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PubMed1983

Protoplasts of filamentous fungi in genetics and metabolite production.

Fungal protoplasts are interesting structures for genetic studies. They can be fused and their fusion products can revert to osmotic stable cells. The ability to fuse protoplasts of diverse origin makes them versatile tools in fundamental and applied genetics. Intraspecies protoplast fusion provides an efficient method to induce the parasexual cycle, making genetic analysis as well as strain breeding through mitotic recombination feasible in all kinds of species, regardless the occurrence of parasexuality by conventional means. Interspecies protoplast fusion allows the generation of different types of hybrids, depending on somatic and/or nuclear compatibility. Crosses between closely related species resulted in the formation of stable haploid recombinants as the consequence of genetic processes similar as occurring in the intraspecies parasexual cycle. From fusions between less related species hybrid progeny with differences in morphology and stability arose. Unstable hybrids segregated to other hybrid progeny or to one of the parental species, without recovery of the other parent. The exact genetic background of these hybrids remained obscure. Several studies demonstrated that the novel genetic combination arisen after interspecies protoplast fusion could result in changed gene expression and in the synthesis of novel or hybrid molecules.

Crosses, Genetic↗
PubMed1990

mRNAs newly synthesized by tobacco mesophyll protoplasts are wound-inducible.

We have used 2-dimensional (2D) non-equilibrium pH gradient gel electrophoresis (NEPHGE) of in vitro synthesized proteins and northern hybridization with labelled cDNAs coding for three pathogenesis related (P.R.) proteins, to analyze the shift in mRNA content induced by the isolation and culture of tobacco mesophyll protoplasts. The in vitro protein pattern of mRNAs from freshly isolated protoplasts is characterized by the absence of most leaf spots and the appearance of 19 new spots. After 6 hours of culture, the mRNAs coding for the P.R. proteins become detectable and after 12 hours the protoplasts contain an mRNA population almost typical of callus cells. The different steps involved in the isolation and culture of protoplasts were analysed. Cutting off the leaf and sterilization do not change the mRNA set. In contrast, the mechanical injury applied to the leaf in order to facilitate the penetration of the enzymatic mixture induces a modification of the mRNA content identical to that resulting from protoplast isolation. Wounding is the essential event inducing dedifferentiation. Varying the culture medium and conditions leads to only limited modifications of the mRNA pattern. These results are discussed on the basis of present knowledge of the reaction of the plant to wounding and we suggest that wound healing callus and in vitro callus correspond to the same differentiation state.

Cell Differentiation↗
PubMed1990

Linear DNA introduced into carrot protoplasts by electroporation undergoes ligation and recircularization.

The integrated DNA in stable transformants formed by direct gene transfer often shows complex restriction patterns. One cause of these complex restriction patterns could be the ligation of plasmid fragments prior to their integration. This paper provides evidence for the ligation of plasmid fragments by plant cells. Carrot protoplasts were electroporated in the presence of pCaMVCATM and assayed for chloramphenicol actyltransferase (CAT) activity 24 h later. Linear and supercoiled forms of pCaMVCATM supported similar levels of CAT expression. Surprisingly, digestion of the plasmid at a site between the CaMV 35S promoter and the CAT coding region reduced expression by only 40-50%. Electroporation carried out in the presence of isolated plasmid fragments suggested that this result was due to ligation of the linearized plasmid by the protoplasts. CAT expression was obtained with a mixture of isolated CaMV 35S promoter and the CAT coding region; neither fragment alone supported expression. Further evidence of ligation was provided by electroporation of protoplasts in the presence of a mixture of linearized pGEM and the 1.5-kb Hind III fragment of pCaMVCATM. DNA isolated from nuclei of the protoplasts was used to transform competent cells of Escherichia coli, and colonies were recovered that carried pGEM with Hind III-CaMVCAT inserts. Electroporation of protoplasts in the presence of linear and supercoiled pGEM and use of DNA isolated from nuclei to transform E. coli yielded an estimate of the frequency of plasmid ligation. A maximum of only 4% of the input linear DNA was recovered as circular molecules. This result suggests the frequency of ligation is low, but examination of the plasmid DNA in the plant nuclei by electrophoresis indicates extensive degradation of the plasmid and preferential loss of the circular forms. Thus, the ligated plasmids may be converted to the linear form and hence rendered unrecoverable by cloning into E. coli.

Chloramphenicol O-Acetyltransferase↗
PubMed1992

Maize polyubiquitin genes: structure, thermal perturbation of expression and transcript splicing, and promoter activity following transfer to protoplasts by electroporation.

