Effects of Benlate 50 DF on microtubules of cucumber root tip cells and on growth of cucumber seedlings

The aim of this study was to determine whether the fungicide Benlate 50 DF has adverse effects on the microtubules of cucumber root tip cells and on growth of the young seedlings. Germinating cucumber seeds and young seedlings were exposed to six concentrations of Benlate ranging from 1 mg/liter to 10 g/liter. Although seed germination was not affected by Benlate, seedling growth, especially number and length of branch roots, was reduced in proportion to the concentration of Benlate and length of treatment. Significantly lower mitotic indices were obtained from root tips exposed to 0.1, 0.6, 1, or 10 g/liter of Benlate. By means of immunofluorescence microscopy, the major microtubule arrays of root tip cells exposed to Benlate were compared and contrasted to those of controls. A few abnormalities in microtubule arrays were found at or after cytokinesis, and were of two types: 1) persistence of phragmoplast microtubules; and 2) presence of an array of microtubules between two recently divided daughter nuclei in the plane that would normally be occupied by the new cell wall. These abnormalities are somewhat similar to those induced by caffeine, and, as with caffeine treatment, possibly reflect impaired or/and incomplete cytokinesis that results in production of binucleate cells. A few binucleate cells were observed in root tips exposed to 10 g/liter of Benlate.     

Taxol maintains organized microtubule patterns in protoplasts which lead to the resynthesis of organized cell wall microfibrils

Summary We have investigated the effects of microtubule stabilizing conditions upon microtubule patterns in protoplasts and developed a new method for producing protoplasts which have non-random cortical microtubule arrays. Segments of elongating pea epicotyl tissue were treated with the microtubule stabilizing drug taxol for 1 h before enzymatic digestion of the cell walls in the presence of the drug. Anti-tubulin immunofluorescence showed that 40 M taxol preserved regions of ordered microtubules. The microtubules in these regions were arranged in parallel arrays, although the arrays did not always show the transverse orientation seen in the intact tissue. Protoplasts prepared without taxol had microtubules which were random in distribution. Addition of taxol to protoplasts with random microtubule arrangements did not result in organized microtubule arrays. Taxol-treated protoplasts were used to determine whether or not organized microtubule arrays would affect the organization of cell wall microfibrils as new walls were regenerated. We found that protoplasts from taxol-treated tissue which were allowed to regenerate cell walls produced organized arrays of microfibrils whose patterns matched those of the underlying microtubules. Protoplasts from untreated tissue synthesized microfibrils which were disordered. The synthesis of organized microfibrils by protoplasts with ordered microtubules arrays shows that microtubule arrangements in protoplasts influence the arrangement of newly synthesized microfibrils.Abbreviations DIC differential interference contrast - DMSO dimethyl sulfoxide - FITC fluorescein isothiocyanate - IgG immunoglobulin G - PIPES piperazine-N,N-bis[2-ethane-sulfonic acid] - PBS phosphate buffered saline     

Consistent handedness of microtubule helical arrays in maize and Arabidopsis primary roots

Summary The orientation of cortical microtubules in plant cells has been extensively studied, in part because of their influence on the expansion of most plant cell types. Cortical microtubules are often arranged in helical arrays, which are well known to occur with a specific pitch as a function of development or experimental treatment; however, it is not known if the handedness of helical arrays can also be specified. We have studied the handedness of helical arrays by using Vibratome sectioning of maize primary roots and confocal microscopy of Arabidopsis primary roots. In cortical cells of maize roots, the helical array was found to have the same handedness at a given position, not only for the cells of a single root, but also for the cells of more than one hundred roots examined. Quantification of angular distribution of apparent individual microtubules showed that defined regions of the root were composed of cells with highly uniform microtubule orientation. In the region between transverse and longitudinal microtubules (5–10.5 mm from the tip), the array formed a right-handed helix, and basal of cells with longitudinal microtubules (11.5–15 mm from the tip), the array formed a left-handed helix. Similarly, in epidermal cells of Arabidopsis roots right-handed helical arrays were found in the region between transverse and longitudinal microtubules. These results suggest that, in addition to the orientation of microtubules, the handedness of helical microtubule arrays is under cellular control.Abbreviations Cy3 indocarbocyanine - PBS phosphate-buffered saline - PIPES piperazine-N,N-bis-[2-ethanesulfonic acid]     

