Developmental studies on the loricate choanoflagellateStephanoeca diplocostata Ellis

Summary Cell division inStephanoeca diplocostata follows the accumulation of a large number of costal strips in horizontal bundles at the top of the parent collar. Prior to nuclear division the flagellum is lost and the protoplast is large and rectangular. Nuclear division takes place whilst the protoplast undergoes vigorous metabolic movements and subsequent cytokinesis is achieved by equatorial constriction. The anterior of the two daughter protoplasts is the juvenile and is inverted with respect to the sister which remains attached to the parent lorica. The two protoplasts are joined by a cytoplasmic strand that consists of two threads both of which are initially attached to the daughter protoplasts at one side of the collar. Cell separation involves elongation of the strand and after each thread has broken contact with one of the daughter cells the two threads slide over each other until the juvenile is released. The juvenile takes the accumulation of supernumerary strips as it leaves the parent lorica and after release of the juvenile the strips are mobilised to form a new lorica. The collar tentacles of the parent are thought to play a significant role in the movement of strips during division and certain selected tentacles on the juvenile are associated with lorica assembly. Cell separation takes between 9–12 minutes and lorica assembly by the juvenile 2–3 minutes.     

Developmental studies on the loricate choanoflagellate Stephanoeca diplocostata Ellis. III. Growth and turnover of silica,preliminary observations

Growth, turnover of silica and lorica morphology of Stephanoeca diplocostata Ellis have been investigated in batch cultures at 20 °C. Mean cell doubling times for separate experiments ranged from 9.1–13.8 h. During exponential growth, uptake of reactive silicate progressed steadily and throughout this phase the average amount of biogenic silicon per cell was 2.1 pg. Once growth declined, net dissolution of silica from loricae became apparent and progressed steadily throughout the stationary and death phases. The minor difference in solubility between loricae of living cells and costal strips cleaned with acid indicates that even if an organic component is associated with the silica of costal strips it does not inhibit silica dissolution. The first effects of silica dissolution on cells as revealed by electron microscopy are limited to corrosion on the surfaces and the hollowing out of the centres of costal strips but ultimately a consistent pattern of lorica disintegration occurs.     

Developmental studies on the loricate choanoflagellateStephanoeca diplocostata Ellis. V. The cytoskeleton and the effects of microtubule poisons

Summary InStephanoeca diplocostata microtubules are located in four positions namely: within the flagellar axoneme; just beneath the plasmalemma; associated with the silica deposition vesicles (SDVs) during early stages of costal strip deposition; and in the mitotic spindle. At the anterior end of the cell the 50–60 peripheral microtubules, which are organized more or less parallel to the long axis of the cell, converge around the base of the emergent flagellum. A short second flagellar base is positioned between the nucleus and the base of the emergent flagellum. Developing costal strips are located individually within SDVs in the peripheral cytoplasm. During the early stages of silica deposition each SDV is curved and subtended longitudinally on its concave side by two microtubules. When a costal strip has achieved sufficient rigidity to withstand bending the SDV-associated microtubules are depolymerized. Treatment of exponentially growing cells with sublethal concentrations of microtubule poisons, such as colchicine, podophyllotoxin, griseofulvin andVinca alkaloids depresses growth. Treatment with these drugs also affects the length and morphology of developing costal strips perhaps by interfering with the shaping and supporting functions of SDV-associated microtubules. Instead of being long and crescentic with a standard radius of curvature, costal strips of treated cells are usually short and misshapen, with irregular bends. After drug treatment, juveniles produced as a result of cell division do not develop flagella but can still assemble a lorica although it is usually misshapen. The role of microtubules and microfilaments in lorica production is discussed.     