Two genomic clones (lambda Ubi-1 and lambda Ubi-2) encoding the highly conserved 76 amino acid protein ubiquitin have been isolated from maize. Sequence analysis shows that both genes contain seven contiguous direct repeats of the protein coding region in a polyprotein conformation. The deduced amino acid sequence of all 14 repeats is identical and is the same as for other plant ubiquitins. The use of transcript-specific oligonucleotide probes shows that Ubi-1 and Ubi-2 are expressed constitutively at 25 degrees C but are inducible to higher levels at elevated temperatures in maize seedlings. Both genes contain an intron in the 5' untranslated region which is inefficiently processed following a brief, severe heat shock. The transcription start site of Ubi-1 has been determined and a transcriptional fusion of 0.9 kb of the 5' flanking region and the entire 5' untranslated sequence of Ubi-1 with the coding sequence of the gene encoding the reporter molecule chloramphenicol acetyl transferase (CAT) has been constructed (pUBI-CAT). CAT assays of extracts of protoplasts electroporated with this construct show that the ubiquitin gene fragment confers a high level of CAT expression in maize and other monocot protoplasts but not in protoplasts of the dicot tobacco. Expression from the Ubi-1 promoter of pUBI-CAT yields more than a 10-fold higher level of CAT activity in maize protoplasts than expression from the widely used cauliflower mosaic virus 35S promoter of a 35S-CAT construct. Conversely, in tobacco protoplasts CAT activity from transcription of pUBI-CAT is less than one tenth of the level from p35S-CAT.

Amino Acid Sequence↗
PubMed1995

Involvement of host cell energy in the transfection of Lactobacillus casei protoplasts with phage PL-1 DNA.

Transfection of Lactobacillus casei ATCC 27092 protoplasts with phage PL-1 DNA was studied under various conditions. The process of transfection was dependent on the incubation temperature, and the apparent activation energy was calculated to be about 11 kcal/mol. Transfection was inhibited by treating the cells before protoplasting either with monoiodoacetate, N,N'-dicyclohexylcarbodiimide (DCCD), or NaN3, without affecting both the viability of uninfected cells and protoplasting. The addition of DCCD after mixing protoplasts and DNA had no influence on transfection efficiencies. The transfection of L. casei protoplasts with phage PL-1 DNA was considered to require cell energy such as proton-motive force, probably in the initial stages, although the direct involvement of cell energy in the transfer of DNA across the cell membrane is still unclear.

Adenosine Triphosphate↗
PubMed1989

Optimization of protoplast formation, regeneration, and viability in Microsporum gypseum.

Factors affecting high yields, regeneration frequencies, and viability of protoplasts from clonal cultures of Microsporum gypseum were investigated. Maximum yields of protoplasts were obtained after 6 hrs digestion of 2-4 days old mycelium with Novozyme 234 using CaCl2 (0.4 M) as an osmotic stabilizer and glycine + HCl (pH 4.5) as the buffer system. Mercaptoethanol + dithiothreitol (0.01 M) proved to be the best pretreatment of mycelium prior to digestion with enzyme. A regeneration frequency of 94.4% was obtained using the top agar method with complete medium (pH 6.5) containing 0.5% agar and 0.4 M CaCl2 as an osmoticum. Colonies from regenerated protoplasts on medium containing CaCl2 were pigmented and completely powdery with high sporulation. Protoplast viability was studied in osmotic stabilizer supplemented with glucose or glutamine. After 24 hrs, glucose (2%) and glutamine (2%) enhanced protoplast viability by 22% and 23%, respectively. Protein synthesis, as measured by 3H-lysine uptake, matched the viability profile determined by fluorescence microscopy.

Buffers↗
PubMed1976

The infection of cucumber mesophyll protoplasts with tobacco mosaic virus.

Protoplasts from the first leaf mesophyll of cucumber plants were isolated by an 18 hours combined petinase/cellulase treatment. Conditions favouring the infection of these protoplasts with tobacco mosaic virus (TMV), and the accumulation of infective virus up to 96 hours after inoculation have been studied. Infection of approximately 5--10 per cent of the protoplats, revealed by indirect fluorescent antibody staining, was achieved by pre-treatment of the cells in 0.01 M citrate-buffered mannitol (CBM), pH 5.2 with 2 mug/ml poly-L-ornithine followed by centrifugation and direct resuspension of the cells in the same mixture together with 2 to 4 mug/ml TMV. Higher concentrations of the polycation and buffer were toxic to the protoplasts. Under the best conditions, virus yields of approximately 10-20 mug TMV/10(6) protoplasts were attained, while after 72 hours incubation, significant amounts of virus could often be recovered from the incubation medium. Addition of actinomycin D to cultures of protoplasts 2 hours post-inoculation partially inhibited development of infectivity.

