21 in fiber cell development.

CLASP balances two competing cell division plane cues during leaf development

Starting as small, densely packed boxes, leaf mesophyll cells expand to form an intricate mesh of interconnected cells and air spaces, the organization of which dictates the internal surface area of the leaf for light capture and gas exchange during photosynthesis. Despite their importance, little is known about the basic patterns of mesophyll cell division, and how they contribute to cell and intercellular space organization. To address this, we tracked divisions within individual cell lineages in three dimensions over time in Arabidopsis spongy mesophyll. We found that early on, successive cell division planes switch their orientation such that each new cell wall intersects the previous at a right angle, creating a new multi-cell junction (the intersection of three or more cells). These junctions then open to create intercellular spaces. During subsequent enlargement of the spaces, the division planes of the surrounding cells show an increasing tendency to tilt in the direction of their adjacent intercellular spaces. This disrupts the alternating pattern, and by extension, halts the initiation of new multi-cell junctions and intercellular spaces, but allows the expansion of existing spaces. Both division patterns are specified before mitosis by the orientation of interphase cortical microtubules, which gradually narrow to form a preprophase band in the same orientation to establish the future plane of cell division. In the absence of the microtubule-associated protein CLASP, the early alternating division plane and microtubule patterns are compromised, whereas space-oriented divisions are exacerbated. This results in large distortions of the topological relations between cells and intercellular spaces, as well as changes in their relative abundance. Our data reveal the existence of two competing cell division mechanisms that are balanced by CLASP to specify the distribution of cells and intercellular spaces in spongy mesophyll tissue.

Inhibition of cell expansion enhances cortical microtubule stability in the root apex of Arabidopsis thaliana

BackgroundCortical microtubules regulate cell expansion by determining cellulose microfibril orientation in the root apex of Arabidopsis thaliana. While the regulation of cell wall properties by cortical microtubules is well studied, the data on the influence of cell wall to cortical microtubule organization and stability remain scarce. Studies on cellulose biosynthesis mutants revealed that cortical microtubules depend on Cellulose Synthase A (CESA) function and/or cell expansion. Furthermore, it has been reported that cortical microtubules in cellulose-deficient mutants are hypersensitive to oryzalin. In this work, the persistence of cortical microtubules against anti-microtubule treatment was thoroughly studied in the roots of several cesa mutants, namely thanatos, mre1, any1, prc1-1 and rsw1, and the Cellulose Synthase Interacting 1 protein (csi1) mutant pom2-4. In addition, various treatments with drugs affecting cell expansion were performed on wild-type roots. Whole mount tubulin immunolabeling was applied in the above roots and observations were performed by confocal microscopy.ResultsCortical microtubules in all mutants showed statistically significant increased persistence against anti-microtubule drugs, compared to those of the wild-type. Furthermore, to examine if the enhanced stability of cortical microtubules was due to reduced cellulose biosynthesis or to suppression of cell expansion, treatments of wild-type roots with 2,6-dichlorobenzonitrile (DCB) and Congo red were performed. After these treatments, cortical microtubules appeared more resistant to oryzalin, than in the control.ConclusionsAccording to these findings, it may be concluded that inhibition of cell expansion, irrespective of the cause, results in increased microtubule stability in A. thaliana root. In addition, cell expansion does not only rely on cortical microtubule orientation but also plays a regulatory role in microtubule dynamics, as well. Various hypotheses may explain the increased cortical microtubule stability under decreased cell expansion such as the role of cell wall sensors and the presence of less dynamic cortical microtubules.

External Mechanical Cues Reveal a Katanin-Independent Mechanism behind Auxin-Mediated Tissue Bending in Plants.

