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.     

The cyclic reorientation of cortical microtubules in epidermal cells of azuki bean epicotyls: the role of actin filaments in the progression of the cycle

The orientation of microtubules (MTs) was examined in epidermal cells of azuki bean (Vigna angularis Ohwi et Ohashi) epicotyls. The orientation of MTs adjacent to the outer tangential wall of the cells, which has a crossed polylamellate structure with lamellae of longitudinal cellulose microfibrils alternating with lamellae of transverse cellulose microfibrils, differed from one cell to another. Treatment with an auxin-free solution caused the accumulation of cells with longitudinal MTs and subsequent treatment with a solution that contained auxin resulted in the accumulation of cells with transverse MTs, showing that sequential treatments with auxin-free and auxin-containing solutions can synchronize the reorientation of MTs. The MTs, once reoriented from longitudinal to transverse, returned to longitudinal and then back to transverse once again, the duration of the cycle being about 6 h. Gibberellic acid, known to increase the percentage of cells with transverse MTs, promoted reorientation of MTs from longitudinal to transverse and inhibited that from transverse to longitudinal. Cytochalasin D, an agent that disrupts actin filaments, speeded up the reorientation from transverse to longitudinal and slowed down that from longitudinal to transverse. It caused an increase in the percentage of cells with MTs in mixed orientation, and the percentage of such cells was highest when the percentage of cells with longitudinal MTs was decreasing and that of cells with transverse MTs was increasing. Received: 27 November 1997 / Accepted: 7 January 1998     

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.     

Increase in the gibberellin response by low temperature pretreatment of azuki bean epicotyls

Epicotyl segments cut from azuki bean (Vigna angularis) seedlings grown at 27 C for 5 days and then treated at 15 C for 2 days were compared with those cut from seedlings not receiving the 15-C treatment, for their response to auxin and auxin plus gibberellin. The segments cut from 15C-treated seedlings showed greater elongation than the segments cut from untreated seedlings both in the absence and in the presence of gibberellin. The 15-C treatment increased the elongation caused by auxin plus gibberellin to a greater extent than it did the elongation caused by auxin alone; the gibberellin response of epicotyl was increased by the treatment. Diffusates from leaves and buds inhibited both the elongation caused by auxin and that caused by auxin plus gibberellin. The diffusates inhibited the latter more strongly than the former; the gibberellin response was decreased by the diffusates. Leaves or buds appear to supply epicotyls with a substance which decreases the gibberellin response. The supply of this substance was found to be temperature dependent. The diffusates obtained at 15 C caused no inhibition, while those obtained at 27 C decreased the gibberellin response. The lack of the supply of this substance at 15 C may account for the increase in the gibberellin response by the 15-C treatment.     

Effects of temperature on the cell wall and osmotic properties in dark-grown rice and azuki bean seedlings

The mechanism inducing the difference in growth rate under various temperature (10–50 °C) conditions was analyzed using rice and azuki bean seedlings. The growth rate of rice coleoptiles and azuki bean epicotyls increased as temperature increased up to 40 and 30 °C, respectively, and the elongation was retarded at a higher temperature. The cell wall extensibility of rice coleoptiles and azuki bean epicotyls also showed the highest value at 40 and 30 °C, respectively, and became smaller as the temperature rose or dropped from the optimum. The opposite tendency was observed in the minimum stress-relaxation time of the cell wall. On the other hand, the cellular osmotic concentration of rice coleoptiles and azuki bean epicotyls was lower at the temperature optimum for growth at 40 and 30 °C, respectively. When rice and azuki bean seedlings grown at 10, 20, 40, or 50 °C were transferred to the initial temperature (30 °C), the growth rate of coleoptiles and epicotyls was mostly elevated, concomitant with an increase in the cell wall extensibility. The growth rate was correlated with the cell wall mechanical parameters in both materials. These results suggest that the environmental temperature modulates the growth rate of plant shoots by affecting mainly the mechanical properties of the cell wall. Electronic Publication     