Antigens, Viral↗
PubMed1991

Cytoplasmic calcium levels in protoplasts from the cap and elongation zone of maize roots.

Calcium has been implicated as a key component in the signal transduction process of root gravitropism. We measured cytoplasmic free calcium in protoplasts isolated from the elongation zone and cap of primary roots of light-grown, vertically oriented seedlings of Zea mays L. Protoplasts were loaded with the penta-potassium salts of fura-2 and indo-1 by incubation in acidic solutions of these calcium indicators. Loading increased with decreasing pH but the pH dependence was stronger for indo-1 than for fura-2. In the case of fura-2, loading was enhanced only at the lowest pH (4.5) tested. Dyes loaded in this manner were distributed predominantly in the cytoplasm as indicated by fluorescence patterns. As an alternative method of loading, protoplasts were incubated with the acetoxymethylesters of fura-2 and indo-1. Protoplasts loaded by this method exhibited fluorescence both in the cytoplasm and in association with various organelles. Cytoplasmic calcium levels measured using spectrofluorometry, were found to be 160 +/- 40 nM and 257 +/- 27 nM, respectively, in populations of protoplasts from the root cap and elongation zone. Cytoplasmic free calcium did not increase upon addition of calcium to the incubation medium, indicating that the passive permeability to calcium was low.

Calcium↗
PubMed2000

Osmotically evoked shrinking of guard-cell protoplasts causes vesicular retrieval of plasma membrane into the cytoplasm.

The dye FM1-43 was used alone or in combination with measurements of the membrane capacitance (Cm) to monitor membrane changes in protoplasts from Vicia faba L. guard cells. Confocal images of protoplasts incubated with FM1-43 (10 microM) at constant ambient osmotic pressure (pi omicron) revealed in confocal images a slow internalisation of FM1-43-labelled membrane into the cytoplasm. As a result of this process the relative fluorescence intensity of the cell interior (fFM,i) increased with reference to the total fluorescence (fFM,t) by 7.4 x 10(-4) min(-1). This steady internalisation of dye suggests the occurrence of constitutive endocytosis under constant osmotic pressure. Steady internalisation of FM1-43 labelled membrane caused a prominent staining of a ring-like structure located beneath the plasma membrane. Abrupt elevation of pi omicron by 200 mosmol kg(-1) caused, over the first minutes of incubation, a rapid internalisation of FM1-43 fluorescence into the cytoplasm concomitant with a decrease in cell perimeter. Within the first 5 min the cell perimeter decreased by 7.9%. Over the same time fFM,i/fFM,t increased by 0.13, reflecting internalisation of fluorescent label into the cytoplasm. Combined measurements of Cm and total fluorescence of a protoplast (fFM,p) showed that an increase in pi omicron evoked a decrease in Cm but no change in fFM,p. This means that surface contraction of the protoplast is due to retrieval of excess membrane from the plasma membrane and internalisation into the cytoplasm. Further inspection of confocal images revealed that protoplast shrinking was only occasionally associated with internalisation of giant vesicles (median diameter 2.7 microm) with FM 1-43-labelled membrane. But, in all cases, osmotic contraction was correlated with a diffuse distribution of FM1-43 label throughout the cytoplasm. From this, we conclude that endocytosis of small vesicles into the cytoplasm is the obligatory process by which cells accommodate an osmotically driven decrease in membrane surface area.

Cell Membrane↗
PubMed2004

A new tool for plant cell biology: in vivo antibody uptake in plant protoplasts.

We report on the in vivo uptake of antibodies into plant protoplasts. When protoplasts of sunflower, Arabidopsis or tobacco were incubated in vivo with an antibody, this antibody was detected by immunofluorescence in the cytoplasm and/or the nucleus, depending on the location of the target protein. Furthermore, when protoplasts were cultured in the presence of antibodies, specific effects were observed. Incubation with antibodies raised against p34cdc2 led to a strong inhibition of the division rate, and a decrease in the average DNA content of protoplasts. With antibodies against HaWLIM1, a LIM domain protein of the CRP type, a negative effect on actin organisation was observed. We conclude that antibodies can penetrate plant protoplasts in vivo, and thus may be used as powerful tools for the study of protein function.

Animals↗
PubMed2004

Flow cytometric analysis of protein content in Taxus protoplasts and single cells as compared to aggregated suspension cultures.

