Experimental inhibition of cell wall formation and of reversion inNadsonia elongata andSchizosaccharomyces pombe protoplasts

Summary The growth, cell wall regeneration, and the reversion of the protoplasts ofNadsonia elongata andSchizosaccbaromyces pombe cultivated in nutrient media containing snail enzyme was studied by light and electron microscopy. The protoplasts grew in the presence of snail enzyme and an incomplete cell wall composed of fibrils was formed on their surface. Thus, the presence of snail enzyme inhibited the completion of cell wall structure and, consequently, the reversion of the protoplasts to normal cells. The transfer of these protoplasts to medium free from snail enzyme led first to the completion of the cell wall and then to the reversion of the protoplasts to normal cells. The reported experiments confirmed that the regeneration of the complete cell wall preceded the protoplast reversion.     

Comparison of cell wall regeneration on maize protoplasts isolated from leaf tissue and suspension cultured cells

Summary The cell wall regeneration on protoplasts derived from maize mesophyll cells was compared with wall regeneration on protoplasts derived from suspension cultured cells using light microscopy, transmission electron microscopy, and mass spectrometry. The time course of cell wall regeneration has shown that the mesophyll protoplasts regenerated walls much slower than the protoplasts derived from cultured cells. Moreover, cell wall materials on the mesophyll protoplasts were often unevenly distributed. Electron microscopy has further demonstrated that the mesophyll protoplasts have less organized and compact walls than the protoplasts from cultured cells. Chemical analysis revealed that the mesophyll protoplasts had a lower ratio ofβ-(1–3)-glucan toβ-(1–4)-glucan than protoplasts from cultured cells. The significance of these results for the viability and development of protoplasts in culture is discussed. National Research Council of Canada paper no. 32458.     

Sucrose biosynthesis in Dunaliella

Four independent kinds of observations indicate that the cell wall regenerated by oat (Avena sativa L.) and corn (Zea mays L.) protoplasts in culture is less well developed than that regenerated by tobacco (Nicotiana tabacum L.) protoplasts. Following wall regeneration the cereal protoplasts remained susceptible to osmotic shock upon transfer to water, showed great enlargement, stained poorly with calcofluor white, and maintained a positive internal electrical potential. The development of a negative membrane potential by tobacco protoplasts in culture often occurred simultaneously with the onset of cell division. Since division was observed only in protoplasts which had regenerated good cell walls and had re-established negative membrane potentials it is suggested that culture conditions which favor these two processes should improve protoplast viability.     

Observations on the time course of wall development at the surface of isolated protoplasts

The formation of cell wall fibres at the surface of isolated leaf protoplasts has been studied by scanning electron microscopy. Fibres are not formed on incubated protoplasts until a lag period has elapsed. This period is about 8 h for leaf protoplasts of Nicotiana tabacum and about 45 h for leaf protoplasts of Antirrhinum majus. In the case of Antirrhinum protoplasts the length of the lag period is dependent on the concentration of osmoticum present during the incubation period. If regenerating protoplasts are briefly treated with dilute cellulase, the newly formed wall is completely digested. Such protoplasts are capable of producing new fibres at the surface within minutes of their return to a nutrient medium. These results are discussed in terms of the likely source of the lag period and its significance in wall regeneration studies.Abbreviations MS culture medium used at full strength - 0.1 MS culture medium used at one tenth full strength     

Reversion of protoplasts and spheroplasts in the yeast Nadsonia elongata

Summary The time rate of regeneration of the cell wall and reversion of protoplasts of the yeast Nadsonia elongata to cells of normal shape and size has been compared with the capability for regeneration of spheroplasts of this yeast. Nearly all protoplasts in a given culture were able to regenerate new walls and had usually reverted to cells of normal appearance by the 30th h of cultivation. Spheroplasts required only half this time to do this. These results can be interpreted as evidence that regeneration of a wall by protoplasts does not depend upon the presence of a cell wall primer, because the proportion of reverting protoplasts (which lack wall remnants) was the same as that of reverting spheroplasts (which possess them). The presence of wall remnants in spheroplasts appears to have merely an accelerating effect on the formation of a new wall and on subsequent reversion of the spheroplasts to complete cells of normal shape and size.     

