Effect of ultraviolet radiation on cell division and microtubule organization inPetunia hybrida protoplasts

Summary Mesophyll protoplasts isolated fromPetunia hybrida were subjected to UV radiation (280–360 nm) in an attempt to assess whether (a) UV radiation has an effect on cortical microtubule organization, (b) UV radiation affects the progression of protoplasts through the cell cycle, and (c) there is a connection between the effect of UV radiation on cell division and the polymerization state of the microtubules. The proto plasts were irradiated with the following UV doses: 4, 8, 12, and 24mmol photons/m², 30 min after isolation. Cell cycle analysis and immuno-localization of microtubules were carried out 0, 24, 48, and 72 h after irradiation. The length of cortical microtubules was determined after irradiation and in corresponding controls. We found that UV radiation induced breaks in cortical microtubules resulting in shorter fragments with increasing dose. Also, the protoplasts were delayed in their progression through the cell cycle, with G1 and G2 phases being affected as well as the S phase. The commencement of DNA synthesis in the irradiated protoplasts followed the re-establishment of a microtubule network. At 48 h after irradiation the protoplasts in all treatments, except for the 24 mmol/m², had cortical microtubules of similar length, and at 72 h after irradiation only the protoplasts that had received 24 mmol photons/m² had not started dividing.Abbreviations BSA bovine serum albumin - DMSO dimethyl sulfoxide - FDA fluorescein diacetate - MT microtubules - MTSB microtubule stabilizing buffer - PAR photosynthetically active radiation (400–700 nm) - PBS phosphate buffered saline - UV ultraviolet     

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     

Cell proliferation,cell shape,and microtubule and cellulose microfibril organization of tobacco BY-2 cells are not altered by exposure to near weightlessness in space

The microtubule cytoskeleton and the cell wall both play key roles in plant cell growth and division, determining the plant’s final stature. At near weightlessness, tubulin polymerizes into microtubules in vitro, but these microtubules do not self-organize in the ordered patterns observed at 1g. Likewise, at near weightlessness cortical microtubules in protoplasts have difficulty organizing into parallel arrays, which are required for proper plant cell elongation. However, intact plants do grow in space and therefore should have a normally functioning microtubule cytoskeleton. Since the main difference between protoplasts and plant cells in a tissue is the presence of a cell wall, we studied single, but walled, tobacco BY-2 suspension-cultured cells during an 8-day space-flight experiment on board of the Soyuz capsule and the International Space Station during the 12S mission (March–April 2006). We show that the cortical microtubule density, ordering and orientation in isolated walled plant cells are unaffected by near weightlessness, as are the orientation of the cellulose microfibrils, cell proliferation, and cell shape. Likely, tissue organization is not essential for the organization of these structures in space. When combined with the fact that many recovering protoplasts have an aberrant cortical microtubule cytoskeleton, the results suggest a role for the cell wall, or its production machinery, in structuring the microtubule cytoskeleton.     

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

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

Microtubules in maize leaf protoplasts in relation to donor tissue and in vitro culture

Summary Maize (Zea mays) leaf protoplasts were isolated from various leaves of two-week (4-leaf) seedlings and from sections of the third leaf blades. Microtubules (MTs) were visualized using immunofluorescence microscopy. Only freshly isolated protoplasts from the third and fourth leaf blades contained MTs, with protoplasts from the fourth leaf containing the most i.e. 13% of fourth-leaf protoplasts contained MTs. In general, protoplasts with fewer and smaller chloroplasts had more MTs. Initially 90–95% of protoplasts from basal portions of leaves had MTs but the percentage decreased slightly during culture particularly after 10 days. The antioxidant n-propyl gallate was beneficial in maintaining MT content. Few protoplasts from older sections intitially contained MTs but in all sections at least some protoplasts regained a significant MT content during culture (e.g., 10% of protoplast from the tip section possessed microtubules after 7 days of culture). Far fewer MTs were observed in individual leaf protoplasts than those isolated from suspension culture.Abbreviations BMS Black Mexican Sweet - MT microtubule - MtSB microtubule stabilizing buffer - PBS phosphate buffered saline     

