Temporal and spatial changes of cellulose synthesis inClosterium acerosum (Schrank) Ehrenberg during cell growth

The amount and distribution of wall microfibril synthesis were investigated in the cell-division cycle ofClosterium acerosum. Electron-microscopic examination and a methylation analysis of alkali-extracted wall fragments showed that alkali-extracted wall was mainly composed of microfibrils and that the microfibrils ofC. acerosum were 4-linked glucans, i.e., cellulose. Cellulose synthesis was measured as incorporation of¹⁴C, fed to cells as NaHCO₃, into extracted wall fragments. Extensive cellulose synthesis was coincident with septum formation, continued for more than 6 h and then ceased. It was found by microautoradiography that cellulose synthesis after cell division was essentially restricted to the expanding new semicells. Such a restricted distribution of cellulose synthesis was maintained for more than 6 h after septum formation, i.e., for more than 2 h after the cessation of expansion; afterwards, cellulose synthesis in some, but not all, cells became extended to the old semicells, and then ceased. Considerable cellulose synthesis also took place in the band-like expanding part of non-divided cells, indicating that cell division was not necessarily required for the induction of cellulose synthesis and the latter was coupled with cell expansion. Extension of cellulose synthesis to old semicells was brought about in divided cells by treatment with 3 mM colchicine, 28 M vinblastine, 50 M isopropyl-N-phenylcarbamate or 1 M isopropyl-N(3-chlorophenyl)carbamate, indicating that microtubules are involved in the limitation of cellulose synthesis to the new semicells.Abbreviations CIPC isopropyl-N(3-chlorophenyl)carbamate - DPO 2,5-diphenyloxazole - IPC isopropyl-N-phenylcarbamate     

Kopulation von Schwesterzellen bei Closterium acerosum (Schrank) Ehrenberg

Ohne ZusammenfassungMit 2 Textabbildungen (6 Einzelbildern).     

Re-formation of microtubules in Closterium ehrenbergii Meneghini after cold-induced depolymerization

Immunofluorescence microscopy was used to examine the re-formation of microtubules (MT), after cold-induced depolymerization, in Closterium ehrenbergii. The C. ehrenbergii cells undergo cell division followed by semicell expansion in the dark period of daily light-dark cycles. Five types of MTs, namely the MT ring, hair-like MTs around the nuclei, spindle MTs, radially arranged MTs and transverse wall MTs, appeared and disappeared sequentially during and following cell division. The wall MTs were distributed transversely only in the expanding new semicells. When cells were chilled in ice water, wall MTs in expanding cells were fragmented, and then disappeared as did the other types of MTs, within 5 min. When cells were warmed at 20°C after 2 h chilling, wall MTs and the other types of MTs re-formed. At the early stage of wall-MT re-formation in expanding cells, small, star-like MTs were formed, and then randomly oriented MTs developed in both the expanding new and the old semicells. The MT ring was also re-formed at the boundary between the new and old semicells. There were no obvious MT-organizing centers in the random arrangement. As time passed, the randomly oriented wall MTs in the old semicells disappeared and those in the expanding new semicells gradually assumed a transverse orientation. These results indicate that wall MTs can be rearranged transversely after they have been re-formed and that nucleation of wall MTs is separable from the mechanism for ordering them.Abbreviations MT(s) microtubule(s) - MTOC(s) microtubule-organizing center(s)     

Effects of colchicine on cell shape and on microfibril arrangement in the cell wall of Closterium acerosum

Cell morphogenesis in Closterium acerosum (Schrank) Ehrenberg was greatly influenced by colchicine. Addition of colchicine to the medium led to production of tadpole-shaped cells, by decreasing the length and increasing the thickness of the new semicells. Transversely oriented wall microtubules and microfibrils, characteristic of normally elongating semicells, were not observed in colchicine-treated semicells, randomly oriented microfibrils being present instead. About 3.5 h after septum formation, the randomly oriented microfibrils began to be overlaid by bundles of microfibrils as seen in normal semicells at the later stage of elongation. When colchicine treatment was terminated 1 h after septum formation, cell elongation was partially restored and microfibrils were deposited parallel to each other and transversely to the cell axis, indicating that the effect of colchicine on microfibril arrangement in growing semicells is reversible.     