Regenerated cell wall of carrot protoplasts isolated from suspension-cultured cells

Protoplasts of Daucus carota L. cultured in a synthetic liquid medium resumed cell division after about 4 days of cultivation. During this lag period, nucleic acid and protein showed only slight increases but the protoplasts commenced cell-wall regeneration soon after the removal of lytic enzymes. The originally spherical protoplasts became ellipsoidal before they underwent division. Radioactive glucose and myo-inositol were readily utilized by the protoplasts. Most of the radioactivity, however, appeared in extracellular polysaccharides and only a small portion was deposited in the regenerated wall. The sugar composition of new cell wall, as studies by chemical analysis and incorporation of labelled precursors, was shown to be considerably different from that of normal cell wall.     

Co-culture ofFrankia alni subsp.pommerii (strain ACN1 AG ) with birch (Betula papyrifera) protoplasts

The present study was undertaken to set up an experimental system in which barriers to infection of a non-host plant related to the presence of the cell wall, at the level of recognition and/or the necessity of penetrating the cell wall, might be bypassed. Co-cultures betweenFrankia alni subsp.pommerii (strain ACN1 ^AG ) andBetula papyrifera protoplasts were established. Betula protoplasts remained viable after 2 weeks with no substantial cell wall regeneration. Suppression of the wall barrier was not sufficient to allowFrankia infection under the conditions tested. The non-infectivity ofFrankia on Betula protoplasts may also reflect difficulties inherent to thein vitro environment, which might not permit duplication of infection mechanisms.     

Electron microscopic observations of cell regeneration from cultured protoplasts ofAmmi visnaga

Summary Protoplasts ofAmmi visnaga initiated cell wall formation within 2 days in culture; after 13 days the new cells were enclosed by a cell wall similar to the walls on the original cultured cells. Budding occurred in protoplasts with little or no detectable cell wall. No evidence was obtained for direct participation of any organelle in cell wall formation. The cytoplasm of regenerating cells contained numerous organelles and appeared typical of actively growing plant cells; they were easily distinguished from degenerate cells and protoplasts. While coated vesicles were common, spiny vesicles occurred in only a few cells. Sustained cell division yielded multicellular aggregates. Multinucleate protoplasts, formed by spontaneous fusion, did not divide; some of them contained annulate lamellae with few pore complexes.Supported by the National Research Council of Canada, Grant A6304.     

Karyokinesis in growing and reverting protoplasts ofSchizosaccharomyces pombe

Division of nuclei without cytokinesis proceeds in growing protoplasts ofSchizosaccharomyces pombe. Prior to regeneration of the complete cell wall and reversion the protoplasts contain 1–7 nuclei, protoplasts with 1–2 nuclei are most frequent. When regeneration of the wall is postponed by adding snail enzymes to the growth medium, protoplasts with a higher number of nuclei (2–4) occur. Multinuclear protoplasts can revert to cells. During the first cytokinesis the protoplast with the regenerated cell wall is divided into two cells by a septum, distribution of nuclei between the two cells being probably incidental. More than only a single nucleus can pass to the revertants even during the second cytokinesis. Septation of protoplasts occurs also during a partial blockage of the wall formation by the snail enzyme preparation, however, reversion to cells can never be observed here (it occurs only after transfer of protoplasts to the medium without the enzyme preparation). The growing and reverting protoplasts represent a very good model system for studying relations among individual processes of the cell cycle, primarily growth of the cell, nuclear cycle and cytokinesis. Yeast protoplasts are often utilized as models for studying morphogenic processes, relations among regeneration of the cell wall, including division of the nucleus (karyokinesis) and cytokinesis.     

Morphological and ultrastructural studies on protoplast production and reversion of the higher basidiomyceteAgaricus bisporus

Protoplasts have been isolated from young vegetative mycelia ofAgaricus bisporus by an enzyme mixture of novozym and chitinase. Protoplasts were released through ruptures in the wall, initially at the apices, but also later from older parts of the hyphae, indicating that they may lack the cell wall. The process of regeneration of these protoplasts has been investigated in liquid medium in which the protoplasts produced short chains of convoluted cells that finally produced a hypha. Electron microscopy has shown that at the start of regeneration two different kinds of fibrils were produced at the external surface of the protoplasts. Later, the thickness of the cell wall increased, and there was a deposit of amorphous material giving rise to a complete new wall.     