Role of plasma membrane proteins and cell wall in meridional arrangement of cortical microtubules inBoodlea coacta

Summary The effects of 2,6-dichlorobenzonitrile (DCB, an agent which inhibits cellulose synthesis) and cycloheximide (CHI, a known inhibitor of protein synthesis) on the construction and stability of the cortical microtubule (MT) cytoskeleton in two kinds of protoplasts (smaller protoplasts and larger ones) prepared fromBoodlea coacta (Dickie) Murray et De Toni were examined by immunofluorescence microscopy. In smaller protoplasts which develop from released protoplasmic masses in culture media, parental cortical MTs assume a convoluted configuration, but new cortical MTs appear following disassembly of convoluted MTs. New cortical MTs initially have a random arrangement but later, a rough meridional arrangement following development of cell polarity and finally, a high density meridional arrangement. In larger protoplasts which are formed within cell wall cylinders of thalli cut at 500 m length, longitudinally oriented parental cortical MTs are preserved. Each exhibits a curving configuration just after protoplast formation, but a straight configuration after 3 h of culture. In smaller protoplasts, cortical MT orientation changes from random to rough meridional orientation but never to a high density meridional orientation following treatment with 10 M CHI, and MT density decreases after 12 h. However, rough meridional and high density meridional arrangements of MTs ceased to be formed and MT density decreased following treatment with 10 M DCB. In larger protoplasts, high density meridional arrangements of MTs were noted not to be affected by treatment with CHI; instead, they continued to remain oriented meridionally, but the length and density were decreased after treatment with DCB for 3–4 h. After 10 h, the MTs became fragmented and orientation was random. From these findings it is summarized that: (1) There are no putative anchors in the plasma membrane of nascent smaller protoplasts, but the meridional orientation of cortical MTs requires anchors which may be distributed in the plasma membrane following the establishment of cell polarity. (2) Plasma membranes in larger protoplasts contain parental anchors oriented meridionally. Anchors stabilize cortical MTs via their close relation to cell walls (especially to cellulose). Anchors are detached from the plasma membrane when cellulose is not formed. (3) Cellulose regeneration may be indispensable to the formation and stabilization of the MT cytoskeleton inBoodlea.Abbreviations CHI cycloheximide - DCB 2,6-dichlorobenzonitrile - DMSO dimethylsulfoxide - MT microtubule     

Changes in the organization of the tubulin cytoskeleton during the early stages of<Emphasis Type="Italic"> Solanum lycopersicoides</Emphasis> Dun. protoplast culture

Changes in the tubulin cytoskeleton during protoplast culture and plant regeneration of Solanum lycopersicoides Dun. were analyzed using an immunodetection method. Directly after isolation, four groups of protoplasts were distinguished: (1) mononuclear, (2) polynuclear, (3) homogeneous, (4) anuclear. The tubulin cytoskeleton of the protoplasts underwent rearrangements, correlating to the number and structure of cell nuclei in the protoplast. All protoplast groups with the exception of mononuclear were characterized by perturbations in the organization of the tubulin cytoskeleton. Anuclear and homogeneous protoplasts did not have a tubulin cytoskeleton. Polynuclear protoplasts had cortical microtubules, but were not capable of re-forming their original arrangement and did not possess a radial or perinuclear cytoskeleton. Irregularities in microtubule arrangement of these three groups of protoplasts caused their inability to regenerate a cell wall and to divide. Anuclear, polynuclear and homogeneous protoplasts were eliminated from the culture. Mononuclear protoplasts rearranged their cortical microtubules and reestablished the radial and perinuclear tubulin cytoskeleton. Re-formation of the cell suspension and subsequent regeneration of plants occurred exclusively from mononuclear protoplasts, which were able to regenerate cell walls and to divide.Abbreviations 2,4D 2,4-Dichlorophenoxyacetic acid - BA Benzyloadenine - DAPI 4,6 Diamidino-2-phenylindole - DMSO Dimethyl sulfoxide - EGTA Ethylene glycol-bis(2-aminoethylether)-N, N, N, N-tetraacetic acid - FITC Fluorescein isothiocyanate - MS Murashige and Skoog medium - MSB Microtubule stabilizing buffer - PBS Phosphate-buffered saline - PIPES Piperazine-N, N-bis(2-ethane sulfonic acid) - PPB Preprophase bandCommunicated by H. Lörz     