Plant suspension cultures are highly aggregated, preventing the direct application of flow cytometry for the study of population dynamics. The utility of single cells to accurately represent aggregated suspension cultures was tested through the analysis of total protein content. Specifically, protein content of two Taxus cuspidata suspension culture lines was studied using the Bradford assay for aggregated suspension cultures, and flow cytometry with fluorescein isothiocyanate staining for protoplasts and single cells. Taxus protein levels were measured at 75-160 mg per gram dry weight via the Bradford assay. Aggregated suspension cultures, protoplasts, and single cells predicted the same trend of protein content over the culture period (21 days). Normalized protein content of isolated single cells was statistically equivalent to aggregated suspensions for both cell lines. However, normalized protein content of isolated protoplasts showed significant differences from aggregated suspensions for one of the two cell lines. Elicitation with methyl jasmonate (MJ) is commonly utilized to increase paclitaxel accumulation in suspension cultures, and therefore the effect of MJ elicitation on protein content in aggregated suspensions, isolated single cells and protoplasts was assessed. Aggregated suspension cultures, protoplasts, and single cells did not show any change in total protein content following elicitation with MJ at 200 microM on day 7. This study illustrates the usefulness of flow cytometry for obtaining culture population information and the value of using intact single cells for the study of plant metabolism.

Cell Aggregation↗
PubMed2006

Function of mitochondria during the transition of barley protoplasts from low light to high light.

Mitochondrial contribution to photosynthetic metabolism during the transition from low light (25-100 micromol quanta m(-2) s(-1), limiting photosynthesis) to high light (500 micromol quanta m(-2) s(-1), saturating photosynthesis) was investigated in protoplasts from barley (Hordeum vulgare) leaves. After the light shift, photosynthetic oxygen evolution rate increased rapidly during the first 30-40 s and then declined up to 60-70 s after which the rate increased to a new steady-state after 80-110 s. Rapid fractionation of protoplasts was used to follow changes in sub-cellular distribution of key metabolites during the light shift and the activation state of chloroplastic NADP-dependent malate dehydrogenase (EC 1.1.1.82) was measured. Although oligomycin (an inhibitor of the mitochondrial ATP synthase) affected the metabolite content of protoplasts following the light shift, the first oxygen burst was not affected. However, the transition to the new steady-state was delayed. Rotenone (an inhibitor of mitochondrial complex I) had similar, but less pronounced effect as oligomycin. From the analysis of metabolite content and sub-cellular distribution we suggest that the decrease in oxygen evolution following the first oxygen burst is due to phosphate limitation in the chloroplast stroma. For the recovery the control protoplasts can utilize ATP supplied by mitochondrial oxidative phosphorylation to quickly overcome the limitation in stromal phosphate and to increase the content of Calvin cycle metabolites. The oligomycin-treated protoplasts were deficient in cytosolic ATP and thereby unable to support Calvin cycle operation. This resulted in a delayed capacity to adjust to a sudden increase in light intensity.

Adenosine Diphosphate↗
PubMed2005

Extra thin alginate film: an efficient technique for protoplast culture.

This paper reports an efficient protoplast culture technique, the "extra thin alginate film" technique. The development of this improved method of protoplast culture was an outcome of an assessment of the efficiency and shortcomings of various protoplast culture techniques. The efficiency of this technique was evaluated with two model plant systems, viz., Nicotiana tabacum and Lotus corniculatus, and a comparison was made with the "thin alginate layer" technique, another efficient protoplast culture system. Results indicate that the culture technique with extra thin alginate film is as efficient as the technique with thin alginate layer, with many additional advantages. The present innovation overcomes most of the limitations of protoplast culture techniques described so far and can now be applied to a wide variety of crops to check its general applicability.

Alginates↗
PubMed2003

A rapid and reliable flow cytometric method for determining Hsp70 levels in tobacco protoplasts.

Current methods to determine heat shock protein (Hsp) synthesis or accumulation in plant cells, such as Western blotting and biometabolic labelling are either indirectly quantitative, labour-intensive or biohazardous. An optimal flow cytometric protocol was developed to measure the intracellular Hsp70/Hsc70 levels in tobacco protoplasts. After heat treatments, protoplasts were fixed in 2% paraformaldehyde-phosphate-buffered saline and dehydrated overnight in methyl cellusolve, followed by permeabilization with Triton X-100 (0.1% in Protoplast Wash Fluid). Immunolabelling of Hsp70/Hsc70 was done for 1 hour with a mouse monoclonal antibody and detected by R-Phycoerythrin-conjugated goat anti-mouse IgG using flow cytometry. Flow cytometry detected a significant 1.2-fold increase in Hsp70/Hsc70 accumulation (P < 0.001) in protoplasts, while Western blotting, quantified by image analysis, showed induction under similar conditions but at lower significance (P < 0.05). The coefficients of variance for flow cytometry and Western blotting were 30.7 and 49.8 respectively. Optimum temperature of heat-induced Hsp70/Hsc70 accumulation in tobacco protoplasts occurred at 40 degrees C. Flow cytometry is proposed as a quantitative, more reproducible and rapid alternative to Western blotting for the detection of Hsp70 accumulation in plant cells.