Microtubule organization and morphogenesis of stomata in caffeine-affected seedlings ofZea mays

Summary Treatment ofZea mays seedlings with a 5 mM caffeine solution inhibits cytokinesis in guard cell mother cells (GMCs), producing unicellular, binucleate aberrant stomata (a-stomata). Ventral wall (VW) strips of limited length, which usually meet the wall portions of GMCs adjoining the cortical zone of the preprophase microtubule band (PMB), are laid down in many a-stomata.In a-stomata with or without VW-strips, the periclinal walls are lined by numerous microtubules (Mts) converging on their mid-region, where local wall thickenings are deposited. When the VW-strips reach the mid-region of the periclinal walls, thickenings lined by numerous Mts rise at their free margins. In certain a-stomata an anticlinal wall column, surrounded by a dense Mt bundle, grows centripetally from either or both of the periclinal wall thickenings. In wall thickenings, the cellulose microfibrils are co-aligned with the adjacent Mts. Pore formation is initiated in all a-stomata. Deposition of an electron dense intra-wall material followed by lysis precedes pore opening. This process is closely related to the a-stornata morphogenesis. These observations show that the primary morphogenetic phenomenon in a-stomata is the establishment of an intense and stable polarity in the cytoplasm abutting on the mid-region of the periclinal walls and/or the adjacent plasmalemma area. Prime morphogenetic factor(s), including microtubule organizing centres (MTOCs), seem to function in these sites. Morphogenesis in a-stomata is a Mt-dependent process that is carried out as in normal stomata but in the absence of a VW.Abbreviations a-stomata unicellular binucleate aberrant stomata - CIPC chlorisopropyl-N-phenyl carbamate - GC guard cell - GMC guard cell mother cell - Mt microtubule - MTOC microtubule organizing centre - PMB preprophase microtubule band - VW ventral wall     

Aberrant microtubule organization can result in genetic abnormalities in protoplast cultures of Vicia hajastana Grossh

Protoplast cultures of Vicia hajastana have a high division frequency. However, 20–40% of the microcolonies fail to develop beyond the 20-30-cell stage. Aneuploids and polyploids were found in early divisions and persisted in older cultures. The resulting protoplast-derived suspension culture differed karyologically from the original culture. Karyokinesis and cytokinesis were studied using simultaneous staining of microtubules (MT) by immunofluorescence, DNA by Hoechst 33258 (2-[2-(4-hydroxyphenyl)-6-benzimidazoyl]-6-[1-methyl-4-piperazyl]benzimidazole) and cell walls by Calcofluor. Freshly prepared protoplasts showed mitoses and high frequencies of binucleate cells, which probably resulted mainly from failure of cytokinesis. In early divisions, many mitoses showed metaphase chromosomes with kinetochore MT but lacking polar MT. These aberrant mitoses probably accounted for an increase in hyperploid cells observed in protoplast cultures. Multipolar spindles, which gave rise to hypoploid cells, were also seen in the early divisions. Telophase abnormalities included dislocated phragmoplasts and incomplete formation of cross walls. Many divisions resulted in daughter nuclei of unequal size. Unequal segregation of chromosomes was detected by cytofluorimetric measurements of telophase nuclei stained with Hoechst. After 5 d of culture, 91% of the divisions with incomplete cross walls also contained different-size nuclei; conversely, 78% of the divisions with fully formed cross walls contained nuclei of equal size. The malfunctioning of spindles and phragmoplasts in the same cells indicates a functional interdependence of the different MT configurations in mitosis. During the first 24 h of culture, a high frequency of abnormalities was found in spindles, cross-wall formation and chromosome segregation; this was reduced substantially in the cells undergoing first division by 48 h. The data indicate that it may be possible to manipulate the frequency of abnormalities by controlling the onset of the first division in protoplast cultures.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MT microtubule(s) - PB prophase band(s) - PNF perinuclear fluorescence - PPB pre-prophase band     