Extending the Microtubule/Microfibril Paradigm : Cellulose Synthesis Is Required for Normal Cortical Microtubule Alignment in Elongating Cells

The cortical microtubule array provides spatial information to the cellulose-synthesizing machinery within the plasma membrane of elongating cells. Until now data indicated that information is transferred from organized cortical microtubules to the cellulose-synthesizing complex, which results in the deposition of ordered cellulosic walls. How cortical microtubules become aligned is unclear. The literature indicates that biophysical forces, transmitted by the organized cellulose component of the cell wall, provide a spatial cue to orient cortical microtubules. This hypothesis was tested on tobacco (Nicotiana tabacum L.) protoplasts and suspension-cultured cells treated with the cellulose synthesis inhibitor isoxaben. Isoxaben (0.25–2.5 μm) inhibited the synthesis of cellulose microfibrils (detected by staining with 1 μg mL⁻¹ fluorescent dye and polarized birefringence), the cells failed to elongate, and the cortical microtubules failed to become organized. The affects of isoxaben were reversible, and after its removal microtubules reorganized and cells elongated. Isoxaben did not depolymerize microtubules in vivo or inhibit the polymerization of tubulin in vitro. These data are consistent with the hypothesis that cellulose microfibrils, and hence cell elongation, are involved in providing spatial cues for cortical microtubule organization. These results compel us to extend the microtubule/microfibril paradigm to include the bidirectional flow of information.     

The role of microtubules and cell-wall deposition in elongation of regenerating protoplasts of Mougeotia

Protoplasts of the filamentous green alga Mougeotia sp. are spherical when isolated and revert to their normal cylindrical cell shape during regeneration of a cell wall. Sections of protoplasts show that cortical microtubules are present at all times but examination of osmotically ruptured protoplasts by negative staining shows that the microtubules are initially free and become progressively cross-bridged to the plasma membrane during the first 3 h of protoplast culture. Cell-wall microfibrils areoobserved within 60 min when protoplasts are returned to growth medium; deposition of microfibrils that is predominantly transverse to the future axis of elongation is detectable after about 6 h of culture. When regenerating protoplasts are treated with either colchicine or isopropyl-N-phenyl carbamate, drugs which interfere with microtubule polymerization, they remain spherical and develop cell walls in which the microfibrils are randomly oriented.     

Microtubule rearrangements accompanying dedifferentiation in mesophyll cultures ofZinnia elegans L.

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

Plasma Membrane Ghosts Form Differently When Produced from Microtubule-Free Tobacco BY-2 Cells

When lysed in an actin stabilizing buffer, protoplasts madefrom tobacco BY-2 suspension culture cells formed plasma membraneghosts that retained both cortical actin and microtubules. Distinctcytoskeletal arrays occurred: the most common ghost array (typeI) derived from protoplasts in interphase and had random actinand microtubules, although the alignment of the actin was dependent,at least partially, on microtubule organization. Type II ghostswere larger and more irregular in shape than type I ghosts,and were characterized by a lack of microtubules and the presenceof distinctive arrays of actin bundles in concentric arcs. Theseghosts derived from protoplasts lacking cortical microtubulesproduced when wall digestion occurred while the cells were incell division, or from protoplasts isolated in the presenceof 100 µM propyzamide. Because type II ghosts derivedfrom protoplasts of similar size to those that give rise totype I ghosts, and because type II ghosts retained ordered actinarrays while the parent protoplasts had random cortical actin,type II ghosts apparently form differently to type I ghosts.We speculate that instead of the protoplast being sheared offto produce a round ghost, the plasma membrane tears and collapsesonto the slide, ordering the actin bundles in the process. Oneimplication of this model would be that cortical microtubulesprovide structural support to the plasma membrane of the protoplastso that only in their absence do the type II ghosts form. (Received May 26, 1998; Accepted October 26, 1998)     