The change of pattern in microfibril arrangement on the inner surface of the cell wall of Closterium acerosum during cell growth

Closterium acerosum (Schrank) Ehrenberg cells cultured on cycles of 16 h light and 8 h dark, undergo cell division synchronously in the dark period. After cell division, the symmetry of the daughter semicells is restored by controlled expansion, the time required for this restoration, 3.5–4 h, being relatively constant. The restoration of the symmetry is achieved by highly oriented surface expansion occurring along the entire length of the new semicell. During early semicell expansion, for about 2.5 h, microfibrils are deposited parallel to one another and transversely to the cell axis on the inner surface of the new wall. Wall microtubules running parallel to the transversely oriented microfibrils are observed during this period. About 2.5 h after septum formation, preceding the cessation of cell elongation, bundles of 7–11 microfibrils running in various directions begin to overlay the parallel-arranged microfibrils already deposited. In the fully elongated cells, no wall microtubules are observed.     

Spatial relationship between microtubules and plasma-membrane rosettes during the deposition of primary wall microfibrils in Closterium sp.

The mechanism by which cortical microtubules (MTs) control the orientation of cellulose microfibril deposition in elongating plant cells was investigated in cells of the green alga, Closterium sp., preserved by ultrarapid freezing. Cellulose microfibrils deposited during formation of the primary cell wall are oriented circumferentially, parallel to cortical MTs underlying the plasma membrane. Some of the microfibrils curve away from the prevailing circumferential orientation but then return to it. Freeze-fracture electron microscopy shows short rows of particle rosettes on the P-face of the plasma membrane, also oriented perpendicular to the long axis of the cell. Previous studies of algae and higher plants have provided evidence that such rosettes are involved in the deposition of cellulose microfibrils. The position of the rosettes relative to the underlying MTs was visualized by deep etching, which caused much of the plasma membrane to collapse. Membrane supported by the MTs and small areas around the rosettes resisted collapse. The rosettes were found between, or adjacent to, MTs, not directly on top of them. Rows of rosettes were often at a slight angle to the MTs. Some evidence of a periodic structure connecting the MTs to the plasma membrane was apparent in freeze-etch micrographs. We propose that rosettes are not actively or directly guided by MTs, but instead move within membrane channels delimited by cortical MTs attached to the plasma membrane, propelled by forces derived from the polymerization and crystallization of cellulose microfibrils. More widely spaced MTs presumably allow greater lateral freedom of movement of the rosette complexes and result in a more meandering pattern of deposition of the cellulose fibrils in the cell wall.Abbreviations E-face exoplasmic fracture face - MT microtubule - P-face protoplasmic fracture-face     

Distribution and Local Activity of Particle Complexes Synthesizing Cellulose Microfibrils in the Plasma Membrane of Closterium acerosum (Schrank) Ehrenberg

At the early stage of expansion in Closterium acerosum, cellulose-synthesizingcomplexes are distributed only in new semicells. At the laterstage, when cellulose synthesis remains restricted, many complexesenter the old semicells from the new ones. This suggests thatthe complexes in old semicells become inactivated. (Received October 13, 1982; Accepted March 3, 1983)     

Role of the microtubule cytoskeleton in alternating changes in cellulose-microfibril orientation in the coenocytic green alga,Chaetomorpha moniligera

