Hypersensitivity to cytoskeletal antagonists demonstrates microtubule-microfilament cross-talk in the control of root elongation in Arabidopsis thaliana

Elongation of diffusely expanding plant cells is thought to be mainly under the control of cortical microtubules. Drug treatments that disrupt actin microfilaments, however, can reduce elongation and induce radial swelling. To understand how microfilaments assist growth anisotropy, we explored their functional interactions with microtubules by measuring how microtubule disruption affects the sensitivity of cells to microfilament-targeted drugs. We assessed the sensitivity to actin-targeted drugs by measuring the lengths and diameters of expanding roots and by analysing microtubule and microfilament patterns in the temperature-sensitive Arabidopsis thaliana mutant microtubule organization 1 (mor1-1), along with other mutants that constitutively alter microtubule arrays. At the restrictive temperature of mor1-1, root expansion was hypersensitive to the microfilament-disrupting drugs latrunculin B and cytochalasin D, while immunofluorescence microscopy showed that low doses of latrunculin B exacerbated microtubule disruption. Root expansion studies also showed that the botero and spiral1 mutants were hypersensitive to latrunculin B. Hypersensitivity to actin-targeted drugs is a direct consequence of altered microtubule polymer status, demonstrating that cross-talk between microfilaments and microtubules is critical for regulating anisotropic cell expansion.     

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.     

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     

Mutation or drug-dependent microtubule disruption causes radial swelling without altering parallel cellulose microfibril deposition in Arabidopsis root cells

As critical determinants of growth anisotropy in plants, cortical microtubules are thought to constrain the movement of cellulose synthase complexes and thus align newly deposited cellulose microfibrils. We tested this cellulose synthase constraint model using the temperature-sensitive mor1-1 mutant of Arabidopsis. Contrary to predictions, the disruption of cortical microtubules in mor1-1 root epidermal cells led to left-handed root twisting and radial swelling but did not alter the transverse orientation of cellulose microfibrils. We also found that drug-dependent disassembly or hyperstabilization of cortical microtubules did not alter the parallel order of cellulose microfibrils. By measuring cellulose content in mor1-1 seedlings, we verified that cellulose synthesis is not reduced at the restrictive temperature. The independence of cortical microtubule organization and cellulose microfibril alignment was supported by the observation that double mutants of mor1-1 and rsw1-1, the cellulose-deficient mutant with misaligned microfibrils, had additive phenotypes. Our results suggest that cortical microtubules regulate growth anisotropy by some mechanism other than cellulose microfibril alignment or synthesis.     

Plant-specific microtubule-associated protein SPIRAL2 is required for anisotropic growth in Arabidopsis

In diffusely growing plant cells, cortical microtubules play an important role in regulating the direction of cell expansion. Arabidopsis (Arabidopsis thaliana) spiral2 (spr2) mutant is defective in directional cell elongation and exhibits right-handed helical growth in longitudinally expanding organs such as root, hypocotyl, stem, petiole, and petal. The growth of spr2 roots is more sensitive to microtubule-interacting drugs than is wild-type root growth. The SPR2 gene encodes a plant-specific 94-kD protein containing HEAT-repeat motifs that are implicated in protein-protein interaction. When expressed constitutively, SPR2-green fluorescent protein fusion protein complemented the spr2 mutant phenotype and was localized to cortical microtubules as well as other mitotic microtubule arrays in transgenic plants. Recombinant SPR2 protein directly bound to taxol-stabilized microtubules in vitro. Furthermore, SPR2-specific antibody and mass spectrometry identified a tobacco (Nicotiana tabacum) SPR2 homolog in highly purified microtubule-associated protein fractions from tobacco BY-2 cell cultures. These results suggest that SPR2 is a novel microtubule-associated protein and is required for proper microtubule function involved in anisotropic growth.     

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.     

