Gravitropic microtubule reorientation can be uncoupled from growth

The causal relationship between gravitropic growth responses and microtubule reorientation has been studied. Growth and microtubule reorientation have been uncoupled during the gravitropic response of maize (Zea mays L.) coleoptiles. Microtubule orientation and growth were measured under three different conditions: (i) a gravitropic stimulation where the growth response was allowed to be expressed (intact seedlings were displaced from the vertical position by 90°), (ii) a gravitropic stimulation where the growth response was suppressed (coleoptiles were attached to microscope slides and kept in a horizontal position), (iii) suppression of growth in the absence of gravitropic stimulation (coleoptiles were attached to microscope slides and kept in a vertical position). It was found that (i) gravitropic stimulation can induce a microtubular reorientation from transverse to longitudinal in the upper (slower growing) flank of the coleoptile, and an inhibition of growth; (ii) the reorientation of microtubules precedes the inhibition of growth; (iii) the gravitropic response of microtubules is weaker, not elevated, when the inhibition of growth is artificially enhanced by attaching the coleoptiles to a slide; and (iv) artificial inhibition of growth in the absence of gravitropic stimulation cannot induce a microtubular response. Thus, the extent of microtubule reorientation is not correlated with the extent of growth inhibition. Moreover, these findings demonstrate that microtubules do not reorient passively after growth changes, but actively in response to gravitropic stimulation. Received: 23 November 1999 / Accepted: 10 May 2000     

Mechanosensory microtubule reorientation in the epidermis of maize coleoptiles subjected to bending stress

Summary Plants respond to mechanical stress by adaptive changes in growth. Although this phenomenon is well established, the mechanism of the perception of mechanical forces by plant cells is not yet known. We provide evidence that the cortical microtubules sub-adjacent to the growth-controlling outer epidermal cell wall of maize coleoptiles respond to mechanical extension and compression by rapidly reorientating perpendicular to the direction of the effective force change. These findings shed new light on many seemingly unrelated observations on microtubule reorientation by growth factors such as light or phytohormones. Moreover, our results suggest that microtubules associated with the plasma membrane are causally involved in sensing vectorial forces and provide vectorial information to the cell that can be utilized in the orientation of plant organ expansion.Abbreviation MT cortical microtubule     

Microtubule reorientation in shoots precedes bending during the gravitropic response of cut snapdragon spikes

The microtubule reorientation during the gravitropic bending of cut snapdragon (Antirrhinum majus L.) spikes was investigated. Using indirect immunofluorescence methods, we examined changes in microtubule orientation in the cortex, endodermis and pith tissues of the shoot bending zone, in response to gravistimulation. Our results show that dense microtubule arrays were visible throughout the cortical, endodermal and pith shoot tissues, and that the transverse orientation of the microtubules (perpendicular to the growth axis) was specifically associated with the shoot growing bending zone. Microtubules showed gravity-induced kinetics of changes in their orientation, which occurred only in the upper stem flank and preceded shoot bending. While this observation, that the gravity-induced microtubule orientation precedes bending, was previously reported only in special above-ground organs such as coleoptiles and hypocotyls, our present study is the first to show that such patterns of change occur in mature flowering shoots. These changes were exhibited first in the upper flank of the cortex and then in the upper flank of the endodermis. No changes in microtubule orientation were observed in the cortex or endodermis tissues of the lower flanks or in the pith, suggesting that these tissues continue to grow during shoot gravistimulation. Our results imply that microtubules may be involved in growth cessation of the upper shoot flank occurring during the gravitropic bending of snapdragon cut spikes.     