Microtubule patterning in apical epidermal cells ofVinca minor preceding leaf emergence

Summary Serial thin sectioning for electron microscopy was carried out on the cortical cytoplasm of surface cells of the apical dome ofVinca minor. The cellulose reinforcement pattern in the outer epidermal walls forming this surface is known to correlate well with the decussate phyllotaxis pattern. The purpose of this study was to determine the location of microtubules immediately under these epidermal walls as a first step toward finding out how the cellulose pattern arises. First, correspondence between the patterns of microtubules and cellulose was checked. Second, the role of potential orienting cues for the alignment of microtubule arrays in specific cells was evaluated.Microtubule arrays which were well or moderately ordered (70% of the total interphase cells) generally had alignment parallel to the adjacent leaf base, as has been seen for cellulose. The aligned features or cues potentially correlating with a given array were: (1) orientation and length of the previous anticlinal cross-wall, (2) alignment of microtubules in adjacent cells, and (3) direction of inferred stretch, parallel to the nearby leaf bases. All three features were found to agree with the microtubule alignment in 17 of 34 cells with ordered arrays. At least two features agreed in 33 of the 34 cases. All 34 cells with ordered arrays had at least one feature parallel to the array. Random association between microtubule orientation and these features would lead to such correlations less than 0.01% of the time. Thirty percent of the interphase cells showed no obvious order. Most of these cells were located in the central linear corridor region of the apex. The unordered cells were more likely than the ordered cells to have more than one orientation specified by the potential cues; i.e., no single orientation parallel to all of the cues existed. This indicates that uniformity of the orientation cues may be as important as their direction.     

Mitotic disrupter herbicides act by a single mechanism but vary in efficacy

Summary Although there are numerous herbicides that disrupt mitosis as a mechanism of action, to date not one has compared the effects of these disrupters on a single species and over a range of concentrations. Oat seedlings, treated with a range of concentrations of nine different mitotic disrupter herbicides, were examined by immunofluorescence microscopy of tubulin in methacrylate sections. All herbicides caused the same kinds of microtubule disruption, although the concentrations required to cause the effects differed markedly between the herbicides. Effects on spindle and phragmoplast mitotic microtubule arrays were seen at the lowest concentrations and manifested as multipolar spindles and bifurcated phragmoplasts (which subsequently resulted in abnormal cell plate formation). At increasing concentrations, effects on mitotic microtubule arrays manifested as microtubule tufts at kinetochores and reduction of cortical microtubules resulting in arrested prometaphase figures and isodiametric cells. These data indicate that all mitotic disrupter herbicides have a common primary mechanism of action, inhibition of microtubule polymerization, and that marginal effects observed in the past were the result of incomplete inhibition and/or differential sensitivity of the microtubule arrays.Abbreviations DCPA 2,3,5,6-tetrachloroterephthalic acid dimethyl ester - APM amiprophosmethyl - DAPI 4,6-diamidino-2-phenyl indole - MTOC microtubule organizing center     

Heat stress affects the organization of microtubules and cell division in Nicotiana tabacum cells