Tissue folding is a central building block of plant and animal morphogenesis. In dicotyledonous plants, hypocotyl folds to form hooks after seedling germination that protects their aerial stem cell niche during emergence from soil. Auxin response factors and auxin transport are reported to play a key role in this process. Here, we show that the microtubule-severing enzyme katanin contributes to hook formation. However, by exposing hypocotyls to external mechanical cues mimicking the natural soil environment, we reveal that auxin response factors ARF7/ARF19, auxin influx carriers, and katanin are dispensable for apical hook formation, indicating that these factors primarily play the role of catalyzers of tissue bending in the absence of external mechanical cues. Instead, our results reveal the key roles of the non-canonical TMK-mediated auxin pathway, PIN efflux carriers, and cellulose microfibrils as components of the core pathway behind hook formation in the presence or absence of external mechanical cues.

Cortical tension overrides geometrical cues to orient microtubules in confined protoplasts

In plant cells, cortical microtubules (CMTs) generally control morphogenesis by guiding cellulose synthesis. CMT alignment has been proposed to depend on geometrical cues, with microtubules aligning with the cell long axis in silico and in vitro. Yet, CMTs are usually transverse in vivo, i.e., along predicted maximal tension, which is transverse for cylindrical pressurized vessels. Here, we adapted a microwell setup to test these predictions in a single-cell system. We confined protoplasts laterally to impose a curvature ratio and modulated pressurization through osmotic changes. We find that CMTs can be longitudinal or transverse in wallless protoplasts and that the switch in CMT orientation depends on pressurization. In particular, longitudinal CMTs become transverse when cortical tension increases. This explains the dual behavior of CMTs in planta: CMTs become longitudinal when stress levels become low, while stable transverse CMT alignments in tissues result from their autonomous response to tensile stress fluctuations.

KATANIN-dependent mechanical properties of the stigmatic cell wall mediate the pollen tube path in Arabidopsis

Successful fertilization in angiosperms depends on the proper trajectory of pollen tubes through the pistil tissues to reach the ovules. Pollen tubes first grow within the cell wall of the papilla cells, applying pressure to the cell. Mechanical forces are known to play a major role in plant cell shape by controlling the orientation of cortical microtubules (CMTs), which in turn mediate deposition of cellulose microfibrils (CMFs). Here, by combining imaging, genetic and chemical approaches, we show that isotropic reorientation of CMTs and CMFs in aged Col-0 and katanin1-5 (ktn1-5) papilla cells is accompanied by a tendency of pollen tubes to coil around the papillae. We show that this coiled phenotype is associated with specific mechanical properties of the cell walls that provide less resistance to pollen tube growth. Our results reveal an unexpected role for KTN1 in pollen tube guidance on the stigma by ensuring mechanical anisotropy of the papilla cell wall.

Maize ZmRPH1 encodes a microtubule-associated protein that controls plant and ear height.

Maize ZmRPH1 encodes a microtubule-associated protein that controls plant and ear height.

Localization of actin filaments and cortical microtubules in wood-forming tissues of conifers

ABSTRACT The aim of the present study was to investigate the orientation and localization of actin filaments and cortical microtubules in wood-forming tissues in conifers to understand wood formation. Small blocks were collected from the main stems of Abies firma, Pinus densiflora, and Taxus cuspidata during active seasons of the cambium. Bundles of actin filaments were oriented axially or longitudinally relative to the cell axis in fusiform and ray cambial cells. In differentiating tracheids, actin filaments were oriented longitudinally relative to the cell axis during primary and secondary wall formation. In contrast, the orientation of well-ordered cortical microtubules in tracheids changed from transverse to longitudinal during secondary wall formation. There was no clear relationship between the orientation of actin filaments and cortical microtubules in cambial cells and cambial derivatives. Aggregates of actin filaments and a circular band of cortical microtubules were localized around bordered pits and cross-field pits in differentiating tracheids. In addition, rope-like bands of actin filaments were observed during the formation of helical thickenings at the final stage of formation of secondary walls in tracheids. Actin filaments might not play a major role in changes in the orientation of cortical microtubules in wood-forming tissues. However, since actin filaments were co-localized with cortical microtubules during the formation of bordered pits, cross-field pits and helical thickenings at the final stage of formation of the secondary wall in tracheids, it seems plausible that actin filaments might be closely related to the localization of cortical microtubules during the development of these modifications of wood structure.