Polarity induction versus phototropism in maize: Auxin cannot replace blue light

In a previous study (Nick and Schäfer 1991, Planta 185, 415–424), unilateral blue light had been shown, in maize coleoptiles, to induce phototropism and a stable transverse polarity, which became detectable as stable curvature if counteracting gravitropic stimulation was removed by rotation on a horizontal clinostat. This response was accompanied by a reorientation of cortical microtubules in the outer epidermis (Nick et al. 1990, Planta 181, 162–168). In the present study, this stable transverse polarity is shown to be correlated with stability of microtubule orientation against blue light and changes of auxin content. The role of auxin in this stabilisation was assessed. Although auxin can induce reorientation of microtubules it fails to induce the stabilisation of microtubule orientation induced by blue light. This was even true for gradients of auxin able to induce a bending response similar to that ellicited by phototropic stimulation. Experiments involving partial irradiation demonstrated different perception sites for phototropism and polarity induction. Phototropism starts from the very coleoptile tip and involves transmission of a signal (auxin) towards the subapical elongation zone. In contrast, polarity induction requires local action of blue light in the elongation zone itself. This blue-light response is independent of auxin.This work was supported by the Deutsche Forschungsgemeinschaft and two grants of the Studienstiftung des Deutschen Volkes and the Human Frontier Science Program Organization to P.N.     

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.     

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.     

Cellular basis for the automorphic curvature of rice coleoptiles on a three-dimensional clinostat: possible involvement of reorientation of cortical microtubules

Coleoptiles of rice (Oryza sativa L.) show a spontaneous (automorphic) curvature toward the caryopsis under microgravity conditions. The possible involvement of the reorientation of cortical microtubules in automorphic curvature was studied in rice coleoptiles grown on a three-dimensional clinostat. When rice seedlings that had been grown in the normal gravitational field were transferred to the clinostat in the dark, cortical microtubules of epidermal cells in the dorsal side of the coleoptiles oriented more transversely than the ventral side within 0.5 h. The rotation on the clinostat also increased the cell wall extensibility in the dorsal side and decreased the extensibility in the ventral side, and induced automorphic curvature. The reorientation of cortical microtubules preceded the changes in the cell wall extensibility and the curvature. The irradiation of rice seedlings with white light from above inhibited microtubule reorientation and changes in the cell wall extensibility, as well as curvature of coleoptiles. Also, colchicine, applied to the bending region of coleoptiles, partially inhibited the automorphic curvature. These results suggest that reorientation of cortical microtubules is involved in causing automorphic curvature in rice coleoptiles on the clinostat.     

Position and cell type-dependent microtubule reorientation characterizes the early response of the Arabidopsis root epidermis to ethylene

The involvement of cortical microtubules in the control of plant cell expansion was studied in the Arabidopsis root epidermis. In the zone of fast elongation microtubules were transverse to the root axis in all epidermal cells. However when cells entered the differentiation zone cell type-specific microtubule reorientation took place. In the trichoblasts that were then approximately 130 µm long and formed the root hair bulge, the microtubules switched to a random distribution. In the adjoining atrichoblasts microtubules adopted a slightly oblique orientation. In more proximal parts of the differentiation zone atrichoblast microtubules were found in a more oblique and finally in a longitudinal orientation. Upon exposure to ethylene or 1-aminocyclopropane-1-carboxylic acid (ACC – the precursor of ethylene) at a saturating dose, cell elongation abruptly stopped. From then on trichoblast cells reached only a length of about 35 µm, and developed root hairs. Cortical microtubules changed orientation within 10 min. In trichoblasts they adopted the typical random orientation, in atrichoblasts however, they took up a longitudinal orientation. Microtubule reorientation was complete within 60 min. The possible role of microtubules in the control of cell elongation is discussed.     

Organization of cortical microtubules in graviresponding maize roots

Immunofluorescence labeling of cortical microtubules (MTs) was used to investigate the relationship between MT arrangement and changes in growth rate of the upper and lower sides of horizontally placed roots of maize (Zea mays L. cv. Merit). Cap cells and cells of the elongation zone of roots grown vertically in light or darkness showed MT arrangements that were transverse (perpendicular) to the growth direction. Microtubules of cells basal to the elongation zone typically showed oblique orientation. Two hours after horizontal reorientation, cap cells of gravicompetent, light-grown and curving roots contained MTs parallel to the gravity vector. The MT arrangement on the upper side of the elongation zone remained transverse but the MTs of the outer four to five layers of cortical cells along the lower side of the elongation zone showed reorientation parallel to the axis of the root. The MTs of the lower epidermis retained their transverse orientation. Dark-grown roots did not curve and did not show reorientation of MTs in cells of the root cap or elongation zone. The data indicate that MT depolymerization and reorientation is correlated with reduction in growth rate, and that MT reorientation is one of the steps of growth control of graviresponding roots.Abbreviations MT microtubule - QC quiescent center This work was supported by National Science Foundation grant IBN-9118094.     

