Chemical analysis of cell wall regeneration and reversion of protoplasts from Schizophyllum commune

In the presence of MgSO₄ as osmotic stabilizer, nucleated protoplasts of Schizophyllum commune developed a large vacuole and could be isolated on the basis of their low buoyant density. All these protoplasts were capable of wall regeneration and about 50 percent reverted to the hyphal mode of growth in liquid medium. The kinetics of the formation of three main cell-wall components, S-glucan (α-1,3-glucan), R-glucan (β-1,3, β-1,6-glucan) and chitin were studied from the onset of regeneration. S-glucan and chitin accumulation as well as RNA and protein synthesis started simultaneously after a short lag, but R-glucan formation was delayed. The reversion to hyphal tubes only began after several hours of rapid R-glucan synthesis. Cycloheximide (0.5 μg/ml), inhibiting protein synthesis by 98% inhibited the formation of R-glucan and the reversion to hyphal growth but the formation of chitin and S-glucan did start and continued seemingly unimpaired for several hours. This indicates that the enzymes responsible for the synthesis of S-glucan and chitin remained intact during protoplast preparation. Polyoxin D inhibited both the synthesis of chitin and R-glucan and also the reversion to hyphal growth. However, the synthesis of S-glucan was not suppressed. These inhibitor studies as well as the kinetics of R-glucan formation during normal regeneration suggest that the synthesis of R-glucan is required for the initiation of hyphal morphogenesis.     

Chemical analysis of cell wall regeneration and reversion of protoplasts from Schizophyllum commune.

In the presence of MgSo4 as osmotic stabilizer, nucleated protoplasts of Schizophyllum commune developed a large vacuole and could be isolated on the basis of their low buoyant density. All these protoplasts were capable of wall regeneration and about 50 percent reverted to the hyphal mode of growth in liquid medium. The kinetics of the formation of three main cell-wall components, S-glucan (alpha-1,3-glucan), R-glucan (beta-1,3, beta-1,6-glucan) and chitin were studied from the onset of regeneration. S-glucan and chitin accumulation as well as RNA and protein synthesis started simultaneously after a short lag, but R-glucan formation was delayed. The reversion of hyphal tubes only began after several hours of rapid R-glucan synthesis. Cycloheximide (0.5 mug/ml), inhibiting protein synthesis by 98% inhibited the formation of R-glucan and the reversion to hyphal growth but the formation of chitin and S-glucan did start and continued seemingly unimpaired for several hours. This indicates that the enzymes responsible for the synthesis of S-glucan and chitin remained intact during protoplast preparation. Polyoxin D inhibited both the synthesis of chitin and R-glucan and also the reversion to hyphal growth. However, the synthesis of S-glucan was not suppressed. These inhibitor studies as well as the kinetics of R-glucan formation during normal regeneration suggest that the synthesis of R-glucan is required for the initiation of hyphal morphogenesis.     

Formation and regeneration of protoplasts in the moldRhizopus nigricans

Wall-less protoplasts of the moldRhizopus nigricans were obtained by means of enzymic digestion of the walls of vegetative hyphae by Helix pomatia digestive juice. After removing the enzymes the protoplasts transferred to a nutrition medium regenerate. In the first stage of this process the protoplasts from a firm wall and then the hyphae begin to grow. Regeneration results in the formation of a normal mycelium. If in the course of regeneration of the protoplasts snail enzymes are present, they partially block the synthesis of the wall and the protoplasts transform into incessantly growing giant formations. Re-establishment of morphogenesis begins only after eliminating the effect of the snail enzymes.     

Yeast protoplasts immobilized in alginate: Cell wall regeneration and reversion to cells

Yeast protoplasts may regenerate the cell wall and revert to cells if immobilized in a 2%–5% Ca-alginate gel and cultured in an osmotically stabilized medium. The method of protoplast immobilization and subsequent isolation from the gel is described in detail. The reversion yield is dependent of the actual gel concentration, gel shape (beads vs. sheets) and of a medium molarity, and it may be up to 90%. The morphology of the cell wall regeneration and morphology of reversion to the cell forms correspond to protoplast development in gelatin or agar gels.     