The functions of the microtubule (MT) cytoskeleton in changing the orientation of microfibrils (MFs) in the cell walls of the coenocytic green alga Chaetomorpha moniligera Kjellman were investigated by electron microscopy. The cortical MT cytoskeleton in Chaetomorpha was comprised of longitudinally oriented MTs. Cellulose MFs, however, alternately changed their orientation longitudinally and transversely to form crisscross MF textures. Microtubules were parallel to longitudinally oriented MFs but never to those that were transversely oriented. The average density of MTs during the formation of longitudinally oriented MFs was 216 per 50 m of wall and that of transversely oriented MFs 170/50 m. To determine exactly the MT-density dependency of each MF orientation, changes in MF orientation were examined by changing MT density after treating and removing amiprophos-methyl (APM). Microtubules were reduced in number by a half (100/50 m) after 2 h and by 3/4 (50/50 m) after 3 h of treatment with APM (3 mM). This reduction was caused by the disappearance of alternating MTs. Microtubules retained this density (50/ 50 m) up to 6 h, and then gradually disappeared within 24 h. Microfibril orientation in the innermost cell wall was transverse after treatment with APM for 2 h but was helicoidal after 6 h. Polymerization of MTs occurred in the longitudinal direction following the removal of APM after treatment for 48 h. Microtubule density rose to about 100/50 m and 200/50 m after 6 h and 24 h, respectively. The orientation of MTs changed from helicoidal to transverse and transverse to longitudinal after 6 h and 24 h, respectively. When APM was removed prior to formation of the helicoidal texture, longitudinally oriented MFs appeared within 6 h. There is thus an alternating cycle of formation of longitudinally and transversely oriented MFs within a 12-h period. Formation of transversely oriented MFs as a result of APM treatment started in the middle of a cell as hoops which then extended in the apical and basal directions. Formation of longitudinally oriented MFs as a result of the removal of APM started from the apical end and proceeded toward the base. It follows from these results that: (1) the point of formation of longitudinally oriented MFs differs from that for transversely oriented MFs, (2) MF orientation in each case depends on a separately functioning mechanism, (3) MT density changes rhythmically to trigger a switch for crisscross orientation of MFs.Abbreviations APM amiprophos-methyl - MF microfibril - MT microtubule - TC terminal complex We thank Dr. K. Okuda for making helpful discussion and Miss. T. Matsuki for assistance with replica preparation.     

Organogenesis in Graptopetalum paraguayense E. Walther: shifts in orientation of cortical microtubule arrays are associated with periclinal divisions

The interior of a new lateral organ, such as a leaf, arises from the products of periclinal divisions of sub-epidermal cells. The biophysical basis of the elongation of such a new axis is transverse (hoop) reinforcement of the cells by cellulose in the primary walls. This structural polarity is associated with transverse alignment of cortical microtubules. We have brought the histological and biophysical views together by showing that the new, periclinal, divisions are a prerequisite for a corresponding change in the orientation of the microtubular array in the daughter cells. Investigation of this relationship required development of criteria for assessing the predominant orientation of a microtubule array in a single section of known orientation. By obtaining information about the predominant orientation of microtubule arrays in the sub-epidermal cells, we were able to study structural polarity shifts which occurred as a detached leaf of Graptopetalum produced a new shoot. During organogenesis, the new polarity is seen only in cells which have divided periclinally. Following single periclinal divisions, cells are seen with microtubules in the old or new orientation or in a mixture of different orientations. Cells with more than one orientation of microtubules are probably at intermediate stages in the shift to the new polarity. Among cells which have undergone two consecutive periclinal divisions, the old polarity is no longer seen, all cells having high frequencies of microtubules in the new orientation. Such cells are either polarized in the new direction or nonpolarized. The shifts in polarity of the cells in the interior anticipate the appearance of the first leaf primordia. However, contrary to the expectations from the histological view of organogenesis, these shifts do not dominate the process. Concurrent polarity changes in the epidermis appear at least as important.     

Effect of antibiotics on cell proliferation in the Closterium peracerosum-strigosum-littorale complex (Charophyceae, Chlorophyta)

The effects of several antibiotics on the proliferation of cells of the Closterium peracerosum-strigosum-littorale complex, a unicellular charophycean alga, were examined. When cells were cultured on solid medium containing hygromycin B and phleomycin the proliferation of cells was inhibited at low concentrations of these antibiotics, with a minimum inhibitory concentration of 5.0 and 0.2 μg/mL, respectively. By contrast, kanamycin sulfate was less effective at concentrations up to 50 μg/mL. When cells were incubated in liquid medium containing hygromycin B and phleomycin, cell proliferation was severely inhibited at concentrations of 5.0 and 0.01 μg/mL, respectively. It is concluded that hygromycin B and phleomycin are highly effective for inhibiting the proliferation of C. psl. complex both on solid and in liquid medium and thus are useful for the selection of the cells transformed by selectable marker genes. Presented at the International Symposium Biology and Taxonomy of Green Algae V, Smolenice, June 26–29, 2007, Slovakia.     

