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.     

Interference of cell wall regeneration ofBoergesenia forbesii protoplasts by Tinopal LPW,a fluorescent brightening agent

Summary Wounding cells ofBoergesenia forbesii (Harvey) Feldmann induces the synchronous formation of numerous protoplasts which synthesize large cellulose microfibrils within 2–3 hours after wounding. The microfibrils appear to be assembled by linear terminal synthesizing complexes (TCs). TC subunits appear on both E- and P-faces of the plasma membrane, thus suggesting the occurrence of a transmembrane complex. The direction of microfibril synthesis is random during primary wall assembly and becomes ordered during secondary wall assembly. The average density of TCs during secondary wall deposition is 1.7/m², and the average length of the TC is 510 nm. TC organization is similar to that ofValonia macrophysa; however, the larger TCs ofBoergesenia (510 nm vs. 350 nm) produce correspondingly larger microfibrils (30 nm vs. 20 nm).The effects of a fluorescent brightening agent (FBA), Tinopal LPW, on cell wall regeneration ofBoergesenia protoplasts was investigated. The threshold level of Tinopal LPW for interfering with microfibril assembly is 1.5 M. At 95 M Tinopal (for short periods up to 15 minutes), microfibril impressions have atypical spherical impressions at their termini. At longer incubations (24 hours), TCs and microfibril impressions are absent. When washed free of Tinopal, the protoplasts eventually resume normal wall assembly; however, TCs do not reappear until at least 30 minutes after the removal of Tinopal. In consideration of the presence of ordered TCs before FBA treatment, their random distribution upon recovery implies an intermediate stage of assembly or possiblyde novo synthesis.     

Cell development of a green alga,Pleurotaenium nodosum,which was tied by a fiber

Summary Cells of a green alga,Pleurotaenium nodosum, were tied off with thin fibers, after which development of daughter semicells from the small tied mother semicells was examined with a light microscope. Semicells slightly shortened due to tying mostly developed normal sized daughter semicells with four nodes. However, semicells shorter than 1/2 the normal size developed small daughter semicells with fewer nodes. This was interpreted that the daughter semicells of shortened semicells had not enlarged sufficiently to allow formation of four nodes on the cell surface at the initial stage of node formation. The initiation of node formation was recognized as taking place in the contact region between the plasma membrane and the cell wall in plasmolyzed cells. The distance between two neighbouring contact regions was consistent at about 6 m.     

Ultrastructural aspects of cell wall synthesis inSpirogyra

Summary Filaments ofSpirogyra were fixed in 2% osmium tetroxide dehydrated in alcohol and embedded in Araldite. The fine structure of cells with regard to wall synthesis was studied. The cell wall was shown to have four layers. The inner one contains microfibrils and is considered to be the cell wall proper. The outer three layers are components of the slime layer. The innermost of these, the second layer of the wall, was shown to be between 1m to 3m and the third 0.3m to 1m. The fourth layer appears as no more than a dark black line measuring 10 nm across. In the cytoplasm two types of vesicles were seen. The largest of these has contents similar in appearance to the slime layer of the wall. This same material was also seen in the large vesicles attached to the Golgi bodies. It is suggested that the smaller vesicles are derived from the larger vesicles and later fuse with the cell membrane. The Golgi bodies were found to be fairly large measuring up to 5m across. Small electron opaque blobs and flecks on the outside of the plasmalemma and in between the microfibrils of the cell wall proper are considered to be mucilage droplets travelling to the slime layer. It cannot be excluded that some of the material of the large vesicles is released directly into the cytoplasm and is transferred without vesicles through the plasma membrane. The negative contrast appearance of the microfibrils seen in the cell wall is thought to be due to the spaces between them being filled with this electron opaque mucilage.Intercisternal rodlets measuring 2.5 nm across were seen in the Golgi bodies.Transverse microtubules were found to occur near the plasmalemma having the same orientation as some of the microfibrils.Lomasome-like structures sometimes with many 5 nm fibrils in their vicinity were seen.     

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.     

Wall texture in the spore of a vesicular-arbuscular mycorrhizal fungus

Summary InGlomus epigaeum Daniels and Trappe, a vesicular-arbuscular mycorrhizal fungus, the mature spore has a complex multi-layered wall containing a regular pattern of wall subunits.The outer wall (2–4 m thick) consists of a simple layer of parallel microfibrils. The inner wall (5–6 m thick) is built from two layers possessing different organization. The innermost layer, near the plasmalemma has a texture of apparently dispersed fibrils, whereas the second layer is regularly organized with an arced texture. Ten to twelve bundles of fibrils connected by apparently bow-shaped fibrils are consistently observed. The appearance of this arced organization depends on the section plane and on the angle of observation in the electron microscope as confirmed by tilting experiments. Wall subunits are evident as straight electron transparent fibrils; particularly well-defined in negatively stained frozen sections: their diameter is about 3.5nm.The regular pattern of wall subunits in this fungal cell wall is compared with the textures shown by cellulose fibrils in algae or higher plants and by chitin fibrils in arthropod cuticle.Research work supported by CNR, Italy. Special grant I.P.R.A.—Sub-project 1. Paper No. 55.     