An Induction System for Clustered Stomata by Sugar Solution Immersion Treatment in Arabidopsis thaliana Seedlings.

Stomatal movement mediates plant gas exchange, which is essential for photosynthesis and transpiration. Stomatal opening and closing are accomplished by a significant increase and decrease in guard cell volume, respectively. Because shuttle transport of ions and water occurs between guard cells and larger neighboring epidermal cells during stomatal movement, the spaced distribution of plant stomata is considered an optimal distribution for stomatal movement. Experimental systems for perturbing the spaced pattern of stomata are useful to examine the spacing pattern's significance. Several key genes associated with the spaced stomatal distribution have been identified, and clustered stomata can be experimentally induced by altering these genes. Alternatively, clustered stomata can be also induced by exogenous treatments without genetic modification. In this article, we describe a simple induction system for clustered stomata in Arabidopsis thaliana seedlings by immersion treatment with a sucrose-containing medium solution. Our method is easy and directly applicable to transgenic or mutant lines. Larger chloroplasts are presented as a cell biological hallmark of sucrose-induced clustered guard cells. In addition, a representative confocal microscopic image of cortical microtubules is shown as an example of intracellular observation of clustered guard cells. The radial orientation of cortical microtubules is maintained in clustered guard cells as in spaced guard cells in control conditions.

Cortical Microtubule Reorientation and Its Relation to Cell Surface Texture of Epidermal Cells of <i>Arabidopsis Thaliana</i> Hypocotyls under Simulated Microgravity Conditions

Gravitropic curvature growth of Arabidopsis hypocotyls mainly occurred in the rapid growing Elongation Zone (EZI), not in the slow-growing Elongation Zone (EZⅡ). By examining reorientation of Microtubules (MT) and phenotype of the cell wall in the EZI and the EZⅡ of Arabidopsis hypocotyls under normal gravitational condition, it is found that MTs in the rapid growing epidermal cells were mainly in the transverse direction, while those in the non-growing epidermal cells were in the longitudinal directions. However, this difference in cortical MT arrays between the EZI and EZⅡ cells disappeared when the seedlings were exposed to the simulated microgravity condition on a horizontal clinostat. Field emission scanning electron microscopy revealed that the surface texture of epidermal cells, like the direction of the MT, in the EZI and the EZⅡ also became similar when exposed to the simulated microgravity condition. This result indicated that simulate microgravity could modify the potential differentiation between the EZI and the EZⅡ by affecting the orientation of cortical MT in the epidermal cells.

Cortical microtubule orientation in Arabidopsis thaliana root meristematic zone depends on cell division and requires severing by katanin

BackgroundTransverse cortical microtubule orientation, critical for anisotropic cell expansion, is established in the meristematic root zone. Intending to elucidate the possible prerequisites for this establishment and factors that are involved, microtubule organization was studied in roots of Arabidopsis thaliana, wild-type and the p60-katanin mutants fra2, ktn1-2 and lue1. Transverse cortical microtubule orientation in the meristematic root zone has proven to persist under several regimes inhibiting root elongation. This persistence was attributed to the constant moderate elongation of meristematic cells, prior to mitotic division. Therefore, A. thaliana wild-type seedlings were treated with aphidicolin, in order to prevent mitosis and inhibit premitotic cell elongation.ResultsIn roots treated with aphidicolin for 12 h, cell divisions still occurred and microtubules were transverse. After 24 and 48 h of treatment, meristematic cell divisions and the prerequisite elongation ceased, while microtubule orientation became random. In meristematic cells of the p60-katanin mutants, apart from a general transverse microtubule pattern, cortical microtubules with random orientation were observed, also converging at several cortical sites, in contrast to the uniform transverse pattern of wild-type cells.ConclusionTaken together, these observations reveal that transverse cortical microtubule orientation in the meristematic zone of A. thaliana root is cell division-dependent and requires severing by katanin.