Protoplasts formation inFusarium species

Formation of protoplasts from four species ofFusarium genus is described. Protoplasts were isolated from mycelium by enzymatic digestion of the cell wall in the presence of an osmotic stabilizer. The results obtained differed between the studied species. Best yields of protoplasts were obtained fromF. moniliforme (90 % cells as protoplasts).     

A study of cell wall regeneration of protoplasts inMarchantia polymorpha L.

Protoplasts ofMarchantia polymorpha L. were isolated from suspension cells. Regeneration of cell walls on the surface of the protoplasts began within a few hr of cultivation. New cell walls completely covered the surface of the protoplasts within 48 hr. Coumarin and 2,6-dichlorobenzonitrile treatment inhibited the formation of the new cell wall. In the initial stage of cell wall regeneration, endoplasmic reticula developed remarkably close to the plasma membrane in the protoplasts, but no development of Golgi bodies was observed at the same locus. This may suggest that the Golgi bodies do not play an active role in the cell wall formation, at least not in very early periods of cell wall regeneration. The development of endoplasmic reticula and an ultrastructural change of plasma membrane from smooth to rough may be important in the cell wall formation of protoplasts.     

PREPARATION AND CHARACTERIZATION OF PROTOPLASTS OBTAINED FROM THE PRASINOPHYTE SCHERFFELIA DUBIA (CHLOROPHYTA)1

The prasinophyte genera Scherffelia and Tetraselmis are the only genera that form a cell wall by an extracellular fusion of scales called a theca. We established a protocol for the production of protoplasts from Scherffelia dubia Pascher emend. Melkonian et Preisig using 3 mM Ellman's reagent (5,5′‐dithio‐bis‐2‐nitrobenozoic acid [DTNB]). Protoplasts analyzed by EM lacked flagella and thecae but were otherwise similar to control cells. In response to treatment with DTNB, many protoplasts synthesized new thecal scales in the Golgi apparatus, indicating that cells attempted to regenerate new cell walls. However, complete regeneration of the thecae only occurred once DTNB was washed out from the medium. At higher DTNB concentrations (5 mM), two protoplasts were found within the parental cell wall and scales accumulated between the plasma membrane of the protoplasts and the original theca but failed to form a new theca.     

Conditions that affect binding of DNA by cultured plant cells and protoplasts: An autoradiographic analysis

Summary The binding of the¹⁴C-labelledSalmonella typhimurium DNA or³H-labelled soybean SB-1 DNA to cultured soybean cells (Glycine max L. Merr.) (SB-1) could be increased at least 100-fold by choosing the proper incubation conditions. The uptake of DNA by cells could completely be inhibited by the addition of an excess of unlabelled thymidine, indicating that the observed uptake of DNA by cells most probably is simply uptake of DNA degradation products. Autoradiograms, prepared from SB-1 protoplasts that were previously incubated with DNA, showed that the DNA was not associated with the protoplasts, but only with aggregates of cell wall material contaminating the protoplast preparation. When protoplasts and DNA were incubated in the presence of DEAE-dextran, the amount of DNAse resistant radioactivity increased 40 times. Again, the autoradiograms showed that most if not all DNAse-resistant material was associated with cell wall materials. Our observation that it is cell wall contaminants in protoplast preparations which account for most of the DNA binding demonstrates the need for caution in interpreting experiments on the binding and uptake of DNA by plant protoplasts.NRCC No. 16353.     

Inhibition of the cell wall regeneration and reversion in protoplasts ofRhizopus nigricans

During cultivation in a nutrient medium with snail gastric juice the protoplasts ofRhizopus nigricans produce an incomplete cell wall and grow. A true growth, associated with nuclear division, is involved. Morphology of growth of the formations is determined by the structure of the incomplete cell wall. When the incomplete wall is formed by a thin fibrillar net the growing formation assumes the physically optimal shape—i.e. a sphere; when the net is dense polar growth predominates. The degree of construction of the new wall depends on the activity of snail gastric juice enzymes which decreases during the cultivation. When fresh snail enzymes were added at certain intervals, only a fine fibrillar net was formed on the surface of growing protoplasts. The formations grew for up to 8 d under these conditions, reached several hundred μm in size and the number of nuclei increased up to 80-fold. When the blocking of the wall synthesis was interrupted, a complete cell wall regenerated on the surface of these giant formations and a reversion to hyphae was observed. The incomplete cell wall functions as a passive morphogenetic factor: It can influence the morphology of the growing protoplasts but it cannot induce reversion to hyphase and secure the permanent existence of these structures.     