Role of the cytoskeleton in choanoflagellate lorica assembly

ABSTRACT. Cell division in Acanthoeca spectabilis produces a "naked" motile daughter cell (juvenile) that settles onto a surface and deposits siliceous costal strips that are stored extracellularly in bundles. When complete, the bundles of strips are assembled in a single continuous movement to form a basket-like lorica. Assembly can be divided into four overlapping stages. Stage 1 entails the left-handed rotation of strips at the anterior end while the posterior end remains stationary. Stage 2 includes the posterior protrusion of the cell to form a stalk. Stage 3 involves the anterior extension of the spines, and Stage 4 the dilation of the lorica chamber and deposition of the organic investment. Scanning electron microscopic images reveal a one-to-one association between the moving bundles of strips and the anterior ring of lorica-assembling tentacles. Treatment with microtubule inhibitors produces "dwarf" cells that lack stalks, have their spines extended, and possess collars but lack flagella. Treatment with microfilament (actin) inhibitors prevents extension of the anterior spines. These experiments demonstrate that posterior cell extension is primarily mediated by microtubules whereas extension of the spines is controlled by the actin cytoskeleton. The processes of cytoskeletal rotation and extracellular costal strip movement are compared, respectively, with rotation of nuclei in animal embryos and movement of mammalian cells over surfaces.     

Developmental studies on the loricate choanoflagellateStephanoeca diplocostata ellis

Summary Cells ofStephanoeca diplocostata comprise a colourless, flagellated, protoplast lodged in a lorica made of siliceous costae. The single anterior flagellum creates a water current from which bacteria and other food particles are filtered by the collar and ingested by linguiform pseudopodia that arise from the protoplast at the base of the collar. A waist divides the lorica into two chambers, the anterior of which contains three transverse and 17–20 longitudinal costae whereas the posterior chamber comprises two systems of spirally deflected costae and on some cells a pedicel at the hind end. Between 150–185 costal strips of similar length form the lorica. A thin investment covers the inner surface of the posterior chamber and lower part of the anterior chamber and joins with the protoplast at the level of the waist. Costal strips are produced within membrane-bounded vesicles in the peripheral cytoplasm and, although the origin of these vesicles is unknown, there is usually a close association with the Golgi apparatus. Once complete, strips are apparently released sideways through the plasmalemma into the cavity of the posterior lorica chamber. Later, bundles of strips are transferred to the top of the inner surface of the collar where they collectively form a horizontal ring. When sufficient strips to form a lorica have been accumulated at the top of the collar, cell division proceeds.     

Kakoeca antarctica gen. et sp.n., a loricate choanoflagellate (Acanthoecidae, Choanoflagellida) from Antarctic sea ice with a unique protoplast suspensory membrane

A new genus and species of loricate choanoflagellate, Kakoeca aniarctica Buck & Marchant gen. et sp.n. grown in rough culture from an Antarctic sea ice innoculum is described. This organism has a distinctive lorica morphology consisting of more than 200 costal strips arranged in transverse and longitudinal costae that arc perpendicular to one another in the posterior portion of the lorica. The transverse costae show declination with respect to the lorica axis in the anterior part of the lorica. The cell is suspended in the lorica by a robust protoplast suspensory membrane. This membrane blocks water flow from the posterior of the lorica necessitating water entry through the side of the lorica, an area where the maximum sized apertures in the lorica are found. Terminology (lorica lining and protoplast suspensory) is suggested for the two types of lorica membranes which have been found associated with loricas.     

Costal strip production and lorica assembly in the large tectiform choanoflagellate Diaphanoeca grandis Ellis

Diaphanoeca grandis posseses a voluminous flask-shaped lorica comprising an outer layer of 12 longitudinal costae and an inner layer of four transverse costae. The cell is suspended just above the centre of the lorica chamber by tentacles that are attached to the anterior transverse ring. The component costal strips are superficially similar although four different strip categories can be distinguished on the basis of length and morphology. Costal strips are produced ‘upside-down’ within the parent cell and accumulated in a close-packed horizontal ring at the top of the inner surface of the collar. The order in which costal strips are produced is consistent, starting with those for the transverse rings, basal to anterior, and then the longitudinal costae, again with the posterior first and the anterior later. Cell division is of the classical tectiform variety with the juvenile cell being inverted and pushed backwards out of the parent lorica. Lorica assembly entails firstly the rotation of the anterior vertical strips so they become horizontal and then their movement backwards under the posterior layer of longitudinal strips. From this time onwards, lorica assembly proceeds in a standard manner with the lorica-assembling tentacles providing a forward and left-handed rotational movement.     

