MDP25, a novel calcium regulatory protein, mediates hypocotyl cell elongation by destabilizing cortical microtubules in Arabidopsis

The regulation of hypocotyl elongation is important for plant growth. Microtubules play a crucial role during hypocotyl cell elongation. However, the molecular mechanism underlying this process is not well understood. In this study, we describe a novel Arabidopsis thaliana microtubule-destabilizing protein 25 (MDP25) as a negative regulator of hypocotyl cell elongation. We found that MDP25 directly bound to and destabilized microtubules to enhance microtubule depolymerization in vitro. The seedlings of mdp25 mutant Arabidopsis lines had longer etiolated hypocotyls. In addition, MDP25 overexpression resulted in significant overall shortening of hypocotyl cells, which exhibited destabilized cortical microtubules and abnormal cortical microtubule orientation, suggesting that MDP25 plays a crucial role in the negative regulation of hypocotyl cell elongation. Although MDP25 localized to the plasma membrane under normal conditions, increased calcium levels in cells caused MDP25 to partially dissociate from the plasma membrane and move into the cytosol. Cellular MDP25 bound to and destabilized cortical microtubules, resulting in their reorientation, and subsequently inhibited hypocotyl cell elongation. Our results suggest that MDP25 exerts its function on cortical microtubules by responding to cytoplasmic calcium levels to mediate hypocotyl cell elongation.     

Phospholipase Dδ negatively regulates plant thermotolerance by destabilizing cortical microtubules in Arabidopsis

The pattern of cortical microtubule arrays plays an important role in plant growth and adaptation in response to hormonal and environmental changes. Cortical microtubules are connected with the plasma membrane (PM); however, how the membrane affects cortical microtubule organization is not well understood. Here, we showed that phospholipase Dδ (PLDδ) was associated with the PM and co‐localized with microtubules in cells. In vitro analysis revealed that PLDδ bound to microtubules, resulting in microtubule disorganization. Site‐specific mutations that decreased PLDδ enzymatic activity impaired its effects on destabilizing microtubule organization. Heat shock transiently activated PLDδ, without any change of its PM localization, triggering microtubule dissociation from PM and depolymerization and seedling death in Arabidopsis, but these effects were alleviated in pldδ knockout mutants. Complementation of pldδ with wild‐type PLDδ, but not mutated PLDδ, restored the phenotypes of microtubules and seedling survival to those of wild‐type Arabidopsis. Thus, we conclude that the PM‐associated PLDδ negatively regulates plant thermotolerance via destabilizing cortical microtubules, in an activity‐dependent manner, rather than its subcellular translocation.     

Regulation of secondary cell wall development by cortical microtubules during tracheary element differentiation in Arabidopsis cell suspensions

Cortical microtubules participate in the deposition of patterned secondary walls in tracheary element differentiation. In this study, we established a system to induce the differentiation of tracheary elements using a transgenic Arabidopsis (Arabidopsis thaliana) cell suspension stably expressing a green fluorescent protein-tubulin fusion protein. Approximately 30% of the cells differentiated into tracheary elements 96 h after culture in auxin-free media containing 1 mum brassinolide. With this differentiation system, we have been able to time-sequentially elucidate microtubule arrangement during secondary wall thickening. The development of secondary walls could be followed in living cells by staining with fluorescein-conjugated wheat germ agglutinin, and the three-dimensional structures of the secondary walls could be simultaneously analyzed. A single microtubule bundle first appeared beneath the narrow secondary wall and then developed into two separate bundles locating along both sides of the developing secondary wall. Microtubule inhibitors affected secondary wall thickening, suggesting that the pair of microtubule bundles adjacent to the secondary wall played a crucial role in the regulation of secondary wall development.     