Evacuolation and enucleation of mesophyll protoplasts in self-generating percoll gradients

Mesophyll protoplasts from Brassica oleracea, B. napus, Nicotiana tobaccum and Solanum tuberosum were isolated and subjected to uttracentrifugation at 65000g for 30 min in percoll solutions containing various strengths of salt and osmotic stabilizing agents. After centrifugation, the self-generated percoll gradients were evaluated for their effectiveness in protoplast evacuolation and enucleation. The vacuoles, cell debris, evacuolated protoplasts and enucleated protoplasts were separated. Factors that affected evacuolation and enucleation in the percoll gradients were described. Mesophyll protoplasts produced by epidermis peeling and short enzyme incubation periods were more easily evacuolated and enucleated than those produced by leaf-slicing and long incubation periods. Lower centrifugal force at 25000g for 80 min was also successful in evacuolating and enucleating the mesophyll protoplasts. A green band that contained nearly pure evacuolated protoplasts, of which 45% were enucleated protoplasts, was obtained from the self-generated percoll gradient. Rhodamine 123 staining of mitochondria indicated that the evacuolated protoplasts were metabolically active and were capable of regenerating the vacuole and cell wall. Cell divisions were also observed when the evacuolated protoplasts were cultured.     

Concanavalin A binding sites on the plasma membrane of leek stem protoplasts

The binding of concanavalin A (con A) to leek (Allium porrum L.) stem protoplasts has been investigated using sequential treatment with con A and haemocyanin and using con A covalently linked to ferritin. Prefixed protoplasts were evenly labelled. Unfixed protoplasts showed a clustered distribution of label. Low temperature and lanthanum reduced the clustering. Bound con A was lost from unfixed protoplasts incubated for 5 h after treatment, but con A/haemocyanin was not bound to nascent wall materials. Prefixed protoplasts treated with wall-removing enzymes before labelling showed only a small reduction of con A binding. These results indicate that con A is bound to plasma membrane components, but that binding is reduced by competition of nascent wall precursors.Abbreviations con A concanavalin A - con A-H sequential treatment with con A and haemocyanin - con A-F con A covalently linked to ferritin     

Release ofAspergillus niger protoplasts byPenicillium purpurogenum enzymes

Enzymes formed by the fungusPenicillium purpurogenum destroy theAspergillus niger cell wall and if a suitable stabilizing solution is used, protoplasts are released from the hyphae. The rate of release and the quantity and properties of the protoplasts are similar to those of protoplasts obtained by means of snail digestive enzymes. The lytic enzymes also destroy the cell walls of some other filamentous fungi.     

Formation,growth, and regeneration of protoplasts of the green alga,Uronema gigas

Summary Separate protoplasts were obtained by the action of snail gut juice enzymes on the cell walls of the green algaUronema gigas. The cultivation of the protoplasts in mineral media caused only their enormous growth; in the presence of glucose a fibrillar network was formed on the surfaces of the growing protoplasts. Only after the addition of pectin the regeneration of the cell wall and the renewal of their morphogenesis could be observed.     

Stabilization of cortical microtubules by the cell wall in cultured tobacco cells

Cortical microtubules (MTs) in protoplasts prepared from tobacco (Nicotiana tabacum L.) BY-2 cells were found to be sensitive to cold. However, as the protoplasts regenerated cell walls they became resistant to cold, indicating that the cell wall stabilizes cortical MTs against the effects of cold. Since poly-l-lysine was found to stabilize MTs in protoplasts, we examined extensin, an important polycationic component of the cell wall, and found it also to be effective in stabilizing the MTs of protoplasts. Both extensin isolated from culture filtrates of tobacco BY-2 cells and extensin isolated in a similar way from cultures of tobacco XD-6S cells rendered the cortical MTs in protoplasts resistant to cold. Extensin at 0.1 mg·ml⁻¹ was as effective as the cell wall in this respect. It is probable that extensin in the cell wall plays an important role in stabilizing cortical MTs in tobacco BY-2 cells.