Cortical microtubules and fusicoccin response in clustered stomatal guard cells induced by sucrose solution immersion

ABSTRACTWe previously found that sucrose solution immersion treatment permitted ectopic guard cell differentiation, resulting in clustered stomatal guard cells. Using this system, we examined the effects of sucrose solution-induced stomatal clustering on guard cell cortical microtubules and the stomatal response to fusicoccin. Confocal observation revealed that the radial orientation of cortical microtubules was largely maintained in clustered guard cells. Outward movement of cortical microtubule plus-ends was also kept in the clustered guard cells. Fusicoccin treatment induced stomatal opening in both spaced and clustered stomata, although sucrose solution-treated guard cells had lower stomatal apertures. These results suggested that immersion treatment with sucrose solution perturbed the one-cell spacing of stomata but not the cortical microtubule organization required to open stomatal pores.

Growth and cortical microtubule dynamics in shoot organs under microgravity and hypergravity conditions

ABSTRACTThe body shape of plants varied in proportion to the logarithm of the magnitude of gravity in the range from microgravity to hypergravity to resist the gravitational force. Here we discuss the roles of cortical microtubule and 65 kDa microtubule-associated protein-1 (MAP65-1) in gravity-induced modification of growth anisotropy. Microgravity stimulated elongation growth and suppressed lateral expansion in shoot organs, such as hypocotyls and epicotyls. On the other hand, hypergravity inhibited elongation growth and promoted lateral expansion in shoot organs. The number of cells with transverse microtubules was increased by microgravity, but decreased by hypergravity. Furthermore, the levels of MAP65-1, which is involved in the maintenance of the transverse microtubule orientation, were increased by microgravity, but decreased by hypergravity. Therefore, the regulation of orientation of cortical microtubules via changes in the levels of MAP65-1 may contribute to the modification of the body shape of plants to resist the gravitational force.

Reorganization of interphase microtubules in root cells of Medicago sativa L. during acclimation to osmotic and salt stress

We have investigated the organization of microtubule system in interphase cells of Medicago sativa L. roots during acclimation to salt and osmotic stress at different concentrations of NaCl, Na2SO4 and mannitol. We have identified several morphological changes in tubulin cytoskeleton that appear during the acclimation to salt and osmotic stress in the cells of different root tissues: 1) decreased density of cortical microtubule network, 2) random orientation of cortical microtubule bundles, 3) non-uniform density of the bundles, 4) thickening of the bundles, 5) fragmentation of the bundles, 6) formation of centers of converging microtubule. Reduced density of the microtubule network and thickening of the bundles was detected during osmotic and salt stress, yet random orientation of cortical microtubules was observed under osmotic stress and not found during salt stress. Fragmentation of microtubule bundles was apparent during salt stress and less evident at high concentration of mannitol. Formation of centers of converging microtubule was common under prolonged action of sodium sulfate, less common under sodium chloride and not found after mannitol treatment. Our data show that cortical microtubules in alfalfa root cells rearrange not only in response to different ions, but also to osmotic pressure. Thus, the signaling pathways and molecular mechanisms inducing reorganization of the microtubule system may be triggered not only by sodium cations but also by sulfate and chloride anion at the concentrations that do not cause irreversible cell damage. Our study show that the osmotic and salt stress differently affect the cortical microtubules, and their reorganization in response to stress depends on the salt cations as well as anions might also show additional effect under salt stress.

Environmental and Endogenous Control of Cortical Microtubule Orientation.

Plant growth requires a tight coordination of cell shape and anisotropic expansion. Owing to their immobility, plant cells determine body architecture through the orientation of cell division and cell expansion. Microtubule cytoskeleton represents a versatile cellular structure essential for coordinating flexible cell morphogenesis. Previous studies have identified a large number of microtubule-associated regulators that control microtubule dynamics; however, the mechanisms by which microtubule reorientation responds to exogenous and environmental stimuli are largely unknown. In this review, we describe the molecular details of microtubule dynamics that are required for cortical microtubule array pattern formation, and recapitulate current knowledge on the mechanisms by which various environmental and endogenous stimuli control cortical microtubule reorientation.