Analysis of binding of biotinylated protoplast-release-inducing protein that induces release of gametic protoplasts in the Closterium peracerosum-strigosum-littorale complex

A protoplast-release-inducing protein (PR-IP) which is released from mating-type plus (mt⁺) cells and induces the release of gametic protoplasts from matingtype minus (mt⁻) cells of Closterium was biotinylated and then used to examine the interaction of this protein with mt⁻ cells. The protoplast-release-inducing activity of PR-IP was not altered after the biotinylation. When mt⁻ cells that had been pre-cultured for 24 h were incubated with biotinylated PR-IP for 6 h in nitrogen-deficient medium that contained 1% (w/v) bovine serum albumin, and then washed with the same medium, only a 19-kDa polypeptide, the smaller subunit of PR-IP, was detected in cells by the avidin and biotinylated horseradish-peroxidase macromolecular complex system. The amount of bound 19-kDa polypeptide increased with increasing doses of PR-IP and reached a maximum at around 10 nM, reflecting the protoplast-release-inducing activity. From a Scatchard plot, the dissociation constant of the polypeptide was calculated to be 2.7 · 10⁻⁸ M. The binding of the polypeptide proceeded only after an appropriate period of pre-culture in the light, and the polypeptide was competitively displaced by non-biotinylated PR-IP. From these results, it appears that the PR-IP induces the release of protoplasts from mt⁻ cells by binding of a polypeptide of relative molecular mass 19000 to the receptor on the cell surface in a manner analogous to the binding of peptide hormones in animals.     

Microtubules and possible microtubule nucleation centers in the cortex of stomatal cells as visualized by high voltage electron microscopy

Summary Thick sections of fixed, embedded stomatal cells ofPhleum pratense were examined using high voltage electron microscopy and stereological procedures. The cortex of guard cells and subsidiary cells throughout differentiation contains numerous microtubules adjacent to the plasmalemma. Although microtubules are usually aligned in one net direction, individual microtubules may diverge from this orientation in various ways, producing anastomosing or crossed arrays. Also present in the cortex of both guard and subsidiary cells are collections of membranous elements and amorphous material upon which microtubules seem to focus, terminate or overlap. Such structures may constitute microtubule nucleation centers. The significance of these observations is discussed in terms of the control of microtubule development, wall microfibril deposition and cell morphogenesis.     

Aberrant nuclear migration and microtubule arrangement in a defect mutant cell ofMicrasterias thomasiana

Summary Nuclear migration and the spatial arrangement of the participating microtubules are studied inMicrasterias thomasiana and in the defect mutant cellMicrasterias thomasiana f. uniradiata.In both of these cell types the two microtubule systems, the posttelophase system of MT (PTS) and the isthmus system of MT (IS)—which are known to be involved in nuclear migration and anchoring from earlier studies onMicrasterias denticulata—are present in the vicinity of the nucleus. In the mutant cell, however, the orientation of these two MT systems as well as their MT arrangement differ from those in the normalMicrasterias cells. Nuclear migration in the mutant is characterized by a turn of the nucleus and the associated PTS around one of the isthmus invaginations of the cell while in the normalMicrasterias cells it occurs as a straight-lined motion along the longitudinal axis of the cell.The results indicate that the reduction of cell pattern inMicrasterias caused by mutation is attended by a disoriented establishment of the cytoskeleton involved in nuclear migration. From comparison of the nuclear behavior and the MT arrangement in the mutant with that of the normalMicrasterias cells further information on the mechanism of nuclear migration inMicrasterias is obtained. It is suggested that interactions between the microtubule center (MC), the nuclear envelope and areas of the plasma membrane are functional in the formation, orientation and localization of the nucleus associated microtubule-microfilament complex.     