Do Microtubules Control Growth in Tropism? Experiments with Maize Coleoptiles

A recently proposed hypothesis [Nick et al. (1990) Planta 181:162] suggests that, in maize coleoptiles, tropistic curvaturemight be caused by a stimulus-induced trans-organ gradient overthe orientation of cortical microtubules adjacent to the outercell wall of the outer epidermis. This gradient, in turn, iscontrolled by a light-induced redistribution of auxin. The hypothesiswas tested by following the behaviour of microtubules for variouslight stimuli using indirect immuno-fiuorescence in epidermalstrips as assay. Analysis of gravitropic straightening, nasticcurvature on the horizontal clinostat, effects of tonic irradiationwith red and/or blue light, and experiments involving opposinglight pulses demonstrate that bending direction and microtubuleorientation gradients are not as closely linked as predicted:Considerable bending can be produced without detectable gradientsof microtubule orientation, and conspicuous gradients of microtubuleorientation are not necessarily expressed as corresponding curvature.Thus, a monocausal relationship between microtubules and tropismis excluded. Furthermore, a comparison of tonic light effectson microtubules to earlier studies into the impact of lightupon auxin content indicate that the relationship between auxinand microtubules might be more complex than hitherto assumed.It is concluded that, at least in maize coleoptiles, growthcan be regulated by various mechanisms, and that microtubules,although somehow related to tropism, are probably not the causeof the fast tropistic responses. (Received April 26, 1991; Accepted July 17, 1991)     

Unilateral reorientation of microtubules at the outer epidermal wall during photo- and gravitropic curvature of maize coleoptiles and sunflower hypocotyls

Auxin (indole-3-acetic acid) controls the orientation of cortical microtubes (MT) at the outer wall of the outer epidermis of growing maize coleoptiles (Bergfeld, R., Speth, V., Schopfer, P., 1988, Bot. Acta 101, 57-67). A detailed time course of MT reorientation, determined by labeling MT with fluorescent antibodies, revealed that the auxin-mediated movement of MT from the longitudinal to the transverse direction starts after less than 15 min and is completed after 60 min. This response was used for a critical test of the functional involvement of auxin in tropic curvature. It was found that phototropic (first phototropic curvature) as well as gravitropic bending are correlated with a change of MT orientation from transverse to longitudinal at the slower-growing organ flank whereas the transverse MT orientation is maintained (or even augmented) at the faster-growing organ flank. These directional changes are confined to the MT subjacent to the outer epidermal wall. The same basic results were obtained with sunflower hypocotyls subjected to phototropic or gravitropic stimulation. It is concluded that auxin is, in fact, involved in asymmetric growth leading to tropic curvature. However, our results do not allow us to discriminate between an uneven distribution of endogenous auxin or an even distribution of auxin, the activity of which is modulated by an unevenly distributed inhibitor of auxin action.     

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     

Arabidopsis AUGMIN Subunit8 Is a Microtubule Plus-End Binding Protein That Promotes Microtubule Reorientation in Hypocotyls

In plant cells, cortical microtubules provide tracks for cellulose-synthesizing enzymes and regulate cell division, growth, and morphogenesis. The role of microtubules in these essential cellular processes depends on the spatial arrangement of the microtubules. Cortical microtubules are reoriented in response to changes in cell growth status and cell shape. Therefore, an understanding of the mechanism that underlies the change in microtubule orientation will provide insight into plant cell growth and morphogenesis. This study demonstrated that AUGMIN subunit8 (AUG8) in Arabidopsis thaliana is a novel microtubule plus-end binding protein that participates in the reorientation of microtubules in hypocotyls when cell elongation slows down. AUG8 bound to the plus ends of microtubules and promoted tubulin polymerization in vitro. In vivo, AUG8 was recruited to the microtubule branch site immediately before nascent microtubules branched out. It specifically associated with the plus ends of growing cortical microtubules and regulated microtubule dynamics, which facilitated microtubule reorientation when microtubules changed their growth trajectory or encountered obstacle microtubules during microtubule reorientation. This study thus reveals a novel mechanism underlying microtubule reorientation that is critical for modulating cell elongation in Arabidopsis.     