Mechanism of dynamic reorientation of cortical microtubules due to mechanical stress

Directional growth caused by gravitropism and corresponding bending of plant cells has been explored since 19th century, however, many aspects of mechanisms underlying the perception of gravity at the molecular level are still not well known. Perception of gravity in root and shoot gravitropisms is usually attributed to gravisensitive cells, called statocytes, which exploit sedimentation of macroscopic and heavy organelles, amyloplasts, to sense the direction of gravity. Gravity stimulus is then transduced into distal elongation zone, which is several mm far from statocytes, where it causes stretching. It is suggested that gravity stimulus is conveyed by gradients in auxin flux. We propose a theoretical model that may explain how concentration gradients and/or stretching may indirectly affect the global orientation of cortical microtubules, attached to the cell membrane and induce their dynamic reorientation perpendicular to the gradients. In turn, oriented microtubule arrays direct the growth and orientation of cellulose microfibrils, forming part of the cell external skeleton and determine the shape of the cell. Reorientation of microtubules is also observed in reaction to light in phototropism and mechanical bending, thus suggesting universality of the proposed mechanism.

Cortical microtubule patterning in roots of Arabidopsis thaliana primary cell wall mutants reveals the bidirectional interplay with cell expansion

Cell elongation requires directional deposition of cellulose microfibrils regulated by transverse cortical microtubules. Microtubules respond differentially to suppression of cell elongation along the developmental zones of Arabidopsis thaliana root apex. Cortical microtubule orientation is particularly affected in the fast elongation zone but not in the meristematic or transition zones of thanatos and pom2–4 cellulose-deficient mutants of Arabidopsis thaliana. Here, we report that a uniform phenotype is established among the primary cell wall mutants, as cortical microtubules of root epidermal cells of rsw1 and prc1 mutants exhibit the same pattern described in thanatos and pom2–4. Whether cortical microtubules assume transverse orientation or not is determined by the demand for cellulose synthesis, according to each root zone's expansion rate. It is suggested that cessation of cell expansion may provide a biophysical signal resulting in microtubule reorientation.

Auxin inhibits expansion rate independently of cortical microtubules.

A recent publication announces that auxin inhibits expansion by a mechanism based on the orientation of cortical microtubules. This is a textbook-revising claim, but as I argue here, a claim that is supported by neither the authors' data nor previous research, and is contradicted by a simple experiment.

The Control of Growth Symmetry Breaking in the Arabidopsis Hypocotyl

Complex shapes in biology depend on the ability of cells to shift from isotropic to anisotropic growth during development. In plants, this growth symmetry breaking reflects changes in the extensibility of the cell walls. The textbook view is that the direction of turgor-driven cell expansion depends on the cortical microtubule (CMT)-mediated orientation of cellulose microfibrils. Here, we show that this view is incomplete at best. We used atomic force microscopy (AFM) to study changes in cell-wall mechanics associated with growth symmetry breaking within the hypocotyl epidermis. We show that, first, growth symmetry breaking is preceded by an asymmetric loosening of longitudinal, as compared to transverse, anticlinal walls, in the absence of a change in CMT orientation. Second, this wall loosening is triggered by the selective de-methylesterification of cell-wall pectin in longitudinal walls, and, third, the resultant mechanical asymmetry is required for the growth symmetry breaking. Indeed, preventing or promoting pectin de-methylesterification, respectively, increased or decreased the stiffness of all the cell walls, but in both cases reduced the growth anisotropy. Finally, we show that the subsequent CMT reorientation contributes to the consolidation of the growth axis but is not required for the growth symmetry breaking. We conclude that growth symmetry breaking is controlled at a cellular scale by bipolar pectin de-methylesterification, rather than by the cellulose-dependent mechanical anisotropy of the cell walls themselves. Such a cell asymmetry-driven mechanism is comparable to that underlying tip growth in plants but also anisotropic cell growth in animal cells.