Arabidopsis mitogen-activated protein kinase MPK18 mediates cortical microtubule functions in plant cells

Mitogen-activated protein kinase (MAPK) signalling networks are important regulators of environmental responses and developmental processes in plants. To understand the role of MAPK signalling modules in the regulation of plant microtubule functions, we searched for MAPKs that interact with the dual-specificity MAPK phosphatase, PROPYZAMIDE HYPERSENSITIVE 1 ( PHS1 ), whose mutation has previously been reported to confer hypersensitivity to microtubule-disrupting drugs in Arabidopsis. Yeast two-hybrid assays demonstrated that PHS1 specifically interacts with two MAPKs, MPK12 and MPK18. Bimolecular fluorescence complementation (BiFC) studies confirmed that the PHS1 and MPK18 proteins are physically coupled, and that this interaction occurs in the cytoplasm. At the biochemical level, in vitro dephosphorylation assays indicated that phospho-MPK18 can be dephosphorylated by recombinant PHS1. Mutant mpk18 seedlings show defects in microtubule-related functions, and have moderately stabilized microtubules. Absence of MPK18 in the phs1-1 background partially complements the phs1-1 root growth phenotypes, providing genetic evidence for involvement of MPK18 signalling in microtubule-related functions. We propose a model whereby the PHS1–MPK18 signalling module is involved in a phosphorylation/dephosphorylation switch that regulates cortical microtubule functions.     

Organization of cortical microtubules at the plasma membrane in Arabidopsis

 To understand the role of microtubules in the regulation of cell elongation, we characterized microtubule patterns in fass, a cell shape mutant of Arabidopsis thaliana (L.) Heynh. Examining microtubule patterns via immunocytochemistry, we found that fass cells were able to organize their microtubules into mitotic spindles and phragmoplasts. During interphase or preprophase, fass cells had cortical microtubules, verified by transmission electron microscopy, but these microtubules were not organized into the cortical array or preprophase band. Using chromatin condensation and tubulin localization on the nuclear envelope as preprophase stage markers, we found that although fass cells lacked the preprophase band and cortical array, their cell division cycle appeared normal. To pinpoint the defect in fass cells, we delineated the sequential events leading to cortical array formation in Arabidopsis cells and found that fass cells initiated and recolonized cortical microtubules in the same manner as wild-type cells, but failed to order them into the cortical array. Taken together, these results suggest fass cells are impaired in a component of the microtubule organizing center(s) required for the proper ordering of cortical microtubules at the plasma membrane. Received: 23 August 1996 / Accepted: 25 September 1996     

Twisted growth and organization of cortical microtubules

In plants, directional cell expansion greatly contributes to the final shape of mature cells, and thus to organ architecture. A particularly interesting mode of cell expansion is helical growth in which the growth axis is continuously tilted either to the right or to the left as the cell grows. Fixed handedness of helical growth raises fundamental questions on the possible origin of left–right asymmetry. Twisting mutants of Arabidopsis thaliana offer unique opportunities to study the cellular basis of helical growth. Most of the twisting mutants with fixed handedness have been shown to have defects in microtubule functions, whereas mutants that twist in non-fixed directions appear to be defective in auxin response or transport. Good correlations have been found between the tilted growth direction and alignment of cortical microtubule arrays in twisting mutants with compromised microtubule functions. The present challenge is to understand how particular array patterns are organized during progression of the interphase in rapidly expanding cells. Molecular and cell biological studies on twisting mutants will lead to better understanding on how wild-type plant cells utilize the microtubule cytoskeleton to initiate and rigorously maintain straight growth. An erratum to this article can be found at     

BOTERO1 is required for normal orientation of cortical microtubules and anisotropic cell expansion in Arabidopsis

Mutants at the BOTERO1 locus are affected in anisotropic growth in all non-tip-growing cell types examined. Mutant cells are shorter and broader than those of the wild type. Mutant inflorescence stems show a dramatically reduced bending modulus and maximum stress at yield. Our observations of root epidermis cells show that the cell expansion defect in bot1 is correlated with a defect in the orientation of the cortical microtubules. We found that in cells within the apical portion of the root, which roughly corresponds to the meristem, microtubules were loosely organized and became much more highly aligned in transverse arrays with increasing distance from the tip. Such a transition was not observed in bot1. No defect in microtubule organization was observed in kor-1, another mutant with a radial cell expansion defect. We also found that in wild-type root epidermal cells, cessation of radial expansion precedes the increased alignment of cortical microtubules into transverse arrays. Bot1 roots still show a gravitropic response, which indicates that ordered cortical microtubules are not required for differential growth during gravitropism. Interestingly, the fact that in the mutant, these major changes in microtubule organization cause relatively subtle changes in cell morphology, suggest that other levels of control of growth anisotropy remain to be discovered. Together, these observations suggest that BOT1 is required for organizing cortical microtubules into transverse arrays in interphase cells, and that this organization is required for consolidating, rather than initiating, changes in the direction of cell expansion.     