Microtubule cortical array organization and plant cell morphogenesis

Plant cell cortical microtubule arrays attain a high degree of order without the benefit of an organizing center such as a centrosome. New assays for molecular behaviors in living cells and gene discovery are yielding insight into the mechanisms by which acentrosomal microtubule arrays are created and organized, and how microtubule organization functions to modify cell form by regulating cellulose deposition. Surprising and potentially important behaviors of cortical microtubules include nucleation from the walls of established microtubules, and treadmilling-driven motility leading to polymer interaction, reorientation, and microtubule bundling. These behaviors suggest activities that can act to increase or decrease the local level of order in the array. The SPIRAL1 (SPR1) and SPR2 microtubule-localized proteins and the radial swollen 6 (rsw-6) locus are examples of new molecules and genes that affect both microtubule array organization and cell growth pattern. Functional tagging of cellulose synthase has now allowed the dynamic relationship between cortical microtubules and the cell-wall-synthesizing machinery to be visualized, providing direct evidence that cortical microtubules can organize cellulose synthase complexes and guide their movement through the plasma membrane as they create the cell wall.     

Gibberellin-induced changes in growth anisotropy precede gibberellin-dependent changes in cortical microtubule orientation in developing epidermal cells of barley leaves. Kinematic and cytological studies on a gibberellin-responsive dwarf mutant, M489

We conducted kinematic and cytological studies on "between vein" epidermal cells of the gibberellin (GA)-deficient M489 dwarf mutant of barley (Hordeum vulgare L. Himalaya). GAs affect radial and axial components of cell expansion and cortical microtubule orientation. Adaxial cells in particular expand radially after leaving the elongation zone (EZ), probably as part of leaf unrolling. Exogenous gibberellic acid corrects the mutant's short, wide blades, short EZ, and slow elongation rate. Cell production rates increase more on the adaxial than on the abaxial surface. Cells spend equal periods of time elongating in dwarf and tall plants, but relative elemental growth rates start to decline sooner in the dwarf. GA increased the rate at which longitudinal wall area increased because the increased axial growth more than compensated for reduced radial growth. In dwarf leaves, increased radial expansion was detected in basal parts of the EZ before cortical microtubules lost transverse orientation in the distal elongation zone. We conclude that loss of microtubule orientation is not required for low GA levels to reduce growth anisotropy.     

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.     

Progressive Transverse Microtubule Array Organization in Hormone-Induced Arabidopsis Hypocotyl Cells

The acentriolar cortical microtubule arrays in dark-grown hypocotyl cells organize into a transverse coaligned pattern that is critical for axial plant growth. In light-grown Arabidopsis thaliana seedlings, the cortical array on the outer (periclinal) cell face creates a variety of array patterns with a significant bias (>3:1) for microtubules polymerizing edge-ward and into the side (anticlinal) faces of the cell. To study the mechanisms required for creating the transverse coalignment, we developed a dual-hormone protocol that synchronously induces ∼80% of the light-grown hypocotyl cells to form transverse arrays over a 2-h period. Repatterning occurred in two phases, beginning with an initial 30 to 40% decrease in polymerizing plus ends prior to visible changes in the array pattern. Transverse organization initiated at the cell’s midzone by 45 min after induction and progressed bidirectionally toward the apical and basal ends of the cell. Reorganization corrected the edge-ward bias in polymerization and proceeded without transiting through an obligate intermediate pattern. Quantitative comparisons of uninduced and induced microtubule arrays showed a limited deconstruction of the initial periclinal array followed by a progressive array reorganization to transverse coordinated between the anticlinal and periclinal cell faces.     

Microtubule reorganization,cell wall synthesis and establishment of the axis of elongation in regenerating protoplasts of the algaMougeotia

Summary Microtubule reorganization and cell wall deposition have been monitored during the first 30 hours of regeneration of protoplasts of the filamentous green algaMougeotia, using immunofluorescence microscopy to detect microtubules, and the cell-wall stain Tinopal LPW to detect the orientation of cell wall microfibrils. In the cylindrical cells of the alga, cortical microtubules lie in an ordered array, transverse to the long axis of the cells. In newly formed protoplasts, cortical microtubules exhibit some localized order, but within 1 hour microtubules become disordered. However, within 3 to 4 hours, microtubules are reorganized into a highly ordered, symmetrical array centered on two cortical foci. Cell wall synthesis is first detected during early microtubule reorganization. Oriented cell wall microfibrils, co-aligned with the microtubule array, appear subsequent to microtubule reorganization but before cell elongation begins. Most cells elongate in the period between 20 to 30 hours. Elongation is preceded by the aggregation of microtubules into a band intersecting both foci, and transverse to the incipient axis of elongation. The foci subsequently disappear, the microtubule band widens, and microfibrils are deposited in a band which is co-aligned with the band of microtubules. It is proposed that this band of microfibrils restricts lateral expansion of the cells and promotes elongation. Throughout the entire regeneration process inMougeotia, changes in microtubule organization precede and are paralleled by changes in cell wall organization. Protoplast regeneration inMougeotia is therefore a highly ordered process in which the orientation of the rapidly reorganized array of cortical microtubules establishes the future axis of elongation.     