Ultrastructure and localization of wall polymers during regeneration and reversion of protoplasts ofSchizophyllum commune

Summary Cell-wall regeneration and reversion of protoplasts ofSchizophyllum commune were investigated using electron microscopic methods and X-ray diffraction.After 3 hours of regeneration protoplasts have formed a loosely organized wall which does not react with Thiéry's stain for periodic acid sensitive carbohydrates. This wall largely consists of chitin microfibrils which are adpressed to the plasmalemma and which are covered by loose aggregates of alkali-soluble S-glucan (-1,3-glucan). Both components are microcrystalline, at least partly. Walls formed in the presence of polyoxin D only consist of thick loose fibers of S-glucan.From 3 hours onward the inner chitin microfibrils of the wall of the primary cells become embedded in alkali-insoluble material that stains heavily with the Thiéry reagent and probably is similar to the R-glucan of the mature wall (i.e., -1,3--1,6-glucan). The outer chitin microfibrils remain free of this matrix and are covered by S-glucan only.Bud-like structures that arise have the same wall architecture as the primary cells,i.e., only the inner chitin microfibrils are embedded in R-glucan and the S-glucan forms a fluffy coat. The walls of hyphal tubes that arise are distinct, however, in that all chitin microfibrils are embedded in R-glucan and the S-glucan forms a compact coat.Cytoplasmic vesicles are sparse in primary cells except at the sites of emergence of budlike structures and hyphae. They continue to be present in the apex of growing hyphae.     

Cell wall regeneration and cell division in isolated tobacco mesophyll protoplasts

Summary Protoplasts were isolated from palisade tissue of tobacco leaves by treatment with pectinase and cellulase under aseptic conditions, and were cultured in a synthetic liquid medium. Calcofluor, a fluorescent brightener, was found to be an excellent stain for plant cell walls and was used to demonstrate regeneration of cell walls in these protoplasts. The cultured protoplasts regenerated cell walls by the 3rd day of culture, giving rise to spherical cells. The majority of the protoplasts regenerating cell walls underwent mitosis and cell division. The cycle of mitosis and cell division was repeated 2–3 times during 2 weeks of culture. Some of the nutritional conditions affecting division in the cultured protoplasts were studied.     

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.     

Chitin contents in different morphological forms ofRhizopus nigricans

Chitin (one of the major cell wall constituents ofZygomycetes) was determined in different morphological forms ofRhizopus nigricans mycelia grown at different cultivating conditions. Microscopic observation and chemical analysis has revealed that at the phase of active growth, long and sparsely branched hyphae of dispersed mycelia contain 2.3% of dry cell mass of this structural polysaccharide, which is higher than the value found in pellets of the same age. A large increase in chitin content up to 9.0% of cell dry mass was determined in older, partially autolyzed mycelia in the form of pellets.     

Agarose embedding affects cell wall regeneration and microtubule organization in sunflower hypocotyl protoplasts

Sunflower hypocotyl protoplasts ( Helianthus annuus L. cv. Emil) divide symmetrically to form loosely associated microcolonies when cultured in liquid medium, whereas when embedded in agarose beads they divide asymmetrically to give rise to embryo-like structures. To understand the relationship between protoplast embedding and cell division patterns, we studied the deposition of β-linked glucan and the dynamics of microtubules during early phases of culture. After one day in culture, under both culture conditions, a small proportion of the protoplasts had already begun to rebuild a β-glucan cell wall and the process reached completion in all protoplasts after 10 days. Callose deposition was faster in agarose than in liquid medium but it concerned only 30–40% of the protoplasts and was not related to either division type. No marked differences were observed in cortical arrays of microtubules. However, in embedded protoplasts perinuclear microtubules formed a well-defined basket around the nucleus; these microtubules were never observed in liquid-cultured protoplasts. A narrow preprophase band was present only in dividing protoplasts cultured in liquid medium. The results suggest that asymmetric division could be related to the lack of a narrow preprophase band and that protoplast embedding enhances nucleation or stabilization of microtubules.     