Stabilization of cortical microtubules by the cell wall in cultured tobacco cells

Cortical microtubules (MTs) in protoplasts prepared from tobacco (Nicotiana tabacum L.) BY-2 cells were found to be sensitive to cold. However, as the protoplasts regenerated cell walls they became resistant to cold, indicating that the cell wall stabilizes cortical MTs against the effects of cold. Since poly-l-lysine was found to stabilize MTs in protoplasts, we examined extensin, an important polycationic component of the cell wall, and found it also to be effective in stabilizing the MTs of protoplasts. Both extensin isolated from culture filtrates of tobacco BY-2 cells and extensin isolated in a similar way from cultures of tobacco XD-6S cells rendered the cortical MTs in protoplasts resistant to cold. Extensin at 0.1 mg·ml⁻¹ was as effective as the cell wall in this respect. It is probable that extensin in the cell wall plays an important role in stabilizing cortical MTs in tobacco BY-2 cells.     

Structural polarity and directional growth of microtubules of Chlamydomonas flagella

A basic question concerning microtubule assembly is the polarity of growth, namely, whether subunits can add to either end of a growing microtubule or whether growth proceeds by subunit addition to only one end. To approach this question in an in vitro system, experiments were carried out on the addition of microtubule subunits to isolated flagellar axonemes. Flagella were detached from Chlamydomonas by brief treatment with non-ionic detergent, isolated by differential centrifugation, and incubated with crude high-speed extracts of porcine brain tissue or with purified tubulin (obtained by repetitive temperature-dependent assembly and disassembly). Electron microscopy of negatively stained samples showed as many as 11 long microtubules added at one end of more than 90% of the axonemes. Colchicine (100 μm), CaCl₂ (2.5 mm), and low temperature (0 °C) both prevented and reversed microtubule assembly but had no effect on axonemal length. In crude extracts microtubules formed on both members of the axonemal central pair but on only the A-tubule of the outer doublets. Flagellar fragments, produced by mechanical shearing, were also incubated with microtubule subunit. Single tubules formed at only one end of outer doublet fragments; the appearance of single tubules on one or both members of central pair fragments was predominantly unidirectional. Structural analysis of frayed axonemes and the asymmetry of side-arm attachments permitted the absolute polarity of the axonemal fragments to be determined and revealed that assembly proceeded by addition of subunits to the distal ends of the axonemal microtubules. Using purified brain tubulin, a limited extent of proximal addition and growth on the B-tubule also occurred. The extent of proximal addition increased with increasing protein concentration and temperature. We conclude that the microtubules of flagella have an intrinsic polarity reflected in their side-arm attachments and in their directionality of growth.     

Disruption of arabinogalactan proteins disorganizes cortical microtubules in the root of Arabidopsis thaliana

The cortical array of microtubules inside the cell and arabinogalactan proteins on the external surface of the cell are each implicated in plant morphogenesis. To determine whether the cortical array is influenced by arabinogalactan proteins, we first treated Arabidopsis roots with a Yariv reagent that binds arabinogalactan proteins. Cortical microtubules were markedly disorganized by 1 microM beta-D-glucosyl (active) Yariv but not by up to 10 microM beta-D-mannosyl (inactive) Yariv. This was observed for 24-h treatments in wild-type roots, fixed and stained with anti-tubulin antibodies, as well as in living roots expressing a green fluorescent protein (GFP) reporter for microtubules. Using the reporter line, microtubule disorganization was evident within 10 min of treatment with 5 microM active Yariv and extensive by 30 min. Active Yariv (5 microM) disorganized cortical microtubules after gadolinium pre-treatment, suggesting that this effect is independent of calcium influx across the plasma membrane. Similar effects on cortical microtubules, over a similar time scale, were induced by two anti-arabinogalactan-protein antibodies (JIM13 and JIM14) but not by antibodies recognizing pectin or xyloglucan epitopes. Active Yariv, JIM13, and JIM14 caused arabinogalactan proteins to aggregate rapidly, as assessed either in fixed wild-type roots or in the living cells of a line expressing a plasma membrane-anchored arabinogalactan protein from tomato fused to GFP. Finally, electron microscopy of roots prepared by high-pressure freezing showed that treatment with 5 microM active Yariv for 2 h significantly increased the distance between cortical microtubules and the plasma membrane. These findings demonstrate that cell surface arabinogalactan proteins influence the organization of cortical microtubules.     