To investigate the effects of heat stress on the plant cytoskeleton, the structure of microtubule arrays in N. tabacum suspension cells incubated at 38 or 42°C was analysed. Whilst incubation at 42 °C resulted in the disruption of the majority of cellular microtubules after 30 min, in cells exposed to 38 °C all the microtubule arrays were preserved even after 12 h of incubation, although their organization was altered. The most susceptible were the microtubules of the mitotic spindle and the phragmoplast. Several abnormalities were observed: (i) splitting of the spindle into several parts; (ii) elongation of the spindles; (iii) formation of microtubule asters in mitotic cells, and (iv) elongation of phragmoplast microtubules. Exposure of cells to 38 °C caused a decrease in the mitotic index but an accumulation of telophase cells. The recovery of normal microtubule organization occurred after 12 h. Treatment of the cells subjected to heat stress conditions with an inhibitor of protein synthesis, cycloheximide, did not prevent either the alterations of microtubule organization or accumulation of cells containing phragmoplasts. Therefore, heat shock proteins do not seem to be directly responsible for the microtubule disorganization induced by heat stress.     

Microspore development inBrassica napus and the effect of high temperature on division in vivo and in vitro

Summary Brassica napus cv. Topas microspores isolated and cultured near the first pollen mitosis and subjected to a heat treatment develop into haploid embryos at a frequency of about 20%. In order to obtain a greater understanding of the induction process and embryogenesis, transmission electron microscopy was used to study the development of pollen from the mid-uninucleate to the bicellular microspore stage. The effect of 24 h of high temperature (32.5 °C) on microspore development was examined by heat treating microspore cultures or entire plants. Mid-uninucleate microspores contained small vacuoles. Late-uninucleate vacuolate microspores contained a large vacuole. The large vacuole of the vacuolate stage was fragmented into numerous small vacuoles in the late-uninucleate stage. The late-uninucleate stage contained an increased number of ribosomes, a pollen coat covering the exine and a laterally positioned nucleus. Prior to the first pollen mitosis the nucleus of the lateuninucleate microspore appeared to be appressed to the plasma membrane; numerous perinuclear microtubules were observed. Microspores developing into pollen divided asymmetrically to form a large vegetative cell with amyloplasts and a small generative cell without plastids. The cells were separated by a lens-shaped cell wall which later diminished. At the late-bicellular stage the generative cell was observed within the vegetative cell. Starch and lipid reserves were present in the vegetative cell and the rough endoplasmic reticulum and Golgi were abundant. The microspore isolation procedure removed the pollen coat, but did not redistribute or alter the morphology of the organelles. Microspores cultured at 25 °C for 24 h resembled late-bicellular microspores except more starch and a thicker intine were present. A more equal division of microspores occurred during the 24 h heat treatment (32.5 °C) of the entire plant or of cultures. A planar wall separated the cells of the bicellular microspores. Both daughter cells contained plastids and the nuclei were of similar size. Cultured embryogenie microspores contained electron-dense deposits at the plasma membrane/cell wall interface, vesicle-like structures in the cell walls and organelle-free regions in the cytoplasm. The results are related to embryogenesis and a possible mechanism of induction is discussed.Abbreviations B binucleate - LU late uninucleate - LUV late uninucleate vacuolate - M mitotic - MU mid-uninucleate - RER rough endoplasmic reticulum - TEM transmission electron micrograph     

γ-Tubulin in tobacco pollen tubes: association with generative cell and vegetative microtubules

-Tubulin was localized in tobacco pollen tubes using an antibody raised against a peptide conserved in all known -tubulins. Antibody staining occurs in a primarily punctate pattern along the length of the microtubule bundles in generative cells and along cortical microtubules in the vegetative cytoplasm. During generative cell division, -tubulin is localized in the forming mitotic apparatus. By metaphase, it is present along kinetochore fibers except at their plus ends located at the kinetochores. By telophase, staining is observed in the phragmoplast, where it again avoids the plus ends of microtubules at the cell plate. -Tubulin is also present at the periphery of the sperm nuclei. A patch of intense staining on the distal side of each nucleus marks the site of assembly of a new population of sperm microtubules. No specific fluorescence is present in control pollen tubes treated with preimmune IgG. These localization patterns bear similarities to those seen in somatic cells and in addition may help explain changes in microtubule arrays between generative cells and sperm.     