Cortical microtubule arrays lose uniform alignment between cells and are oryzalin resistant in the Arabidopsis mutant, radially swollen 6

The coordinated expansion of cells is essential to the formation of correctly shaped plant tissues and organs. Members of the radially swollen (rsw) class of temperature-sensitive arabidopsis mutants were isolated in a screen for reduced anisotropic expansion, by selecting plants with radially swollen root tips. Here we describe rsw6, in which cortical microtubules in the root epidermis are well organized in parallel arrays within cells, but neighboring cells frequently contain arrays differing in their mean orientation by up to 90 degrees. Microtubules in rsw6 are more resistant to oryzalin-induced depolymerization than wild-type microtubules, and their reorientation is accompanied by swelling of the epidermal cells. The reorientation phenotype is blocked by taxol and by the depolymerization of actin filaments. We propose that rsw6 microtubule organization is functional on a local level, but defective on a global scale. The rsw6 mutant provides a unique tool with which to study the coordination of microtubule organization at a multicellular level.     

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.     

Differential Responsiveness of Cortical Microtubule Orientation to Suppression of Cell Expansion among the Developmental Zones of Arabidopsis thaliana Root Apex

Τhe bidirectional relationship between cortical microtubule orientation and cell wall structure has been extensively studied in elongating cells. Nevertheless, the possible interplay between microtubules and cell wall elements in meristematic cells still remains elusive. Herein, the impact of cellulose synthesis inhibition and suppressed cell elongation on cortical microtubule orientation was assessed throughout the developmental zones of Arabidopsis thaliana root apex by whole-mount tubulin immunolabeling and confocal microscopy. Apart from the wild-type, thanatos and pom2-4 mutants of Cellulose SynthaseA3 and Cellulose Synthase Interacting1, respectively, were studied. Pharmacological and mechanical approaches inhibiting cell expansion were also applied. Cortical microtubules of untreated wild-type roots were predominantly transverse in the meristematic, transition and elongation root zones. Cellulose-deficient mutants, chemical inhibition of cell expansion, or growth in soil resulted in microtubule reorientation in the elongation zone, wherein cell length was significantly decreased. Combinatorial genetic and chemical suppression of cell expansion extended microtubule reorientation to the transition zone. According to the results, transverse cortical microtubule orientation is established in the meristematic root zone, persisting upon inhibition of cell expansion. Microtubule reorientation in the elongation zone could be attributed to conditional suppression of cell elongation. The differential responsiveness of microtubule orientation to genetic and environmental cues is most likely associated with distinct biophysical traits of the cells among each developmental root zone.     

Microtubule Cycle in Root Tip Cells of Allium fistulosum L.

Using immunofluorescent localization techniques and TEM methods, the organization of microtubule arrays during the cell cycle of root tip cells of Allium fistulosum L. was studied. There are four basic types of microtubule organization, namely, interphase cortical microtubule, pre-prophase band microtubule, spindle microtubule and phragmoplast microtubule, which constitute the typical microtubule cycle in dividing cells of higher plants. The fluorescent figures of microtubules observed under fluorescent microscope were explained and analysed by the ultrastractural informations of microtubules obtained from TEM.     