Orientation of cellulose microfibrils in cortical cells of tobacco explants

The deposition of nascent cellulose microfibrils (CMFs) was studied in the walls of cortical cells in explants of Nicotiana tabacum L. flower stalks. In freshly cut explants the CMFs were deposited in two distinct and alternating orientations — all given with respect to the longitudinal axis of the cell —, at 75° and 115°, in a left-handed (S-helix) and right-handed (Z-helix) form, respectively. The CMFs deposited in these orientations did not form uninterrupted layers, but sheets in which both orientations were present. After explantation, the synthesis of CMFs and their deposition in bundles continued. New orientations occurred within 6 h. After 6 h a new sheet was deposited, with orientations of 15° (S-helix) and 165° (Z-helix). The changes could be seen as sudden bends in individual CMFs or in small bundles of CMFs. In the next stage, more CMFs were deposited with these new orientations and the bundles became larger. New orientations arose by a shift towards more longitudinal directions, starting from either the S-helix or the Z-helix form. It was only after an almost longitudinal orientation was reached that the CMFs were deposited in two opposing directions again and a new sheet was formed. Neither colchicine nor cremart influenced the changes in CMF deposition. It is concluded that microtubules do not control CMF deposition in cortical cells of tobacco explants; control of CMF deposition and microtubule orientation occurs by factors related to cell polarity.Abbreviations CMF cellulose microfibril - MT microtubule We thank Professor M.M.A. Sassen and Dr. G.W.M. Barendse (Department of Experimental Botany, University of Nijmegen, Nijmegen, The Netherlands) for helpful discussions and Mrs. A. Kemp for her assistance in the ethylene experiments.     

Random arrangement of chromosomes inUvularia (Liliaceae)

Chromosome arrangement in interphase has been inferred from an analysis of the relative positions of the chromosomes and the chromosome arms in untreated haploid pollen grain metaphases ofUvularia grandiflora. The distances between centromeres forming the smallest possible circle were measured in 43 metaphases. The relative positions of the chromosomes did not differ significantly from randomness. Neither did similar-sized chromosome arms show any tendency to be next to each other. The results thus disagree both with the hypothesis ofComings (1968) that each chromosome occupies a definite position in the interphase nucleus and with the claim ofBennett (1982) that similar-sized chromosome arms lie next to each other.     

The assembly of cellulose microfibrils in Valonia macrophysa Kütz

The assembly of cellulose microfibrils was investigated in artificially induced protoplasts of the alga, Valonia macrophysa (Siphonocladales). Primary-wall microfibrills, formed within 72 h of protoplast induction, are randomly oriented. Secondary-wall lamellae, which are produced within 96 h after protoplast induction, have more than three orientations of highly ordered microfibrils. The innermost, recently deposited micofibrils are not parallel with the cortical microtubules, thus indicating a more indirect role of microtubules in the orientation of microfibrils. Fine filamentous structures with a periodicity of 5.0–5.5 nm and the dimensions of actin were observed adjacent to the plasma membrane. Linear cellulose-terminal synthesizing complexes (TCs) consisting of three rows, each with 30–40 particles, were observed not only on the E fracture (EF) but also on P fracture (PF) faces of the plasma membrane. The TC appears to span both faces of the bimolecular leaflet. The average length of the TC is 350 nm, and the number of TCs per unit area during primary-wall synthesis is 1 per m². Neither paired TCs nor granule bands characteristic of Oocystis were observed. Changes in TC structure and distribution during the conversion from primary- to secondary-wall formation have been described. Cellulose microfibril assembly in Valonia is discussed in relation to the process among other eukaryotic systems.Abbreviations TC terminal complex - EF E (outer leaflet) fracture face of the plasma membrane - PF P (inner leaflet) fracture face of the plasma membrane - MT microtubule - PS protoplasmic surface of the membrane     

Lattice images from ultrathin sections of cellulose microfibrils in the cell wall of Valonia macrophysa Kütz.

The crystalline ultrastructure and orientation of cellulose microfibrils in the cell wall of Valonia macrophysa were investigated by means of high-resolution electron microscopy of ultrathin (approx. 28 nm) sections. With careful selection of imaging conditions, ultrastructural aspects of the cell wall that had remained unresolved in previous studies were worked out by direct imaging of crystal lattice of cellulose microfibrils. It was confirmed that each microfibril is a single crystal having a lateral dimension of 20·20 nm², because lattice images of 0.39 nm resolution were clearly recorded with no major disruption in the whole area of the cross section of the microfibril. There was no evidence for the existence of 3.5-nm elementary fibrils which have been considered to be basic crystallographic and morphological units of cellulose in general. It was also confirmed that the axial directions (crystallographic fiber direction) of adjacent microfibrils in each single lamella of the cell wall are opposite to each other.     