The timing of synthesis of proteins required for mitotic spindle and phragmoplast in partially synchronized root meristems of Vicia faba L

After cycloheximide treatment (1 h, 2.5 micrograms/ml) protein synthesis was decreased by 70% and was partially restored after 7 h of postincubation (still 20% decrease). In partially synchronized root meristems of Vicia faba L. treated with cycloheximide at middle G2, a strong decrease of the mitotic index was observed. Exposure to the drug at late G2 did not modify the mitotic index; the changes in the phase indices suggested that the course of mitosis was blocked at prophase-metaphase/anaphase-telophase transitions. The use of indirect immunocytochemical staining of tubulin (second antibody labeled with peroxidase) made it possible to show a decreased number of cells with preprophase bands in cycloheximide-treated meristems and the mitotic spindles and phragmoplasts containing a reduced number of shortened bands of microtubules. As a result of these structural and functional disturbances, binucleate cells and polyploid nuclei were observed.     

The effect of low temperature on the microtubules in root meristem cells of spring and winter cultivars of wheat Triticum aestivum L

We have studied the response of the interphase and mitotis microtubule arrays in root meristem cells of spring and winter cultivars of wheat Triticum aestivum L. (Moskovskaya 35 and Moskovskaya 39) during cold stress (1 h at 0 degrees C) and acclimation to cold (3-48 h at 0 degrees C). Our data show that interphase microtubules are more resistant to cold than mitotic arrays in both cultivars. During cold stress the density of endoplasmic microtubules increases in interphase cells of winter plants, yet no changes are detected in cells of spring plants. In mitotic cells of both wheat cultivars the density of microtubules within the kinetochore fibers decreases, yet this effect is more evident in the cells of spring plants. During acclimation to cold of both cultivars, we have observed the disorganization of the interphase cortical arrays and the enhanced growth of endoplasmic microtubule arrays, composed of microtubule converging centers. However, the reaction of mitotic microtubule arrays differs in the cells of winter and spring plants. In winter plants, during prophase diffuse tubulin "halo" accumulates first at perinuclear area, followed by the appearance of the microtubule converging centers. In spring plants, we have observed the formation of the prophase spindle, yet later the prophase spindle is not detected. Metaphase cells of both cultivars show similar aberrations of the mitotic spindle, accumulation of abnormal metaphases and the excessive formation of microtubule converging centers. In telophase cells of both cultivars, acclimation induces similar reaction, resulting in the disorganization of the phragmoplast and the formation of multiple microtubule converging centers. The latter are detected in the perinuclear areas of the daughter cells in winter plants and in the cortical cytoplasm of cells in spring plants. Our data point to the common pathways of microtubule response to cold treatment (0 degrees C). The excessive formation of the microtubule converging centers indicates the activation of microtubule assembly during prolonged cold treatment.     

The distribution of cytoplasmic microtubules throughout the cell cycle of the centric diatom Stephanopyxis turris: their role in nuclear migration and positioning the mitotic spindle during cytokinesis

The cell cycle of the marine centric diatom Stephanopyxis turris consists of a series of spatially and temporally well-ordered events. We have used immunofluorescence microscopy to examine the role of cytoplasmic microtubules in these events. At interphase, microtubules radiate out from the microtubule-organizing center, forming a network around the nucleus and extending much of the length and breadth of the cell. As the cell enters mitosis, this network breaks down and a highly ordered mitotic spindle is formed. Peripheral microtubule bundles radiate out from each spindle pole and swing out and away from the central spindle during anaphase. Treatment of synchronized cells with 2.5 X 10(-8) M Nocodazole reversibly inhibited nuclear migration concurrent with the disappearance of the extensive cytoplasmic microtubule arrays associated with migrating nuclei. Microtubule arrays and mitotic spindles that reformed after the drug was washed out appeared normal. In contrast, cells treated with 5.0 X 10(-8) M Nocodazole were not able to complete nuclear migration after the drug was washed out and the mitotic spindles that formed were multipolar. Normal and multipolar spindles that were displaced toward one end of the cell by the drug treatment had no effect on the plane of division during cytokinesis. The cleavage furrow always bisected the cell regardless of the position of the mitotic spindle, resulting in binucleate/anucleate daughter cells. This suggests that in S. turris, unlike animal cells, the location of the plane of division is cortically determined before mitosis.     