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

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

Galactose prevents proton excretion but not IAA-induced growth in segments of azuki bean epicotyls

We investigated the effect of galactose on IAA-induced elongation and proton excretion in azuki bean (Vigna angularis Ohwi et Ohashi) segments in order to confirm whether or not protons were involved in auxin-induced growth. Galactose inhibited the IAA-induced decrease in the solution pH but had no inhibitory effect on IAA-induced growth in segments of azuki bean epicotyls. On the other hand, galactose inhibited both IAA-induced growth and proton excretion in oat (Avena sativa L.) coleoptile segments. From these results it is unlikely that IAA-induced growth is mediated by proton excretion at least in azuki bean epicotyls.Abbreviations IAA indole-3-acetic acid - FC fusicoccin     

Effects of abscisic acid on the orientation and cold stability of cortical microtubules in epicotyl cells of the dwarf pea

Summary The effects of abscisic acid (ABA) on the orientation and cold stability of cortical microtubules (MTs) in epidermal cells of epicotyls of the dwarf pea,Pisum sativum L. cv. Little Marvel, were examined by immunofluorescence microscopy. The effect of ABA on the elongation of epicotyls and on the orientation of cortical MTs was opposite to that of gibberellin A₃ (GA₃). Treatment with ABA, which reduced the promotion of epicotyl elongation by GA₃, eliminated the GA₃-induced predominance of transverse MTs and resulted in a predominance of longitudinal MTs. The effect of ABA on the cold stability of cortical MTs was also opposite to that of GA₃. ABA increased the cold stability of MTs, while GA₃ decreased it. The predominance of longitudinal MTs brought about by ABA may have some relationship to ABA-induced inhibition of the elongation of the epicotyl. ABA may alter membrane proteins to stabilize cortical MTs and induce cold hardiness of plants.Abbreviations ABA abscisic acid - DMSO dimethylsulfoxide - FITC fluorescein isothiocyanate - GA₃ gibberellin A₃ - MT microtubule - PBS phosphate-buffered saline Dedicated to the memory of Professor Oswald Kiermayer     

Comparative molecular mapping in Ceratotropis species using an interspecific cross between azuki bean (Vigna angularis) and rice bean (V. umbellata)

A genetic linkage map was developed with 86 F₂ plants derived from an interspecific cross between azuki bean (Vigna angularis, 2n=2x=22) and rice bean (V. umbellata, 2n=2x=22). In total, 14 linkage groups, each containing more than 4 markers, were constructed with one phenotypic, 114 RFLP and 74 RAPD markers. The total map size was 1702 cM, and the average distance between markers was 9.7 cM. The loci showing significant deviation from the expected ratio clustered in several linkage groups. Most of the skewed loci were due to the predominance of rice bean alleles. The azuki-rice bean linkage map was compared with other available maps of Vigna species in subgenus Ceratotropis. Based on the lineage of the common mapped markers, 7 and 16 conserved linkage blocks were found in the interspecific map of azuki bean ×V. nakashimae and mungbean map, respectively. Although the present map is not fully saturated, it may facilitate gene tagging, QTL mapping and further useful gene transfer for azuki bean breeding. Received: 20 March 1999 / Accepted: 29 April 1999     

Genome dissection of traits related to domestication in azuki bean (Vigna angularis) and comparison with other warm-season legumes