Auxin-induced longitudinal-to-transverse reorientation of cortical microtubules in nonelongating epidermal cells of azuki bean epicotyls

Summary In epidermal cells of azuki bean (Vigna angularis) epicotyl segments, that were sequentially treated with an auxin-free solution and an auxin solution, cortical microtubules changed their orientation from longitudinal to transverse. Auxin caused the reorientation of microtubules from longitudinal to transverse in segments that were kept under anaerobic conditions and, therefore, showed no elongation, indicating that auxin can regulate the orientation of microtubules by a mechanism that does not involve auxin-induced change in the rate of cell elongation.Abbreviations DMSO dimethylsulfoxide - GA₃ gibberellic acid - IAA indoleacetic acid - MT microtubule - PBS phosphate-buffered saline     

A GFP-MAP4 reporter gene for visualizing cortical microtubule rearrangements in living epidermal cells

Microtubules influence morphogenesis by forming distinct geometrical arrays in the cell cortex, which in turn affect the deposition of cellulose microfibrils. Although many chemical and physical factors affect microtubule orientation, it is unclear how cortical microtubules in elongating cells maintain their ordered transverse arrays and how they reorganize into new geometries. To visualize these reorientations in living cells, we constructed a microtubule reporter gene by fusing the microtubule binding domain of the mammalian microtubule-associated protein 4 (MAP4) gene with the green fluorescent protein (GFP) gene, and transient expression of the recombinant protein in epidermal cells of fava bean was induced. The reporter protein decorates microtubules in vivo and binds to microtubules in vitro. Confocal microscopy and time-course analysis of labeled cortical arrays along the outer epidermal wall revealed the lengthening, shortening, and movement of microtubules; localized microtubule reorientations; and global microtubule reorganizations. The global microtubule orientation in some cells fluctuates about the transverse axis and may be a result of a cyclic self-correcting mechanism to maintain a net transverse orientation during cellular elongation.     

Auxin Redistribution during First Positive Phototropism in Corn Coleoptiles : Microtubule Reorientation and the Cholodny-Went Theory

In red-light grown corn (Zea mays L. cv Brio42.HT) coleoptiles, cortical microtubules adjacent to the outer cell wall of the outer epidermis reorient from transverse to longitudinal in response to auxin depletion and after phototropic stimulation in the lighted side of the coleoptile. This was used as an in situ assay of cellular auxin concentration. The fluence-response relation for the blue light-induced reorientation is compared with that for first positive phototropism and the dose-response relationship for the auxin-dependent reorientation. The result supports the theory by Cholodny and Went, claiming that phototropic stimulation results in auxin displacement across the coleoptile. In terms of microtubule orientation, this displacement becomes even more pronounced after preirradiation with a weak blue light pulse from above.     

MicroFilament Analyzer,an image analysis tool for quantifying fibrillar orientation,reveals changes in microtubule organization during gravitropism

Image acquisition is an important step in the study of cytoskeleton organization. As visual interpretations and manual measurements of digital images are prone to errors and require a great amount of time, a freely available software package named MicroFilament Analyzer (MFA) was developed. The goal was to provide a tool that facilitates high‐throughput analysis to determine the orientation of filamentous structures on digital images in a more standardized, objective and repeatable way. Here, the rationale and applicability of the program is demonstrated by analyzing the microtubule patterns in epidermal cells of control and gravi‐stimulated Arabidopsis thaliana roots. Differential expansion of cells on either side of the root results in downward bending of the root tip. As cell expansion depends on the properties of the cell wall, this may imply a differential orientation of cellulose microfibrils. As cellulose deposition is orchestrated by cortical microtubules, the microtubule patterns were analyzed. The MFA program detects the filamentous structures on the image and identifies the main orientation(s) within individual cells. This revealed four distinguishable microtubule patterns in root epidermal cells. The analysis indicated that gravitropic stimulation and developmental age are both significant factors that determine microtubule orientation. Moreover, the data show that an altered microtubule pattern does not precede differential expansion. Other possible applications are also illustrated, including field emission scanning electron micrographs of cellulose microfibrils in plant cell walls and images of fluorescent actin.     