Costal Strip Accumulation and Lorica Assembly In the Marine Choanoflagellate Diplotheca Costata Valkanov

ABSTRACT The lorica of the tectiform choanoflagellate D. costata contains five categories of costal strips distinguishable from each other on the basis of morphology and patterning. Categories of strips include those forming the anterior transverse costa; the anterior, intermediate, and posterior costal strips, respectively, of the longitudinal costae and those constituting the posterior transverse costa. the distinctive morphology of each class of strips makes it possible to observe their location and orientation within the overall accumulation of strips at the top of the parent cell collar. In Diplotheca costata the orientation and positioning of the different categories of strips in an accumulation anticipates their orientation and imbrication in the mature lorica. Assembly of the lorica from an accumulation of strips involves lateral sliding of costal strips to constitute transverse costae and longitudinal sliding of strips to constitute longitudinal costae. the motive force for lorica assembly is provided by extension of the anterolateral tentacles.     

Developmental and ultrastructural observations on two stalked marine choanoflagellates, Acanthoecopsis spiculifera Norris and Acanthoeca spectabilis Ellis.

Acanthoecopsis spiculifera and Acanthoeca spectabilis are stalked, loricate choanoflagellates found in littoral sea water pools. The two taxa are distinguished from each other by the arrangement of costae forming the lorica chamber. In addition, Acanthoecopsis spiculifera usually has a longer stalk and may be colonial, consisting of two or more attached individuals. Division in Acanthoeca results in the production of a juvenile, flagellated, protoplast without a lorica. After separation, the juvenile protoplast swims away, settles down and produces an accumulation of costal strips. When sufficient strips have been produced the lorica is rapidly assembled.     

Immunological evidence for transfer of the barley nitrate reductase structural gene to Nicotiana tabacum by protoplast fusion

Summary A protoplast fusion experiment was designed in which the selectable marker, nitrate reductase (NR), also served as a biochemical marker to provide direct evidence for intergeneric specific gene transfer. NR-deficient tobacco (Nicotiana tabacum) mutant Nia30 protoplasts were the recipients for the attempted transfer of the NR structural gene from 50 krad -irradiated barley (Hordeum vulgare L.) protoplasts. Barley protoplasts did not form colonies and Nia30 protoplasts could not grow on nitrate medium; therefore, selection was for correction of NR deficiency allowing tobacco colonies to grow on nitrate medium. Colonies were selected from protoplast fusion treatments at an approximate frequency of 10^-5. This frequency was similar to the Nia30 reversion frequency, and thus provided little evidence for transfer of the barley NR gene to tobacco. Plants regenerated from colonies had NR activity and were analyzed by western blotting using barley NR antiserum to determine the characteristics of the NR conferring growth on nitrate. Ten plants exhibited tobacco NR indicating reversion of a Nia30 mutant NR locus. Twelve of 26 regenerated tobacco plants analyzed had NR subunits with the electrophoretic mobility and antigenic properties of barley NR. These included plants regenerated from colonies selected from 1) co-culturing a mixture of Nia30 protoplasts with irradiated barley protoplasts without a fusion treatment, 2) a protoplast fusion treatment of Nia30 and barley protoplasts, and 3) a fusion treatment of Nia30 protoplasts with irradiated barley protoplasts. No barley-like NR was detected in plants regenerated from a colony that grew on nitrate following selfed fusion of Nia30 protoplasts. Because tobacco plants expressing barley-like NR were recovered from mixture controls as well as fusion treatments, explanations for these results other than protoplast fusionmediated gene transfer are discussed.     

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     

Studies on the biochemistry and fine structure of silica shell formation in diatoms

Summary The sequential wall formation in the centric diatom,Ditylum brightwellii (West) Grunow, is described. The silica deposition vesicle is formed by the coalescence of small vesicles. Silicification of the new valve starts from the central labiate process area prior to the completion of cytokinesis, and the developing valve grows in a centrifugal direction. The initiation of the structures on the valve follows the sequence: labiate process, marginal ridge, and rota. A novel labiate process apparatus, which is situated in the cytoplasm close to the developing labiate process, appears prior to the initiation of the labiate process and disappears upon its maturation. Segments of the girdle bands are formed in individual silica deposition vesicles after the valve matures and is exocytosed. Three morphological forms of deposited silica have been determined: thin base layers, microfibrils, and hexagonal columns. The involvement of cytoplasmic structures in the patterning of the siliceous wall is discussed.     