BACKGROUND: The objective of this study was to dissect into quantitative trait loci (QTLs) the large morphological and physiological differences between cultivated azuki bean (Vigna angularis) and a wild relative and to infer the commonalities of the QTLs for domestication-related traits across the Asian Vigna and with other warm-season legumes. METHODS: Two linkage maps, for the BC(1)F(1) and F(2) populations, respectively, from the same cross between azuki bean and V. nepalensis were developed. Using these linkage maps QTLs for 33 domestication-related traits were analysed and mapped. The location of mapped QTLs was compared with locations of similar QTLs in other warm-season legumes. KEY RESULTS: QTLs were detected for seed-, pod-, stem- and leaf-related traits. Most traits were controlled by between two and nine QTLs but several traits, such as pod dehiscence, were controlled by single genes. QTLs for domestication-related traits were restricted to particular regions of the azuki bean genome, especially linkage groups 1, 2, 4, 7 and 9. Linkage groups 1 and 2 had QTLs for a suite of traits including pod size, germination, seed size and lower stem length. QTLs on linkage groups 7 and 9 were associated with upper stem length, maximum leaf size and pod and seed size. Pleiotropy or close linkage of genes for domestication-related traits is suggested in these regions. While some QTLs are common to azuki bean and other warm-season legumes, many are recorded for the first time in azuki bean. CONCLUSIONS: QTLs for a large number of domestication-related traits have been mapped for the first time in azuki bean. QTLs with unexpected effect and new QTLs for traits such as seed size have been found. The results provide a foundation that will be useful for improvement of azuki bean and related legumes.     

Deposition and reorientation of cellulose microfibrils in elongating cells of Petunia stylar tissue

According to Roelofsen and Houwink's (1953, Acta Bot. Neerl. 2, 218–225) multinet growth hypothesis, microfibrils originally deposited transversely in the cell wall become gradually reoriented towards more axial orientations during cell elongation. To establish the extent of reorientation, microfibrils were studied during their deposition and elongation, using stylar parenchyma and transmitting tissue cells of Petunia hybrida L. At the inner surface of very young cells, microfibrils were deposited in alternating Z- and S-helical orientations. The following sequence in deposition, from the exterior to the interior side of the wall, could be inferred: Axial: 150°–180° (Z-helical), 0°–30° (S-helical); oblique: 110°–150° (Z-helical), 30°–70° (S-helical); transverse: 90°–110° (Z-helical), 70°–90° (S-helical). With the increasing pitch, the density of the deposited microfibrils increased as well, giving rise to an alternating helical texture. During elongation, only transversely S- and Z-helically oriented microfibrils were deposited and all microfibrils underwent a certain reorientation as described in the multinet growth hypothesis. The texture resembled that of young cells and the wall maintained its thickness. The extent of passive reorientation was in agreement with the theoretical calculations made by Preston.Dedicated to Professor Dr. A.B. Wardrop, Melbourne, on the occasion of his 70th birthday     

Regulation of the spatial order of cortical microtubules in developing guard cells ofAllium

The organization of microtubules (MTs) in the cortex of cells at interphase is an important element in morphogenesis. Mechanisms controlling the initiation of MTs and their spatial ordering, however, are largely unknown. Our recent study concerning the generation of a radial array of MTs in stomatal guard cells inAllium showed that the MTs initiate in a cortical MT-organizing zone adjacent to the ventral wall separating the two young guard cells (Marc, Mineyuki and Palevitz, 1989, Planta179, 516, 530). In an attempt to detect MT-ordering mechanisms separate from the sites of MT initiation, we now employ various drugs to manipulate the geometry and integrity of the ventral wall and thereby also the associated MT-organizing zone. In the presence of cytochalasin D the ventral wall is tilted away from its normal mid-longitudinal anticlinal alignment, while treatments with the herbicide chloroisopropyl-N-phenylcarbamate (CIPC) induce the formation of a branched ventral wall. Nonetheless, in either case the MTs still form a radial array, although this is asymmetric as it is centered in accordance with the misaligned or branched ventral wall. Since the MTs maintain their original course undisturbed as they extend beyond the abnormal ventral wall, there is no evidence for the presence of an inherent MT-ordering mechanism at locations remote from MT-initiation sites. Following treatments with caffeine, which abolishes the formation of the ventral wall, the MTs revert to a transversely oriented cylindrical array as in normal epidermal cells. Thus the presence of the ventral wall, and presumably also the associated MT-organizing zone, is essential for the establishment of the radial array. The MT-organizing zone is therefore involved not only in the initiation of MTs, but also in determining their spatial order throughout the cell cortex. We thank Drs. Richard J. Cyr and Yoshi Mineyuki for providing valueable suggestions during the course of this work, and Ms. Elizabeth Bruce printing some of the figures. This research was supported by Funds from the National Science Foundation grants DCB-8703292 to B.A.P. and DCB-8803286 to B.A.P. and J.M.     