Regeneration of plants from callus and embryos of ‘Royal’ apricot

Immature embryos of apricot (Prunus armeniaca L.) cv. Royal with a PF index of 25–100 were used to regenerate plants in vitro using two methods. In the first case, callus was initiated on MS medium with 4.5 M 2, 4-D plus 0.44 M BA and regeneration of shoots from the callus occurred on MS medium with 4.4 M BA plus 1.0 M 2, 4-D. In the second case, adventitious buds were directly regenerated from the cotyledons on MS medium with 4.4 M BA plus 1.0 M 2, 4-D.Abbreviations BA 6-benzyladenine - IBA dole-3-butyric acid - NAA -naphthylacetic acid - 2, 4-D 2, 4-dichlorophenoxyacetic acid - PF (embryo length/seed length) x 100     

Changes in cell-wall polysaccharide composition of developingNitella internodes

Changes in the uronide, neutral-polysacharide, and cellulose composition of the cell wall ofNitella axillaris Braun were followed throughout development of the internodes and correlated with changes in growth rate. As the cells increased in length from 4 to 80 mm during development, the relative growth rate decreased. Cell wall thickness, as measured by wall density, increased in direct proportion to diameter, indicating that cell-wall stress did not change during elogation. Cell-wall analyses were adapted to allow determination of the composition of the wall of single cells. The total amounts of uronides, neutral sugars and cellulose all increased during development. However, as the growth rate decreased, the relative proportions of uronides and neutral sugars, expressed as percent of the dry weight of the wall, decreased, while the proportion of cellulose increased. The neutral sugars liberated upon hydrolysis ofNitella walls are qualitatively similar to those found in hydrolysates of higher plant cell walls: glucose, xylose, mannose, galactose, arabinose fucose and rhamnose. Only the percentage of galactose was found to increase in walls of mature cells, while the percentage of all other sugars decreased. The rate of apposition (g of wall material deposited per unit wall surface area per hour) of neutral polysaccharides decreased rapidly with decreasing growth rate during the early stages of development. The rate of apposition of uronides decreased more steadily throughout development, while that of cellulose, after an early decline, remained constant until dropping off at the end of the elongation period. These correlations between decreasing growth rate and decreasing rate of apposition of neutral sugars and uronides indicate that synthesis of these cell-wall components could be involved in the regulation of the rate of cell elongation inNitella.     

Continuous culture and synchronization ofHyphomicrobium sp. B-522

A technique was developed for synchronization ofHyphomicrobium sp. strain B-522. Bacteria were grown in continuous culture with methanol (0.1%; v/v) growth limiting. Vitamin B₁₂ (2.5 g/l) was necessary to obtain steady state growth. The critical dilution rate wasD _c =0.112; maximum cell output occurred atD=0.105 (Dx=30 mg l^-1 h^-1). Continuous cultures ofHyphomicrobium B-522 atD=0.110 were used to obtain cells for synchronization experiments. Synchronization was achieved by trapping young hyphal and budding cells in a glass wool column, while the initial swarmer cells were allowed to pass through. By semicontinuously rinsing the system, newly produced swarmers could be sampled in the effluent. The mean length of these synchronous swarmer cells was 1.25 m (s=±0.13 m; range 0,6 m) as compared to 1.40 m (s=±0.21 m; range 1.2 m) for swarmer cells of the continuous culture. Division of synchronous swarmer populations was completed after 7 h; the synchronization index was 0.76.     

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.     

The organization of cortical microtubule arrays in the radish root hair

Summary Cortical microtubule arrays in the radish root hair were analyzed from reconstructions of serial ultra-thin sections in order to test extant hypotheses concerning the role of microtubules in the deposition of oriented microfibrils of cellulose. Passing away from the tip, root hairs exhibit a transition from random to oriented deposition of microfibrils at approximately 25 m. Along the root hair, passing back from the tip, the microtubules: a) increase in number to a plateau at 25 m; b) change their length profiles from approximately 60% less than 1 m long in the hair tip to approximately 40% less than 1 m long at 60 m; c) maintain a constant pattern of angular deviation from the long axis, which is similar to the deviation pattern of the oriented wall fibrils; d) maintain a constant (approximately 70% of tubules) close (within 50 nm) proximity to the plasma membrane (PM); e) maintain a low (approximately 20%) degree of inter-microtubule proximity (i.e., within 50 nm of one another); f) show evidence for some variable long range (>50 nm) association. Fixation with glutaraldehyde in a complete microtubule polymerization medium (MTPM), or pretreatment with cytochalasin B cause an approximate twofold increase in 1. the proportion of long microtubules in the tip region and 2. microtubules within 50 nm of one another. Fixation in incomplete MTPM (without GTP) produces results similar to phosphate buffer controls. Alternative explanations for these results are examined. A new hypothesis accounting for microtubule involvement in oriented microfibril deposition is described.     