Hypocotyl directional growth in Arabidopsis: a complex trait

The growth direction of the Arabidopsis (Arabidopsis thaliana) etiolated-seedling hypocotyl is a complex trait that is controlled by extrinsic signals such as gravity and touch as well as intrinsic signals such as hormones (brassinosteroid [BR], auxin, cytokinin, ethylene) and nutrient status (glucose [Glc], sucrose). We used a genetic approach to identify the signaling elements and their relationship underlying hypocotyl growth direction. BR randomizes etiolated-seedling growth by inhibiting negative gravitropism of the hypocotyls via modulating auxin homeostasis for which we designate as reset, not to be confused with the gravity set point angle. Cytokinin signaling antagonizes this BR reset of gravity sensing and/or tropism by affecting ethylene biosynthesis/signaling. Glc also antagonizes BR reset but acts independently of cytokinin and ethylene signaling pathways via inhibiting BR-regulated gene expression quantitatively and spatially, by altering protein degradation, and by antagonizing BR-induced changes in microtubule organization and cell patterning associated with hypocotyl agravitropism. This BR reset is reduced in the presence of the microtubule organization inhibitor oryzalin, suggesting a central role for cytoskeleton reorganization. A unifying and hierarchical model of Glc and hormone signaling interplay is proposed. The biological significance of BR-mediated changes in hypocotyl graviresponse lies in the fact that BR signaling sensitizes the dark-grown seedling hypocotyl to the presence of obstacles, overriding gravitropism, to enable efficient circumnavigation through soil.     

An exocyst complex functions in plant cell growth in Arabidopsis and tobacco

The exocyst, an octameric tethering complex and effector of Rho and Rab GTPases, facilitates polarized secretion in yeast and animals. Recent evidence implicates three plant homologs of exocyst subunits (SEC3, SEC8, and EXO70A1) in plant cell morphogenesis. Here, we provide genetic, cell biological, and biochemical evidence that these and other predicted subunits function together in vivo in Arabidopsis thaliana. Double mutants in exocyst subunits (sec5 exo70A1 and sec8 exo70A1) show a synergistic defect in etiolated hypocotyl elongation. Mutants in exocyst subunits SEC5, SEC6, SEC8, and SEC15a show defective pollen germination and pollen tube growth phenotypes. Using antibodies directed against SEC6, SEC8, and EXO70A1, we demonstrate colocalization of these proteins at the apex of growing tobacco pollen tubes. The SEC3, SEC5, SEC6, SEC8, SEC10, SEC15a, and EXO70 subunits copurify in a high molecular mass fraction of 900 kD after chromatographic fractionation of an Arabidopsis cell suspension extract. Blue native electrophoresis confirmed the presence of SEC3, SEC6, SEC8, and EXO70 in high molecular mass complexes. Finally, use of the yeast two-hybrid system revealed interaction of Arabidopsis SEC3a with EXO70A1, SEC10 with SEC15b, and SEC6 with SEC8. We conclude that the exocyst functions as a complex in plant cells, where it plays important roles in morphogenesis.     

NIMA-related kinases regulate directional cell growth and organ development through microtubule function in Arabidopsis thaliana

NIMA-related kinase 6 (NEK6) regulates cellular expansion and morphogenesis through microtubule organizaiton in Arabidopsis thaliana. Loss-of-function mutations in NEK6 (nek6/ibo1) cause ectopic outgrowth and microtubule disorganization in epidermal cells. We recently found that NEK6 forms homodimers and heterodimers with NEK4 and NEK5 to destabilize cortical microtubules possibly by direct binding to microtubules and the β-tubulin phosphorylation. Here, we identified a new allele of NEK6 and further analyzed the morphological phenotypes of nek6/ibo1 mutants, along with alleles of nek4 and nek5 mutants. Phenotypic analysis demonstrated that NEK6 is required for the directional growth of roots and hypocotyls, petiole elongation, cell file formation, and trichome morphogenesis. In addition, nek4, nek5, and nek6/ibo1 mutants were hypersensitive to microtubule inhibitors such as propyzamide and taxol. These results suggest that plant NEKs function in directional cell growth and organ development through the regulation of microtubule organization.     