Changes in microtubule and microfibril arrangement during polarotropism inAdiantum protonemata

Polarotropism was induced inAdiantum (fern) protonemata grown under polarized red light by turning the electrical vector 45 or 70 degrees. One hour after the light treatment, tropic responses became apparent in many cells as a slight distortion of the apical dome. Changes in the position of the circumferentially-arranged cortical microtubule band (Mt-band) (Murataet al., 1987) and the arrangement of microfibrils around the subapical part of protonemata were investigated in relation to the polarotropic responses. Twenty minutes after turning the electrical vector, preceding the morphological change of cell shape, the Mt-band began to change its orientation from perpendicular to oblique to the initial growing axis. After 30 min, the Mt-band changed its orientation further under 45 degrees polarized light, but under light rotated 70 degrees, it began to disappear. In phototropic responses induced by local irradiation of a side of the subapical part of a protonema with a non-polarized red microbeam, the Mt-band on the irradiated side disappeared or became faint within 20 min, but neither disappearance nor a change of orientation of Mts occurred on the non-irradiated side. One hour after turning the electrical vector 45 degrees, in half of the cells tested, the innermost layer of microfibrils in the subapical part of the protonema changed its orientation from perpendicular to oblique to the growing axis, corresponding to the changes in the orientation of the Mt-band. After 2 hr, those changes were obvious in all cells examined. The same basic results on the orientation of microfibrils were obtained with protonemata cultured for 2 hr under 70 degrees polarized light. The role of the Mt-band in tropic responses is discussed.     

Dynamic changes in the arrangement of cortical microtubules in conifer tracheids during differentiation.

The arrangement of cortical microtubules (MTs) in differentiating tracheids of Abies sachalinensis Masters was examined by confocal laser scanning microscopy after immunofluorescent staining. The arrays of MTs in the tracheids during formation of the primary wall were not well ordered and the predominant orientation changed from longitudinal to transverse. During formation of the secondary wall, the arrays of MTs were well ordered and their orientation changed progressively from a flat S-helix to a steep Z-helix and then to a flat S-helix as the differentiation of tracheids proceeded. The orientation of cellulose microfibrils (MFs) on the innermost surface of cell walls changed in a similar manner to that of the MTs. These results provide strong evidence for the co-alignment of MTs and MFs during the formation of the semi-helicoidal texture of the cell wall in conifer tracheids.Abbreviations MT cortical microtubule - MF cellulose microfibril - S1, S2 and S3 the outer, middle and inner layers of the secondary wall The authors thank Mr. T. Itoh of the Electron Microscope Laboratory, Faculty of Agriculture, Hokkaido University, for his technical assistance. This work was supported in part by a Grant-in-Aid from the Ministry of Education, Science and Culture, Japan (no. 06404013).     

Internodal elongation and orientation of cellulose microfibrils and microtubules in deepwater rice

Excised stem sections of deepwater rice (Oryza sativa L.) containing the highest internode were used to study the induction of rapid internodal elongation by gibberellin (GA). It has been shown before that this growth response is based on enhanced cell division in the intercalary meristem and on increased cell elongation. In both GA-treated and control stem sections, the basal 5-mm region of the highest internode grows at the fastest rate. During 24 h of GA treatment, the internodal elongation zone expands from 15 to 35 mm. Gibberellin does not promote elongation of internodes from which the intercalary meristem has been excised. The orientation of cellulose microfibrils (CMFs) is a determining factor in cell growth. Elongation is favored when CMFs are oriented transversely to the direction of growth while elongation is limited when CMFs are oriented in the oblique or longitudinal direction. The orientation of CMFs in parenchymal cells of GA-treated and control internodes is transverse throughout the internode, indicating that CMFs do not restrict elongation of these cells. Changes in CMF orientation were observed in epidermal cells, however. In the basal 5-mm zone of the internode, which includes the intercalary meristem, CMFs of the epidermal cell walls are transversely oriented in both GA-treated and control stem sections. In slowly growing control internodes, CMF orientation changes to the oblique as cells are displaced from this basal 5-mm zone to the region above it. In GA-treated rapidly growing internodes, the reorientation of CMFs from the transverse to the oblique is more gradual and extends over the 35-mm length of the elongation zone. The CMFs of older epidermal cells are obliquely oriented in control and GA-treated internodes. The orientation of the CMFs parallels that of the cortical microtubules. This is consistent with the hypothesis that cortical microtubules determine the direction of CMF deposition. We conclude that GA acts on cells that have transversely oriented CMFs but does not promote growth of cells whose CMFs are already obliquely oriented at the start of GA treatment.