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     

Inhibition of phenylalanine ammonia-lyase activity causes the depression of lignin accumulation of secondary wall thickening in isolatedZinnia mesophyll cells

Summary Isolated mesophyll cells ofZinnia elegans L. cv. Canary Bird differentiate into tracheary elements in differentiation (D) medium. These elements develop lignified secondary wall thickenings. The influence of 2-aminoindan-2-phosphonic acid (AIP), an inhibitor of phenylalanine ammonia-lyase (PAL), on lignification ofZinnia tracheary elements was examined. The mesophyll cells were cultured in D and AIP media. The latter medium, in which 100 M AIP was added to the D medium, inhibited PAL activity, though the differentiation proceeded. Morphological differences of secondary wall thickenings cultured in these two types of media were investigated under an UV microscope and a transmission electron microscope. The secondary wall thickenings at 96 h in the D medium showed strong UV absorption. The fibrillar structure of the thickenings observed clearly at 72 h was covered with electron opaque materials by 96 h. The secondary wall thickenings at 96 h in the AIP medium showed weak UV absorption. The thickenings at 96 h had a cracked appearance. Furthermore, the thickenings showed a little irregular or wavy arrangement of cellulose microfibrils and had many pores and spaces between microfibrils. From these results, the role of lignin accumulation in the formation of secondary wall thickenings was discussed.Abbreviations AIP 2-aminoindan-2-phosphonic acid - PAL phenylalanine ammonia-lyase     

Plant regeneration from embryo-derived callus of oil palm – the effect of exogenous polyamines

Regeneration in oil palm was achieved through somatic embryogenesis/organogenesis from embryo-derived callus. Callus was induced from mature embryos of the cross 281 (D)×18 (P) on modified MS medium supplemented with 2,4-D (113.12 M) and 2-iP (14.76 M). The embryogenic calluses obtained were transferred to Blaydes medium supplemented with 2,4-D (0.045 M) and one of the following growth regulators: TDZ (4.54 M), zeatin riboside (2.85 M), putrescine (1 mM) and spermine (100 M). Secondary somatic embryogenesis was found to occur in media supplemented with polyamines. The efficiency of formation of somatic embryos, secondary somatic embryos and shoot meristemoids were significantly higher in putrescine containing medium. Histological studies were also undertaken.     

A new cellulosic structure, the tunic cord in the ascidianPolyandrocarpa misakiensis

Summary A specialized structure of tunic cord inPolyandrocarpa misakiensis is investigated by electron microscopy. The tunic cord is a cord-like coiled structure of 5–30 m in diameter and 0.1–9.0 mm in length. The tunic cords originate and elongate from the dorsal tunic, and their termini have a swollen and ornamented structure. Scanning and transmission electron micrographs and the electron diffractogram show that the tunic cords are composed of bundled microfibrils of cellulose I with high crystallinity. The tunic cord is completely surrounded by single-layered epidermal cells, which have been found as the site of cellulose biosynthesis. A number of tunic cords are connected to the internal tunic of the siphon by forming eyelet structures at their termini. These observations suggest that the tunic cords act as a connector between dorsal and internal tunic of the siphon.     

The arrangement of microfibrils in sporangial walls of Allomyces

Summary The microfibrillar component of the walls of zoosporangia and resistant sporangia of the phycomycete Allomyces arbusculus has been studied in the electron microscope, after chemical removal of the amorphous wall materials. In the zoosporangium wall the microfibrils are randomly arranged, as in the outer layer of the hyphal walls, and the sporangial wall is of even thickness. In the resistant sporangia the microfibrillar layer of the wall is perforated by numerous pores 0.25 in diameter. The microfibrils are randomly arranged over much of the wall but tend to be concentrically arranged in the vicinity of the pores. On the inside of the wall the microfibrils form a thickened rim around the pore.     