The impact of mechanical compression on cortical microtubules in Arabidopsis: a quantitative pipeline

Exogenous mechanical perturbations on living tissues are commonly used to investigate whether cell effectors can respond to mechanical cues. However, in most of these experiments, the applied mechanical stress and/or the biological response are described only qualitatively. We developed a quantitative pipeline based on microindentation and image analysis to investigate the impact of a controlled and prolonged compression on microtubule behaviour in the Arabidopsis shoot apical meristem, using microtubule fluorescent marker lines. We found that a compressive stress, in the order of magnitude of turgor pressure, induced apparent microtubule bundling. Importantly, that response could be reversed several hours after the release of compression. Next, we tested the contribution of microtubule severing to compression‐induced bundling: microtubule bundling seemed less pronounced in the katanin mutant, in which microtubule severing is dramatically reduced. Conversely, some microtubule bundles could still be observed 16 h after the release of compression in the spiral2 mutant, in which severing rate is instead increased. To quantify the impact of mechanical stress on anisotropy and orientation of microtubule arrays, we used the nematic tensor based FibrilTool ImageJ/Fiji plugin. To assess the degree of apparent bundling of the network, we developed several methods, some of which were borrowed from geostatistics. The final microtubule bundling response could notably be related to tissue growth velocity that was recorded by the indenter during compression. Because both input and output are quantified, this pipeline is an initial step towards correlating more precisely the cytoskeleton response to mechanical stress in living tissues.     

Reorganization of cortical microtubules during cell differentiation in tobacco explants

Summary Using indirect immunofluorescence on polyethylene glycol embedded material, the organization of cortical microtubules (MTs) has been studied in explants ofNicotiana tabacum. Within 6 hours after explantation the orientation of the cortical MTs shifts from transverse to longitudinal to the long axis of the cell in all cells. This change of direction is followed by further shifts that occur only locally and predict the orientation of future cell divisions. These reorientations are independent of the formation of protrusions and buds that will develop in the explants (after 4–7 days) and they represent a stage of de-differentiation of the explants. After two days of culturing clusters of cells can be recognized, at the proximal side of the explants, with randomly oriented cortical MTs. These regions represent the origin of the protrusions from which floral buds will develop. The formation of these clusters represent the first signs of re-differentiation and formation of new polar axes in the explants. The cells thus show a very early commitment (within 2 days) as to their differentiation.Abbreviations BAP benzyl-amino-purine - DMSO dimethylsulfoxid - EGTA ethylene glycol bis(2-aminoethylether)-N,N,N,N-tetraacetic acid - GA glutaraldehyde - MTs microtubules - MTOCs microtubule organizing centres - NAA -naphthalene acetic acid - PEG polyethylene glycol - PFA paraformaldehyde - PPBs preprophase bands     

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.     

Intercourse between cell wall and cytoplasm exemplified by arabinogalactan proteins and cortical microtubules

How does a plant cell sense and respond to the status of its cell wall? Intercourse between cell wall and cytoplasm has long been supposed to involve arabinogalactan proteins, in part because many of them are anchored to the plasma membrane. Disrupting arabinogalactan proteins has recently been shown to disrupt the array of cortical microtubules present just inside the plasma membrane, implying that microtubules and arabinogalactan proteins interact. In this article, we assess possibilities for how this interaction might be mediated. First, we consider microdomains in the plasma membrane (lipid rafts), which have been alleged to link internal and external regions of the plasma membrane; however, the characteristics and even the existence of these domains remains controversial. Next, we point out that disrupting the synthesis of cellulose also can disrupt microtubules and consider whether arabinogalactan proteins are part of a network linking microtubules and nascent microfibrils. Finally, we outline several signaling cascades that could transmit information from arabinogalactan proteins to microtubules through channels of cellular communication. These diverse possibilities highlight the work that remains to be done before we can understand how plant cells communicate across their membranes.     