Ultrastructure of the cell wall regeneration of isolated protoplasts of Skimmia japonica thunb

Isolated protoplasts obtained from leaves and from stem callus cultures of Skimmia japonica were cultivated for 72 h to regenerate a new cell wall. During this process the structural changes in the protoplasts and at the surface of the plasmalemma were studied in ultrathin sections and after freeze-fracturing and deep-etching.The cultured protoplasts show an apparent increase in cell organelles compared to the freshly isolated protoplasts. In particular, mitochondria, endoplasmic reticulum, and ribosomes, many of them appear as polysomes, become numerous. Moreover, special connections between the ER and the plasmalemma are visible. Most important are the fracture faces of the plasmalemma with two different arrangements of membrane-bound particles: (1) particles in hexagonal arrays and (2) rows of ca. 14 particles. Their orientation usually conforms with that of the regenerated microfibrils of the cell wall. According to these results the following model for microfibril synthesis and orientation in higher plants is proposed: While the cytoplasmic activity is involved in the production of cellulose precursors and enzymes, the hexagonal arrays may respresent specialized regions for the outward passage of these cellulose precursors. The rows of membrane-associated particles may function as a linear enzyme complex (matrix) for microfibril biosynthesis and orientation.Abbreviations ER endoplasmic reticulum - IAA -indolylacetic acid - 2,4-D 2,4-dichlorophenoxy acetic acid     

Ultrastructural aspects of wall regeneration byPythium protoplasts

Electron microscope studies were made of wall regeneration byPythium protoplasts. Wall regeneration began with the formation of a loose network of fibrils on the surface of the protoplast followed by increase in density of the fibrillar mesh and deposition of granular matrix material. The majority of the protoplasts did not develop beyond the loose fibrillar network stage, however a small percentage were able to complete wall formation and to form hyphal tubes. A clear zone of demarcation was visible between the fibrillar surface of the protoplast and the smooth surface at the base of the developing hyphal tube.     

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     

Involvement of an acid phosphatase on cell wall regeneration of tobacco protoplasts

Tobacco protoplasts begin to regenerate their own cell walls, the major components of which are β-glucans, soon after they are transferred into an adequate medium. During the cell wall regeneration the protoplasts secrete two isoforms of acid phosphatase (APase) in time-dependent manner. We determined that one of the isoforms, the Brefeldin A (BFA) sensitive one, is the cell wall resident APase (WP-II) by immunoblotting of the isoform with anti-WP-II antibody. We hypothesized that the WP-II may participate in the deposition of β-glucan microfibrils on the protoplast surface during cell wall regeneration. In order to examine this hypothesis, the protoplasts were cultivated in the cell wall regeneration medium containing the same amount of the BFA-sensitive APase (230 µg protein) as is secreted by the observed number of protoplasts (1.4 × 10⁵ protoplasts) per plate (30-mm-diameter) during a 3-h cultivation after transfer to the cell wall regeneration medium. The addition of WP-II to the cell wall regeneration medium stimulated the deposition of β-glucan microfibrils on the surface of the protoplasts during cell wall regeneration. To determine the stimulative effect of the 60 kDa polypeptide of WP-II, protoplasts were cultivated in the medium containing the amount of anti-WP-II IgG (230 µg protein) equivalent to the BFA-sensitive APase. These results suggested that the 60 kDa polypeptide of WP-II is the BFA-sensitive APase which is responsible for the enhanced deposition of β-glucan microfibrils on the surface of the 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.     

Regeneration of the cell wall of protoplasts of the fission yeastSchizosaccharomyces versatilis in liquid media and their reversion to cells

Regeneration of the cell wall and reversion of protoplasts with a completely regenerated cell wall to cells were studied by light and electron microscopy in protoplasts of the fission yeastsSchizosaccharomyces versatilis. On their surface the protoplasts regenerated a complete new wall even m liquid media The wall regeneration began with the formation of a thin irregular net of flat bundles of long microfibrils and the net was gradually filled with aggregates of short straight microfibrils and small piles of amorphous material. Osmotically resistant organisms with regenerated walls were detected after a 4–6 h cultivation Depending on the nutrient medium used 10–80 % of protoplasts with the regenerated wall were obtained that reverted subsequently to cells. The high percentage of the wall regeneration and reversion to cells was reached by combining cultivation in a poor medium with that in a rich medium Reversion to cells could only occur after the protoplasts had regenerated rigid cell walls These walled protoplasts underwent septation, and, by polar growth, produced cylindrical cells, further dividing by fission.     