Tobacco protoplasts differentiate into elongate cells without net microtubule depolymerization

Summary Microtubules (MTs) are important for plant cell morphogenesis because they influence the deposition of cell plate and wall components. It has been observed that tobacco protoplasts contain a disordered MT array in the cortex. Following several days in culture, these protoplasts become elongate cells with an orderly cortical MT array. The transformation of the MT array may occur by net depolymerization of the disordered MTs and repolymerization of MTs into an ordered array, or by movement of the array as an integral unit. To experimentally distinguish between these two possibilities, the drug taxol was used to stabilize MTs. Protoplasts derived from suspension cultured tobacco,Nicotiana tabacum, were grown in a medium containing the two plant hormones -naphthaleneacetic acid and benzyladenine, in the presence or absence of 10M taxol. Changes in cell size and shape were quantified using a video image analysis system. Cell elongation had begun within 48h of protoplast conversion, in both treatments, and continued for 7 days. Immunolocalization of tubulin showed that, in the majority of cells, MTs were disorganized immediately following protoplast conversion. After elongation, the MT arrays were observed to have reoriented to an ordered state. Taxol-treated protoplasts were found to elongate faster and to a greater extent than the non-treated controls. Additionally, the cortical array of taxol-treated protoplasts reorganized more quickly. These data indicate that the net depolymerization of disordered cortical MTs is not necessarily required for the differentiation of a protoplast into an elongate cell.Abbreviations APM amiprophosmethyl - BSA bovine serum albumin - DIC differential interference contrast - DTT dithiothreitol - EGTA ethylenegrycol-bis-(-aminoethyl ether)N,N,N,N-tetra-acetic acid - ELISA enzyme-linked immunosorbent assay - FMS Fukuda, Murashige, and Skoog - MS Murashige and Skoog - MT(s) microtubule(s) - PBS phosphate buffered saline - PIPES piperazine-N,N-bis (2-ethanesulfonic acid, 1.5 sodium) - PM plasma membrane - Tris Tris(hydroxymethyl)amino-methane     

Cytomorphogenesis in cenocytic green algae. V. Segregative cell division and cortical microtubules in Dictyosphaeria cavernosa (Siphonocladales,Chlorophyceae)*

Disassembly and reassembly of cortical microtubules (MT) during and after segregative cell division (SCO) in Dictyosphaeria cavernosa (Forssk.) Børgesen were observed using fluorescence microscopy. Parallel cortical MT in a mother cell were intact just after the initiation of SCD, but soon circular, MT-free patches appeared. Protoplasmic contraction enlarged the patches, and in these areas, the protoplasm eventually became perforated. Long and undulating cortical MT were arranged densely in the reticulate protoplasm. During further protoplasmic contraction, cortical MT appeared to be random and decreased in density. Finally, short and random cortical MT were present in the segregated protoplasts. Parallel cortical MT reassembled in the expanding daughter cells. After the daughter cells came in contact with one another, a radial system of cortical MT was constructed at the side that faced the inside of the mother cell wall. A microtubule inhibitor (amiprophos methyl, APM) had no effect on SCO. Segregative cell division was not induced directly by mechanical wounding. A comparison between SCO and wound-induced protoplasmic contraction was made.     