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.     

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.     

Inhibition of auxin-induced elongation and xyloglucan breakdown in azuki bean epicotyl segments by fucose-binding lectins

Auxin-induced elongation of epicotyl segments of azuki bean ( Vigna angularis Ohwi and Ohashi cv. Takara) was suppressed by fucose-binding lectins from Tetragonolobus purpureus Moench and Ulex europaeus L. These lectins also inhibited auxin-induced cell wall loosening (decrease in the minimum stress-relaxation time of the cell walls) of segments. Auxin caused a decrease in molecular mass of xyloglucans extracted with 24% KOH from the cell walls. The lectins inhibited auxin-induced changes in molecular mass of the xyloglucans. The autolytic release of xylose-containing products from the pectinase-treated cell walls was also suppressed by the lectins. Fucose-binding lectins pretreated with fucose exhibited little or no inhibitory effect on auxin-induced elongation, cell wall loosning, or breakdown of xyloglucans. These results support the view that the breakdown of xyloglucans is involved in the cell wall loosening responsible for auxin-induced elongation in dicotyledons.     

Auxin-induced H+-pump stimulation does not depend on the presence of epidermal cells in corn coleoptiles

Cell-wall acidification and electrical reactions (depolarization and hyperpolarization) are typical auxin responses in maize (Zea mays L.) coleoptiles. In an attempt to test the role of the outer epidermis in these responses, they have been measured and compared in intact and peeled coleoptile fragments. To exclude interactions between parenchymal and epidermal cells, the coleoptile pieces were completely stripped of their outer epidermis. This preparation was monitored by means of a scanning electron microscope. When externally applied indole-3-acetic acid was tested, we found that neither cell-wall acidification nor the electrical membrane responses depended on the presence of intact epidermal cells.Abbreviations IAA Indole-3-acetic acid - MES 2-[N morpholino-ethane-sulfonic acid - TRIS 2-Amino-2-hydroxymethyl-1,3-propanediol We thank Kuki Kaethner for her excellent technical assistance. This work was supported by the Hessische Graduiertenförderung and the Deutsche Forschungsgemeinschaft.     

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.     

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     

Role of xyloglucan breakdown in epidermal cell walls for auxininduced elongation of azuki bean epicotyl segments

Auxin-induced elongation of epicotyl segments of azuki bean ( Vigna angularis Ohwi et Ohashi cv. Takara) was suppressed by a fucose-binding lectin from Tetragonolobus purpureas Moench and by polyclonal antibodies raised against xyloglucan heptasaccharide (Xyl₃Glc₄) when the cuticle present in the outer surface of epicotyls was abraded. In contrast, elongation of non-abraded segments was not influenced by the lectin or the antibodies. Epicotyl segments, from which the epidermal and the outer cortical cells had been removed, elongated rapidly for 2 h and than only slowly. Auxin slightly stimulated elongation of the inner tissue segments in the phase of slow growth. Neither in the presence nor in the absence of auxin did the lectin or the antibodies affect elongation of the inner tissue segments. The split portions of outer surface-abraded epicotyl segments incubated in buffer extended outward, and auxininhibited this outward bending. The lectin and the antibodies reversed the effect of auxin on bending. The fucose-binding lectin pretreated with fucose or the immunoglobulin fraction obtained from preimmune serum exhibited little or no inhibitory effect on auxin-induced elongation of abraded or split segments. These results support the view that a breakdown of xyloglucans in the epidermal cell walls plays an essential role in auxin-induced elongation in dicotyledons.