Ultrastructural research on cell wall regeneration by cultured pollen protoplasts of Lilium longiflorum

Summary Protoplasts from pollen grains of Lilium longiflorum regenerate amorphous cellulosic cell walls in culture, during which some precursors of cellulose are polymerized, thus producing progressively harder cellulosic cell walls as the period of culture continues. It is presumed that the components of the cell wall regenerated during 1 week in culture differ from those of the intine of the pollen grain wall. The regenerated cell wall is formed by means of large smooth vesicles; in addition, numerous coated vesicles and pits aid in wall regeneration. The pollen tube that germinates from the 8-day-old cultured protoplast has numerous Golgi bodies and many vesicles which build the pollen tube wall. The tube wall has two layers just like a normal pollen tube wall.     

Cell wall regeneration and galactomannan biosynthesis in protoplasts from carob

Protoplast isolation from endosperms of developing carob (Ceratonia siliqua L.) seeds is reported for the first time. These protoplasts regenerated cell walls within 12 h. In order to assess their potential for galactomannan biosynthesis, the incorporation of radioactivity in the regenerated cell wall polysaccharides (CWP) and extracellular polysaccharides (ECP), after feeding these protoplasts with D-[U-¹⁴C]glucose or D-[U-¹⁴C]mannose was studied. The pattern of the radioactive label distribution in the neutral sugars of the trifluoroacetic acid (TFA) hydrolysate of CWP was different from that of the ECP. In the TFA hydrolysis products of the CWP, immediately after protoplast isolation, the greatest level of radioactivity (approximately 90%) was detected in glucose, galactose and mannose. After 2 days protoplast culture, the label in mannose increased. In contrast, immediately after protoplast isolation, approximately 90% of radioactivity of the ECP was detected in galactose and mannose. However, during culture, the radioactivity incorporation in mannose dropped to one third, while that in galactose and arabinose increased significantly. Hydrolysis of the CWP and ECP with -galactosidase and endo--mannanase confirmed that, at least part of mannose and galactose belonged to galactomannan molecules. These results were compared with those obtained upon feeding developing endosperm tissue with D-[U-¹⁴C]mannose. From our results we concluded that protoplasts from endosperm tissues of developing carob seeds, retained the ability of their original explant to synthesize galactomannan, making protoplasts candidates for the study of galactomannan biosynthesis.     

A batch assay using Calcofluor fluorescence to characterize cell wall regeneration in plant protoplasts

A batch assay to study and measure the regeneration of cell walls during the early days of culture of primary protoplasts is presented. The assay involves the measurement of Calcofluor White fluorescence on a scanning fluorometer when the Calcofluor is adsorbed to the cellulosic component of the newly synthesized cell walls. The Calcofluor fluorescence, when standardized with microcrystalline cellulose, provided a measure of cell wall cellulose. The assay was used to study cell wall regeneration in Hyoscyamus muticus L. protoplasts during 8 days of culture.     

Ultrastructure of spores ofRhizopus nigricans after repeated freezing and thawing shocks

Disintegration of nuclear envelopes is the only ultrastructural change detectable by freezeetching in dormant spores of the mouldRhizopus nigricans, both dry and swollen, subjected to repeated freezing and thawing. The increase of the number of freeze-inactivated spores corresponds well with the increase of the number of damaged nuclei. This fact led us to formulate a hypothesis that the structure of the nucleus is the primary target of the freezing or thawing damage. As other biomembranes are not damaged it may be assumed that the disintegration of the nuclear membrane is probably secondary. No changes in ultrastructure of metabolically activated spores could be detected, in spite of the fact that the spores lost their germinative ability. Thus, the mechanism of the freeze injury may be different in dormant and growing spores.