Effects of colchicine and amiprophos-methyl on microfibril arrangement and cell shape inAdiantum protonemal cells

Summary 5 mM colchicine and 1 g/ml amiprophos-methyl, known antimicrotubule agents, were applied to fernAdiantum protonemata under red light. Both drugs caused microtubule disruption and subsequent apical swelling of protonemal cells after certain lag periods. While the lag periods for the onset of microtubule disruption after application of the two drugs were different (within 15 minutes in amiprophos-methyl, 1 hour in colchicine), the lag periods of apical swelling after microtubule disruption were nearly the same (approx. 70 minutes). The results suggest that the apical swelling is a consequence of microtubule disruption.In cells examined 1 hour after microtubule disruption by either drug, the microfibril arrangement of the innermost layer of the cell wall was random at the tip, transverse in the subapical region, and roughly longitudinal in the cylindrical region. This pattern of microfibrils was similar to that of untreated cells in which the microtubules show a similar arrangement (Murata and Wada 1989). Surprisingly, even after approx. 4 hours of microtubule disruption, when apical swelling had occurred in most cells, the pattern of microfibril deposition was not altered. The role of microtubules in oriented microfibril deposition and the mechanism of control of cell shape are discussed.Abbreviations APM amiprophos-methyl - DMSO dimethylsulfoxide - MT(s) microtubule(s) - PBS phosphate buffered saline     

Organization of cortical microtubules and microfibril deposition in response to blue-light-induced apical swelling in a tip-growing Adiantum protonema cell

The arrangements of cortical microtubules (MTs) in a tip-growing protonemal cell of Adiantum capillus-veneris L. and of cellulose microfibrils (MFs) in its wall were examined during blue-light (BL)-induced apical swelling. In most protonemal cells which had been growing in the longitudinal direction under red light, apical swelling was induced within 2 h of the onset of BL irradiation, and swelling continued for at least 8 h. During the longitudinal growth under red light, the arrangement of MFs around the base of the apical hemisphere (the subapical region) was perpendicular to the cell axis, while a random arrangement of MFs was found at the very tip, and a roughly axial arrangement was observed in the cylindrical region of most cells. This orientation of MFs corresponds to that of the cortical MTs reported previously (Murata et al. 1987, Protoplasma 141, 135–138). In cells irradiated with BL, a random rather than transverse arrangement of both MTs and MFs was found in the subapical region. Time-course studies showed that this reorientation occurred within 1 h after the onset of the BL irradiation, i.e. it preceded the change in growth pattern. These results indicate that the orientation of cortical MTs and of cellulose MFs is involved in the regulation of cell diameter in a tip-growing Adiantum protonemal cell.Abbreviations BL blue light - MF(s) microfibril(s) - MT(s) microtubule(s)     

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

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

Seed coat cell turgor responds rapidly to air humidity in chickpea and faba bean

The turgor pressure in cells of chickpea (Cicer arietinum L.) and faba bean (Vicia faba L.) seed coats was measured with a pressure probe. Measurements were made under in situ conditions by removing a section of wall from a pod, which remained attached to the plant, and exposing the intact seed. If the pod wall was removed and the turgor measurements made under ambient laboratory conditions of 50% to 70% relative humidity (RH), cell turgor pressure declined over time, typically reaching 0 MPa. If the pod wall was removed and the turgor measurements made under conditions of 100% RH, however, cell turgor pressure was stable over time, relatively uniform within the seed coat tissue, and was found to be 0.1-0.3 MPa for chickpea, and 0.1-0.2 MPa for faba bean. In both species there was a marked decline in cell turgor, beginning within about 60 s, when humidification was discontinued. The decline in cell turgor occurred regardless of the depth of the cell within the seed coat tissue, and this decline could be stopped, but not entirely reversed, when humidification was restored. An increase in cell turgor could also be caused by wetting of the seed. These responses indicate that a very rapid water exchange can occur within the seed coat tissue in situ. The rapid and, in some cases, relatively permanent loss of seed coat cell turgor in the absence of humidification raises serious concerns regarding desiccation artefacts which may be involved in the empty seed coat technique, often used to study seed carbon and water relations in grain legumes.     