Surface charge of protoplasts and their significance in cell-cell interaction

-potential of mesophyll protoplasts of tobacco (Nicotiana tabacum L.), petunia (Petunia hybrida Hort.), turnip (Brassica rapa L.) and cowpea (Vigna unguiculata L. Walpers) was determined by use of a cell electrophoresis apparatus. All protoplasts examined showed a constant negative value of-10 to-35 mV. The addition of CaCl₂ nullified the -potential of tobacco protoplasts. This phenomenon is explained by DLVO theory of colloid science, which has been successfully applied to animal cells. Furthermore, positively charged polymers reversed the -potential to positive values. Treatment of the protoplast surface with several enzymes was carried out to characterize the chemical nature of suface charges. The removal of surface charges was most conspicuous by the treatment of acid phosphatase (EC 3.1.3.2), but did not occur upon treatment with -neuraminidase (EC 3.2.1.18) or Streptomyces griseus pronase. Thus a major part of the surface charge originates from the phosphate groups at the cell membrane. The significance of these studies for the properties of the protoplast surface in cell adhesion is discussed.     

Factors affecting protoplast yield of the carrageenophyte Solieria filiformis (Gigartinales,Rhodophyta)

Summary The effect of age, pH of the culture medium, pre-treatment of tissues, enzymes sources and enzymatic adaptability of phycophages fed with a monospecific diet were analyzed on the protoplast yields of the red seaweed Solieria filiformis (Kützing) Gabrielson. New apices from fast growing plants showed the highest protoplast yields. The protoplast yield decreased when the pH of the culture medium increased from 6.0 to 9.0. Crude extracts from the abalone Haliotis coccinea canariensis Nordsieck, fed with Solieria filiformis thalli for three months in combination with cellulysin, released the highest number of viable cells and protoplasts. Yields ranged from 1.0 to 8.5 x 10⁶ protoplasts per gram of fresh weight.Abbreviations AAP abalone acetone powder - Bis-Tris Bis(2hydroxyethyl)imino-tris(hydroxymethyl)methane - CMC carboxymethyl cellulose - EDTA ethylenediaminetetraacetic acid - EGTA ethylene glycol-bis(ß-aminoethyl ether) NNNN-tetraacetic acid - FDA fluorescein diacetate - FW fresh weight - Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - Tris Tris(hydroxymethyl)aminomethane     

Callus formation from cell culture protoplasts of corn (Zea mays L.)

Summary Routine procedures for the isolation of large numbers of protoplasts from an established cell culture of Zea mays and for the induction of sustained divisions leading to secondary cell cultures have been developed. The critical factors seem to be associated with neither specific enzymatic conditions for the isolation nor specific culture conditions for the protoplasts but with the quality of the culture used for protoplast isolation.     

Further observations on cell-wall formation around isolated protoplasts of tobacco and tomato

Freeze-etch observations of protoplasts isolated from tobacco (Nicotiana tabacum L.) mesophyll tissue and tomato (Lycopersicum esculentum Mill.) fruit locule tissue are described which clarify earlier observations (Burgess, J., Fleming, E.N., Planta 131, 173–178, 1976; Planta 133, 267–273, 1977), obtained using scanning electron microscopy. of fibres associated with projections from these cell surfaces. It is demonstrated (1) that the fibres consist of bundles of small numbers of microfibrils which have become artifactually thickened by the deposition of coating materials, and (2) that the apparent association between fibres and projections results from microfibrils being lifted preferentially from protoplast surfaces in regions rich in projections (plasmalemmasomes). With the higher resolution available using freeze-etching it can be demonstrated that microfibril deposition does not occur in discontinuous zones on these protoplast surfaces. Globules associated with microfibril termini in radish (Raphanus sativus L.) roots are illustrated and it is proposed that turgor pressure differences between isolated protoplasts and intact tissue may account for the absence of similar globules from isolated protoplast surfaces.     