Acetylated tubulin is found in all microtubule arrays of two species of pine

Summary The distribution of acetylated tubulin in microtubule arrays of conifer cells was investigated by immunofluorescence techniques with 6-11B-1, a monoclonal antibody specific for posttranslationally acetylated -tubulins. In methacrylate sections ofPinus radiata andPinus conforta root tip cells, acetylated tubulin was detected in mitotic spindles, phragmoplasts, and cortical microtubules. Furthermore, staining of isolated, intact cells ofP. radiata andP. contorta indicated that all microtubule structures, including preprophase bands, prophase, metaphase and anaphase spindles, and phragmoplasts, contained some acetylated tubulin, and that the intensity of staining with 6-11B-1 was variable. For example, preprophase bands were lightly labelled, kinetochore fibres of anaphase spindles and phragmoplasts were heavily stained, and metaphase spindles had a granular appearance suggesting discontinuous acetylation of their constituent microtubules. This first report of the presence of acetylated tubulin in conifer cells is in contrast to our results with two species of angiosperms where no acetylated tubulin was detected. The significance of this and the variability of the intensity of staining in conifer arrays is discussed in terms of microtubule dynamics.     

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     

Spline and flagellar microtubules are resistant to mitotic disrupter herbicides

Summary Microtubule arrays in developing spermatogenous cells of pteridophytes have unique microtubule organizing centers and post-translation modifications of tubulin. Sensitivity of these arrays to the microtubule-destabilizing effects of the mitotic disrupter herbicides was examined by immunofluorescence, transmission and immunogold electron microscopy. Acetylated, stabilized arrays, such as the spline, and microtubules of the basal bodies and flagella are formed after the final mitotic division and are resistant to these herbicides. Non-acetylated, dynamic arrays that exist prior to the final mitosis, such as interphase and mitotic arrays, are eliminated by all of these herbicides, with symptomology (arrested prometaphase, lobed nuclei, irregular cell plate formation) similar to that observed in other land plants. The only exception to the instability of these mitotic microtubule arrays are the few microtubules that are collected by kinetochores into short tufts. The presence of structurally-distinguishable MTOCs, such as the blepharoplast, did not confer resistance, despite the anchoring of the minus ends of the microtubules. Simultaneous treatment with herbicide and 5-bromodeoxyuridine (BrdU), with subsequent detection with anti-BrdU of cells that had gone through S-phase during the BrdU incubation, reveals that only acetylated arrays formed prior to herbicide treatment are resistant. These data indicate that only actively polymerizing, dynamic microtubule arrays are sensitive to the destabilizing effects of the mitotic disrupter herbicides.Abbreviations MTOC microtubule organizing center - BrdU 5-bromodeoxyuridine     

Effects of the herbicide isopropyl-N-phenyl carbamate on microtubules and MTOCs in lines of Nicotiana sylvestris resistant and sensitive to its action

To clarify the mechanism of isopropyl-N-phenyl carbamate (IPC) action on higher plant cells the sensitivity of microtubules (cortical network and mitotic arrays) and microtubule organizing centers to IPC treatment (30 microM) in IPC-resistant and sensitive Nicotiana sylvestris lines was studied. It was clearly demonstrated that IPC does not depolymerize plant MTs but causes the MTOC damage in cells, which results in MTOC fragmentation, splitting of the spindle poles and in abnormal division spindle formation. It was also found that IPC-resistance of mutant N. sylvestris line correlates not with tubulin resistance to IPC action but possibly with resistance of one of the proteins involved in MTOC composition.     