Pressure Induced Reorientation of Cortical Microtubules in Epidermal Cells of Lolium rigidum Leaves

In order to assess the effect on microtubule arrays of slowlypressurising cells over 50 s from 0.1 MPa (atmospheric pressure)to 55 MPa, microtubules in epidermal cells of Lolium rigidumleaves were visualised by immunofluorescent staining and fluorescencemicroscopy. In both control and pressure-treated leaves cellshape, measured as the ratio of cell length and width, can becorrelated to the arrangement of cortical microtubules. Microtubulearrays change from random to organised in cells whose lengthis greater than their width. In untreated leaves, elongatedcells have microtubules aligned predominantly transversely.In pressure-treated leaves, elongated cells have microtubulesaligned predominantly longitudinally. Thus, pressure treatmentresults in the rapid reorientation of organised cortical microtubulesfrom a transverse to a longitudinal orientation. (Received June 21, 1993; Accepted July 15, 1993)     

Conifer somatic embryogenesis for studies of plant cell biology

Summary Embryogenic cultures have been produced for a wide range of conifers and current methods developed for spruce permit the maturation of high quality embryos that can be desiccated and then germinated to form plantlets. Embryogenic suspensions consisting of immature embryos are an excellent source of regenerable protoplasts. This review considers examples of applications of embryogenic suspension cultures for basic studies in three areas of plant cell biology. a) Immunofluorescence studies of microtubules in mitotic spruce cells reveal focused spindle poles at prophase and anphase, suggesting the presence of microtubule organizing centers (MTOCs). Antibodies known to recognize animal MTOCs do not stain the polar regions but do stain developing kinetochores. b) Embryo-derived protoplasts regenerate directly to somatic embryos. Fluorescence studies of the cytoskeleton in freshly derived protoplasts reveal random cortical microtubules and a fine network of actin filaments. During culture, protoplasts change shape and develop transverse cortical microtubule arrays. Embryonal cells of newly formed embryos possess distinctive arrays of cortical microtubules and networks of fine actin filaments while suspensor cells are characterized by transverse cortical microtubules and longitudinal actin cables. c) Transmission electron microscope studies of endocytosis in spruce protoplasts reveal an endocytotic pathway similar to that described previously for soybean. Uptake results are confirmed using high pressure freeze fixation instead of conventional chemical fixation. Presented in the Session-in-Depth Morphogenesis: Plant Cell and Tissue Differentiation at the 1994 Congress on Cell and Tissue Culture, Research Triangle Park, NC, June 4&#x2013;7, 1994.     

The ultrastructure of cellular division (autosporogenesis) in the coccoid green alga,Trebouxia aggregata,revealed by rapid freeze fixation and freeze substitution

Summary Adequate ultrastructural preservation of cells of the green algaTrebouxia aggregata is achieved by immersion freeze fixation using liquid propane followed by freeze substitution and resin embedding at ambient temperature. Despite differential staining of membranes, using this method we have been able to study plasma membrane biogenesis during cellular division. Daughter protoplasts are separated by an ingrowing septum of plasma membrane that extends into the cell from a particular site at the peripheral plasma membrane marked by centrioles. Septum development involves tip growth followed by lateral growth. This growth seems to involve transfer of membrane from an adjacent partially coated reticulum to the septum plasma membrane. The reticulum which extends from nearby Golgi stacks to the area of septum growth is associated with an extensive array of microtubules. After daughter protoplasts are completely separated, each one becomes surrounded by a cell wall which is distinct from the persisting mother wall. The ultrastructural evidence suggests that cells ofT. aggregata are autospores rather than vegetative cells.Abbreviations C centriole - ER endoplasmic reticulum - G Golgi body - MTOC microtubule organizing center - Mt(s) microtubule(s) - N nucleus - P primary septum - PCR partially coated reticulum - PM plasma membrane - Py pyrenoid - S septum