The dose of 1-naphthaleneacetic acid determines flower-bud regeneration in tobacco explants at a large range of concentrations

Short-term applications of very high concentrations of 1-naphthaleneacetic acid (NAA) to expiants from flower stalks of tobacco (Nicotiana tabacum L. cv. Samsun) induced flower-bud regeneration to the same extent as longer or continuous incubation on lower concentrations. The maximum number of flower buds per explant after 15 d of culture was obtained not only by continuous culturing at 1 mol·l^–1 NAA but also by 12 h of culturing at 22 mol·l^–1 or 0.5 h at 220 mol· l^–1, followed by incubation on medium without auxin for the remaining period. Continuous application of such high concentrations resulted in callus formation or caused the death of the explanted tissue. In all experiments in which auxin concentration and time of application were independently varied, the product of concentration and time determined the number of buds formed. Most, but not all, of the NAA taken up by the tissues was converted into conjugates. In expiants which had received a dose which was optimal for regeneration, the internal concentration of free NAA remaining beyond the pulse period was between 1.7 and 6.2 mol·l^–1. Suboptimal applications led to lower values, supraoptimal treatments to much higher internal concentrations. The physiological effect, which depends on the internal hormone concentration, thus manifested itself as dose-dependent with regard to applied hormone.Abbreviations BAP N⁶-benzylaminopurine - NAA 1-naphthaleneacetic acid     

Cell wall regeneration by protoplasts of Chlamydomonas

Protoplasts from Chlamydomonas smithii prepared by the action of C. reinhardii gamete autolysine have been studied with respect to cell wall regeneration. Natural protoplasts within sporangia were also investigated for purposes of comparison. In both cases a new cell wall is completed within 2–3 h of the onset of regeneration. The first visible stages of wall regeneration are to be seen after 40–60 min as a fine fringe outside of the plasmalemma. The development of the typical central triplet follows within the next 1 h. Cell wall regeneration is reversibly inhibited by cycloheximide (10g ml^-1) and reversibly disturbed by concanavalin A (50 g ml^-1). Actinomycin D at concentration over 100g ml^-1 also inhibit but the inhibition is irreversible and peculiar membrane effects are observed. Chelators (ethylenediamine tetraacetic acid; ethyleneglycol-bis-aminoethyl ether) and 2-deoxyglucose slightly retard or have no effect on cell wall regeneration.Abbreviations EDTA ethylenediamine tetraacetic acid - EGTA ethyleneglycol-bis(aminoethyl ether) - N,N tetraacetic acid     

Localized accumulation of cell wall mannoproteins and endomembranes induced by brefeldin A in hyphal cells of the dimorphic yeastCandida albicans

Summary Candida albicans, a dimorphic yeast, has the abililty to switch its growth form between budding growth and hyphal growth. Since fungal growth involves secretory processes, spatial control of secretion should play a crucial role in such a morphogenetic transition. Brefeldin A (BFA), an inhibitor of the membrane trafficking system of eukaryotes, increases the occurrence of Golgi-like cisternae in the yeast. In the present study, BFA was used to obtain further insights into the spatial organization of secretory processes in hyphal growth ofC. albicans. BFA completely inhibited the formation and growth of germ tubes at a concentration of 35 M or higher. Electron microscopy of BFA-untreated germinated cells revealed many vesicles in the apical region and Golgi-like cisternae in the cytoplasm. In cells treated with 35 M BFA, the vesicles disappeared from the apical region, and, instead, stacked membrane cisternae and membrane-enclosed spherical dense bodies accumulated in the subapical region. These accumulated structures were positive for both polysaccharide staining and immunocytochemical staining with antibodies raised against cell surface antigens ofC. albicans, as were Golgi cisternae in BFA-untreated cells. In cells treated with a higher concentration of BFA (140 M), the structures that appeared in cells treated with 35 M BFA were no longer observed and the endoplasmic reticulum was extended and positive for polysaccharide staining. These results suggested that BFA affects different steps of membrane trafficking in a concentration-dependent manner. The accumulated structures induced by 35 M BFA seemed to be the altered forms of Golgi cisternae. Their accumulation in the subapical region of the germ tube might indicate that the step(s) in membrane trafficking that are associated with the Golgi pathway are vectorially organized in hyphal growth ofC. albicans.Abbrevations BFA brefeldin A - BSA bovine serum albumin - CBB Coomassie brilliant blue - Con A concanavalin A - HRP horseradish peroxidase     

Bioelectric and biosynthetic aspects of cell polarity inAllomyces macrogynus

Summary In contrast to all filamentous fungi examined to date, vegetative hyphae ofAllomyces macrogynus, whether extending or not, produced an outward flow of positive electrical current, at a maximum of 0.16 A cm^–2 around 40 m behind the apex, as measured with a vibrating probe. Inward currents of up to 0.55 A cm^–2 were recorded around the rhizoids. Increases in outward current were observed in hyphae pre-grown under oxygen deficiency and then allowed to widen backwards to the hyphal base in sufficient oxygen. When spores were germinated in an applied electrical field they produced rhizoids predominantly towards the anode. Hyphae were produced initially towards the cathode but later bent around towards the anode. Experiments with a range of chemicals provided no evidence for the involvement of calcium in vegetative growth and development inA. macrogynus. Polyoxin and nikkomycin, inhibitors of chitin synthesis, had no effect on swimming zoospores, but inhibited wall formation of cysts, rhizoids and forward and backward growing hyphae.