The Arabidopsis CLASP gene encodes a microtubule-associated protein involved in cell expansion and division

Controlling microtubule dynamics and spatial organization is a fundamental requirement of eukaryotic cell function. Members of the ORBIT/MAST/CLASP family of microtubule-associated proteins associate with the plus ends of microtubules, where they promote the addition of tubulin subunits into attached kinetochore fibers during mitosis and stabilize microtubules in the vicinity of the plasma membrane during interphase. To date, nothing is known about their function in plants. Here, we show that the Arabidopsis thaliana CLASP protein is a microtubule-associated protein that is involved in both cell division and cell expansion. Green fluorescent protein-CLASP localizes along the full length of microtubules and shows enrichment at growing plus ends. Our analysis suggests that CLASP promotes microtubule stability. clasp-1 T-DNA insertion mutants are hypersensitive to microtubule-destabilizing drugs and exhibit more sparsely populated, yet well ordered, root cortical microtubule arrays. Overexpression of CLASP promotes microtubule bundles that are resistant to depolymerization with oryzalin. Furthermore, clasp-1 mutants have aberrant microtubule preprophase bands, mitotic spindles, and phragmoplasts, indicating a role for At CLASP in stabilizing mitotic arrays. clasp-1 plants are dwarf, have significantly reduced cell numbers in the root division zone, and have defects in directional cell expansion. We discuss possible mechanisms of CLASP function in higher plants.     

Dynamic microtubules under the radial and outer tangential walls of microinjected pea epidermal cells observed by computer reconstruction

By microinjecting rhodamine-conjugated pig brain tubulin into living pea stem epidermal cells it has been possible to follow cortical microtubules beneath the outer tangential wall (OTW) as they re-orientate from a transverse to a longitudinal alignment. Earlier immunofluorescence studies on fixed material have shown that parallel cortical microtubules circumnavigate the cell forming apparently continuous arrays which are transverse, oblique or longitudinal to the cell's long axis. If the array re-orientates as a whole then microtubules along the radial walls would be expected to share the alignment of those on the tangential walls. There are, however, reports that microtubules beneath the outer tangential wall have a different orientation from microtubules at the radial cell walls, raising important questions about the construction and behaviour of the array. Using computer-rotated stacks of optical sections collected by confocal scanning laser microscopy it has been possible to display the microtubules along radial as well as tangential walls of the same microinjected cells. These observations demonstrate for living epidermal cells that when microtubules are aligned longitudinally at the outer epidermal wall they remain oblique or transverse at the radial walls. The array may not therefore re-orientate as a whole but seems to undergo re-organization on only one cell face. However, despite the differing angles between the OTW and radial walls microtubules still form patterns which at the level of the confocal microscope are continuous from one cell face to another, around the cell.
It is concluded that some organizing principle attempts to establish overall organization at the cellular level but that this can be perturbed by local re-organization of dynamic microtubules in subcellular domains. This study emphasizes the importance of the outer epidermal wall and its associated cytoskeleton in initiating changes in the direction of cell expansion.     

NIMA-related kinases 6, 4, and 5 interact with each other to regulate microtubule organization during epidermal cell expansion in Arabidopsis thaliana

NimA-related kinase 6 (NEK6) has been implicated in microtubule regulation to suppress the ectopic outgrowth of epidermal cells; however, its molecular functions remain to be elucidated. Here, we analyze the function of NEK6 and other members of the NEK family with regard to epidermal cell expansion and cortical microtubule organization. The functional NEK6-green fluorescent protein fusion localizes to cortical microtubules, predominantly in particles that exhibit dynamic movement along microtubules. The kinase-dead mutant of NEK6 (ibo1-1) exhibits a disturbance of the cortical microtubule array at the site of ectopic protrusions in epidermal cells. Pharmacological studies with microtubule inhibitors and quantitative analysis of microtubule dynamics indicate excessive stabilization of cortical microtubules in ibo1/nek6 mutants. In addition, NEK6 directly binds to microtubules in vitro and phosphorylates β-tubulin. NEK6 interacts and co-localizes with NEK4 and NEK5 in a transient expression assay. The ibo1-3 mutation markedly reduces the interaction between NEK6 and NEK4 and increases the interaction between NEK6 and NEK5. NEK4 and NEK5 are required for the ibo1/nek6 ectopic outgrowth phenotype in epidermal cells. These results demonstrate that NEK6 homodimerizes and forms heterodimers with NEK4 and NEK5 to regulate cortical microtubule organization possibly through the phosphorylation of β-tubulins.     

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.