Rhinanthus serotinus (Schönheit) Oborny (Scrophulariaceae): immunohistochemical and ultrastructural studies of endosperm chalazal haustorium development

Chalazal endosperm haustorium in Rhinanthus serotinus consists of a single large binucleate cell. It originates from the primary endosperm cell dividing transversely into two unequal cells: a smaller micropylar cell and a larger chalazal cell. The chalazal cell undergoes a single mitotic division, then lengthens significantly during development and functions as a chalazal endosperm haustorium. In this paper, immunofluorescent techniques, rhodamine phalloidin assay, and electron microscopy were used to examine the actin and tubulin cytoskeleton during the development of the chalazal haustorium. During the differentiation stage, numerous longitudinally oriented bundles of microfilaments ran along the axis of transvacuolar strands in haustorium. Microtubules formed intensely fluorescent areas near the nuclear envelope and also formed radial perinuclear microtubule arrays. In the fully differentiated haustorium cell, the actin cytoskeleton formed dense clusters of microfilaments on the chalazal and micropylar poles of the haustorium. Numerous microfilament bundles occurred near wall ingrowths on the chalazal wall. There were numerous clusters of microfilaments and microtubules around the huge lobed polytenic haustorial nuclei. The microfilaments were oriented longitudinally to the long axis of the haustorium cell and surrounded both nuclei. The microtubules formed radial perinuclear systems which were appeared to radiate from the surface of the nuclear envelope. The early stage of degeneration of the chalazal haustorium was accompanied by the degradation of microtubules and disruption of the parallel orientation of microtubules in the chalazal area of the cell. The degree of vacuolization increased, autophagous vacuoles appeared and the number of vesicles decreased.     

Inhibition of ethylene biosynthesis does not block microtubule re-orientation in wounded pea roots

Summary Wounds in pea roots can cause the cortical cells surrounding the wound to change their direction of elongation and division planes in order to replace the removed tissue. These changes in growth polarity are preceded by a re-orientation of microtubules in the affected cells. In an approach to understand the control of this process it was investigated whether or not the plant hormone ethylene plays a role in the re-orientation of microtubules and growth polarity. Our results show that treating pea roots with an inhibitor of ethylene synthesis, L--(2-aminoethyoxyvinyl)-glycine hydrochloride (AVG), did not affect wound-induced microtubule re-orientation. However, the effect of AVG on ethylene synthesis in pea roots was confirmed by its stimulation of root elongation. Therefore we conclude that increased ethylene production, which has been observed previously in wounded tissues, is unlikely to be a control factor in microtubule re-orientation in this system.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG L--(2-aminoethyoxyvinyl)-glycine hydrochloride - MSB microtubule stabilizing buffer     

Electric fields affect the orientation of cortical microtubules and cell expansion in pea callus

Summary Cortical microtubules in callus derived fromPisum sativum roots form parallel arrays within cells but are randomly oriented across the tissue. These arrays align perpendicular to the direction of an applied electric field of 6 mV per cell. Application of a field of 6 mV per cell for 4 days resulted in the co-ordinated expansion of cells parallel to the field direction. Cortical microtubule arrays were still aligned perpendicular to the applied field 24 h after removal of the field. The imposition of a field to callus after the removal of cortical microtubules by oryzalin and in the presence of the herbicide resulted in the orientation of recovering microtubules perpendicular to the direction of the field, indicating that microtubules are not directly involved in the detection of the field.Abbreviations EGTA ethylene glycol-bis (-aminoethyl ether) N,N,N-tetraacetic acid - FITC fluorescein isothiocyanate - MSB microtubule stabilising buffer - PIPES piperazine-N,N-bis(2-ethanesulphonic acid) - oryzalin 3,5-dinitro-N⁴,N⁴ dipropylsulphanil-amide