Mechano-sensitive orientation of cortical microtubules during gravitropism in azuki bean epicotyls

The orientation of cortical microtubules (cMT) during gravitropism was studied in epidermal cells of azuki epicotyls. The relative proportion of cells with longitudinal cMT increased in the upper epidermis, and those with transverse cMT increased in the lower epidermis. When epicotyls were kept straight during gravistimulation, no change in cMT orientation occurred in either the upper and lower epidermis. When epicotyls were forced to bend downward, cells with transverse cMT increased in the upper epidermis, and those with longitudinal cMT increased in the lower epidermis. When epicotyls were loaded with naphthylphthalamic acid, an inhibitor of auxin transport, both gravitropic bending and change in cMT orientation were inhibited. However, when a change in cMT orientation was induced by forced downward bending, cells with longitudinal cMT increased in the compressed (lower) side and those with transverse cMT increased in the extended (upper) side. It was suggested that cMT orientation was controlled by the bending of the epicotyl and not by a gravity signal per se. Loading with Gd³⁺, an inhibitor of the stretch-activated channel, did not inhibit gravitropic bending. However, it inhibited cMT reorientation induced by gravitropic bending and by forced bending. Involvement of the stretch-activated channel in mechano-sensitive orientation of cMT was suggested.     

Microtubule rearrangements accompanying dedifferentiation in mesophyll cultures ofZinnia elegans L.

Summary To determine the orientation of cortical microtubule arrays in mesophyll cells ofZinnia, a new technique designed to increase the rate of fixation of excised leaf tissue and subsequent permeabilization of mesophyll cell walls was developed. This procedure resulted in immunolabeling of high percentages of mesophyll cells, making it possible to quantify cells with different types of cortical microtubule arrays. When developing palisade mesophyll cells were fixed in situ, most of the cells had cortical microtubules organized in parallel arrays oriented transverse to the long axis. Delay in the transfer of leaf tissue to fixative resulted in increased numbers of cells with random cortical microtubule orientations, indicating that arrays may become reoriented rapidly during leaf excision and cell isolation procedures. The role of wound-induced microtubule reorientation in mesophyll dedifferentiation and tracheary element development is discussed.Abbreviations BSA bovine serum albumin - CMT cortical microtubule - TE tracheary element - TBS tris-buffered saline     

On the relation between photo- and gravitropically induced spatial memory in maize coleoptiles

The interaction of photo- and gravitropic stimulation was studied by analysing the curvature of maize (Zea mays L.) coleoptiles subjected to rotation on horizontal clinostats. Gravitropic curvature in different directions with respect to the stimulation plane was found to be transient. This instability was caused by an increasing deviation of response direction from the stimulation plane towards the caryopsis. The bending angle as such, however, increased steadily. This reorientation of the gravitropic response towards the caryopsis is thought to be caused by the clinostat-elicited nastic curvature found in maize coleoptiles. In contrast, the response to phototropic stimulation was stable, in both, orientation and curving. Although stimulation by gravity was not capable of inducing a stable tropistic response, it could inhibit the response to opposing phototropic stimulation, if the counterstimulation was given more than 90 min after the onset of gravistimulation. For shorter time intervals the influence of the phototropic stimulus obscured the response to the first, gravitropic stimulation. For time intervals exceeding 90 min, however, the phototropic effects disappeared and the response was identical to that for gravity stimulation alone. This gravity-induced inhibition of the phototropic response was confined to the plane of gravity stimulation, because a phototropic stimulation in the perpendicular direction remained unaffected, irrespective of the time interval between the stimulations. This concerned not only the stable phototropic curving, but also the capacity of the phototropic induction to elicit a stable directional memory as described earlier (P. Nick and F. Schäfer, 1988b, Planta 175, 380–388). This was tested by a second bluelight pulse opposing the first. It is suggested that gravity, too, can induce a directional memory differing from the blue-light elicited memory. The mechanisms mediating gravi- and phototropic directional memories are thought to branch off the respective tropistic signal chains at a stage where photo- and gravitropic transduction are still separate.This work was supported by the Deutsche Forschungsgemeinschaft and a grant of the Studienstiftung des Deutschen Volkes to P. Nick.