Primary callus as source of totipotent barley (Hordeum vulgare L.) protoplasts

Summary Primary callus of barley (Hordeum vulgare L.) derived from scutella (cv. Dissa) and anthers (cv. Igri) was used for protoplast isolation and plant regeneration. The protoplasts were embedded in agarose and cultured with nurse cells. The plating efficiency varied from 0.1% to 0.7%. Shoots regenerated from the developing callus. Plantlets were transferred to soil and cultivated in the greenhouse three to five months after protoplast isolation. All plants were normal in morphology, and most of them flowered and set seeds.     

Studies of cotyledon protoplast cultures from Brassica napus,B. campestris and B. oleracea. I: Cell wall regeneration and cell division

Protoplasts isolated from cotyledons of a number of cultivars of Brassica napus, B. campestris and B. oleracea were cultured in different media to study the characteristics of cell wall regeneration and cell division at early stages of culture. Time course analysis using Calcolfluor White staining indicated that cell wall regeneration began in some protoplasts 2–4 h following isolation in all cultivars. 30–70% of cultured cotyledon protoplasts exhibited cell wall regeneration at 24 h and about 60–90% at 72 h after the initiation of culture. Results also indicated that a low percentage (0.4–5.4%) of cultured cotyledon protoplasts entered their first cell division one day after initial culture in all twelve cultivars. The percentage of dividing cells increased linearly up to 40% from 1 to 7 day, indicating that cotyledon protoplasts of Brassica had a high capacity for cell division. Factors that influence the level of cell wall regeneration and cell division during cotyledon protoplast culture have been investigated in this study. Cotyledons from seedlings germinated in a dark/dim light regime provided a satisfactory tissue source for protoplast isolation and culture for all Brassica cultivars used. The percentages of protoplasts exhibiting cell wall regeneration and division were significantly influenced by cultivar and species examined, with protoplasts from all five cultivars of B. campestris showing much lower rates of cell wall regeneration than those of B. napus and B. oleracea over 24–120 h, and with the levels of cell division in B. napus cultivars being much higher than those in B. campestris and B. oleracea over 1–9 days. The capacity of cell wall regeneration and cell division in cotyledon protoplast culture of the Brassica species appears under strong genetic control. Cell wall regeneration in protoplast culture was not affected by the culture medium used. In contrast, the composition of the culture medium played an important role in determining the level of cell division, and the interaction between medium type and cultivars was very significant.Abbreviations BA benzylaminopurine - CPW Composition of Protoplast Washing-solution - CW Calcolfluor White - EDTA ethylenediamine-tetraacetic acid - KT Kinetin - Md MS modified Murashige and Skoog medium - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - IAA indole-3-acetic acid - PAR photosynthetically active radiation - SDS sodium dodecyl sulfate     

Phenotypic variation and ploidy level of plants regenerated from protoplasts of tetraploid potato (Solanum tuberosum L. cv. ‘Bintje’)

Summary A wide range of phenotypic variation occurred among protoplast — derived plants of tetraploid potato cultivar Bintje. The variant plants had alterations in growth and vigour, and in leaf and stem characteristics. The results suggest that the altered morphologies are caused predominantly by changes in ploidy levels. Some alterations could be attributed typically to octoploidy and aneuploidy. The occurrence of mixoploidy indicates that at least part of the observed variation arose during culture stage. The exogeneous cytokinin or auxin level and their combination during in vitro phase influenced the frequency of the variants observed. The origin of variation is discussed.     

An improved method for the generation and transfection of protoplasts from Cucumis sativus Cotyledons

Summary A protocol was developed for the preparation of Cucumis sativus var Straight 8 protoplasts that incorporates a two-step Ficoll^® gradient and results in a high percentage of viable, debris-free protoplasts suitable for the transient expression of foreign genes. Polyethylene glycol and electroporation were compared for their effect on protoplast transfection with commonly used reporter genes. Using a polyethylene glycol method, cucumber protoplasts transfected with a plasmid containing the -glucuronidase gene showed high expression levels, while protoplasts transfected with a plasmid containing the chloramphenicol acetyl transferase gene showed levels of activity that were barely distinguishable from mock-transfected controls. Tomato ringspot virus genomic RNA was also transfected into the protoplasts, and the assembly of viral particles was confirmed.