CONSTITUTIVE PHOTOMORPHOGENIC 1 promotes seed germination by destabilizing RGA-LIKE 2 in Arabidopsis

Under favorable moisture, temperature, and light conditions, gibberellin (GA) biosynthesis is induced and triggers seed germination. A major mechanism by which GA promotes seed germination is by promoting the degradation of the DELLA protein RGA-LIKE 2 (RGL2), a major repressor of germination in Arabidopsis (Arabidopsis thaliana) seeds. Analysis of seed germination phenotypes of constitutive photomorphogenic 1 (cop1) mutants and complemented COP1-OX/cop1-4 lines in response to GA and paclobutrazol (PAC) suggested a positive role for COP1 in seed germination and a relation with GA signaling. cop1-4 mutant seeds showed PAC hypersensitivity, but transformation with a COP1 overexpression construct rendered them PAC insensitive, with a phenotype similar to that of rgl2 mutant (rgl2-SK54) seeds. Furthermore, cop1-4 rgl2-SK54 double mutants showed a PAC-insensitive germination phenotype like that of rgl2-SK54, identifying COP1 as an upstream negative regulator of RGL2. COP1 interacted directly with RGL2, and in vivo this interaction was strongly enhanced by SUPPRESSOR OF PHYA-105 1. COP1 directly ubiquitinated RGL2 to promote its degradation. Moreover, GA stabilized COP1 with consequent RGL2 destabilization. By uncovering this COP1–RGL2 regulatory module, we reveal a mechanism whereby COP1 positively regulates seed germination and controls the expression of germination-promoting genes.

A master regulator of photomorphogenesis positively regulates germination in Arabidopsis seeds by directly ubiquitinating and promoting the degradation of a key repressor of seed germination.     

Orientation of cortical microtubules correlates with cell shape and division direction

Summary Cortical microtubules in the epidermis of regeneratingGraptopetalum plants were examined by in situ immunofluorescence. Paradermal slices of tissue were prepared by a method that preserves microtubule arrays and also maintains cell junctions. To test the hypothesis that cortical microtubule arrays align perpendicular to the direction of organ growth, arrays were visualized and their orientation quantified. A majority of microtubules are in transverse orientation with respect to the organ axis early in shoot development when the growth habit is uniform. Later in development, when growth habit is non-uniform and the tissue is contoured, cortical microtubules are increasingly longitudinal and oblique in orientation. Microtubules show only a minor change in orientation at the site of greatest curvature, the transition zone of a developing leaf. To assess the role of the division plane on orientation of arrays, the pattern of microtubules was examined in individual cells of common shape. Cells derived from transverse divisions have predominately transverse cortical arrays, whereas cells derived from oblique and longitudinal divisions have non-transverse arrays. The results show that, regardless of the stage of development, microtubules orient with respect to cell shape and plane of division. The results suggest that cytoskeletal function is best considered in small domains of growth within an organ.Abbrevations DMSO dimethylsulfoxide - EGTA ethylene glycol-bis-(ß-aminoethyl ether)-N, N, N, N-tetra acetic acid - FITC fluorescein isothiocyanate - MTSB microtubule stabilizing buffer - PBS phosphate buffered saline     

Hypergravity induces reorientation of cortical microtubules and modifies growth anisotropy in azuki bean epicotyls

We examined the changes in the orientation of cortical microtubules during the hypergravity-induced modification of growth anisotropy (inhibition of elongation growth and promotion of lateral growth) in azuki bean (Vigna angularis Ohwi et Ohashi) epicotyls. The percentage of cells with transverse microtubules was decreased, while that with longitudinal microtubules was increased, in proportion to the logarithm of the magnitude of gravity. The percentage of cells with longitudinal microtubules showed an increase within 0.5 h of transfer of the 1g-grown seedlings to a 300g-hypergravity condition. Lanthanum and gadolinium, blockers of calcium channels, nullified the modification of growth anisotropy and reorientation of microtubules by hypergravity. Horizontal and acropetal hypergravity modified growth anisotropy and reorientation of microtubules, as did basipetal hypergravity, and these changes were not seen in the presence of lanthanum or gadolinium. These results suggest that hypergravity changes activities of lanthanum- and gadolinium-sensitive calcium channels independently of its direction, which may lead to reorientation of cortical microtubules and modification of growth anisotropy in azuki bean epicotyls.