Blotting, Western↗
PubMed1983

The effect of sucrose on the rate of de novo sucrose biosynthesis in leaf protoplasts from spinach, wheat and barley.

Protoplasts from the leaves of wheat, spinach, and barley were found to synthesize [14C]sucrose from 14CO2 at rates comparable with those of the parent tissue. CO2 fixation and sucrose biosynthesis ceased virtually immediately when the light was switched off. The effect of sucrose pretreatment on the rate of de novo sucrose biosynthesis was found to vary with leaf age and with plant species. Protoplasts from young wheat and spinach leaves showed an apparent stimulation of the rate of sucrose biosynthesis after sucrose pretreatment. In protoplasts from mature leaves of spinach, sucrose pretreatment produced inhibition. After sucrose pretreatment protoplasts from mature spinach leaves showed low rates of CO2 fixation, and sucrose biosynthesis compared with controls. Conversely, with protoplasts from mature leaves of wheat and barley, the rate of CO2 fixation was unchanged and there was little or no effect on the rate of sucrose biosynthesis after sucrose pretreatment. Preincubation with sucrose had no effect on the activity of sucrose-phosphate synthetase (EC 2.4.1.14), cytoplasmic fructose-1,6-bisphosphatase (EC 3.1.3.11), or UDPglucose pyrophosphorylase (EC 2.7.7.9) from spinach leaves. It was concluded that there is no direct feedback inhibition of sucrose on the sucrose biosynthetic pathway in leaves of spinach, wheat, and barley. The mechanism of inhibition of sucrose biosynthesis by sucrose in spinach remains to be elucidated.

Carbon Dioxide↗
PubMed1980

Immobilisation and mechanical, support of individual protoplasts.

Mesophyl cell protoplasts of Vicia faba were suspended in a solution consisting of 10% sodium alginate and 0.4 M mannitol. The protoplasts could be immobilized by cross-linking the alginate in the presence of 100 mM CaCl2. Changes in the osmolarity of the external medium led to reversible shrinkage and swelling of the entrapped protoplasts. It was demonstrated by using the pressure probe technique that a pressure gradient (cell turgor pressure) of several 100 mbar is built up when the immobilized cells were transferred to hypotonic solution. By complexing the Ca2+ in the alginate matrix with sodium citrate buffer the protoplasts could be released from the matrix. No morphological change or alteration of the membrane permeability of the immobilized protoplasts was observed after a storage period of up to 14 days at 4 degrees C in the matrix.

Alginates↗
PubMed1981

Fusion of Avena sativa mesophyll cell protoplasts by electrical breakdown.

Studies with the light microscope were carried out on mesophyll cell protoplasts of Avena sativa which had been made to undergo fusion by reversible electrical breakdown of the cell membrane. In order to establish close membrane contact between the cells, an important prerequisite for fusion, a method known as dielectrophoresis was used. In an inhomogeneous alternating electrical field the protoplasts adhere to the electrodes and to each other in the direction of the field lines. The cells which were thus brought into close contact with each other could be made to fuse by the application of a field pulse of high amplitude (about 750 V/cm) and short duration (20-50 mus). The field strength required for fusion exceeds the value necessary for the electrical breakdown of the cell membrane. Fusion took place within some minutes and led to a high yield of fused protoplasts. The fusion of cells being in the electric field occurred in a synchronous manner. In some of the fusion experiments part of the protoplasts of A. sativa were stained with neutral red. When these cells were fused with unstained protoplasts, the vacuoles from the different cells within the fused aggregate could be shown to remain separate for quite some time.

Cell Fusion↗
PubMed1975

Biosynthesis of acid phosphatase of baker's yeast. Characterization of a protoplast-bound fraction containing precursors of the exo-enzyme.

1. Yeast protoplasts, secreting acid phosphatase (orthophosphoric-monoester phosphohydrolase (acid optimum) EC 3.1.3.2) contain a small amount of firmly bound enzyme, even after lysis (Van Rijn, H.J.M., Boer, P. and Steyn-Parvé, E.P. (1972) Biochim. Biophys. Acta 268, 431-441). The major part (70%) of this protoplast-bound acid phosphatase can be solubilized by nonionic detergents, such as Triton X-100. 2. The kinetics of radioactive amino acid incorporation in the solubilized and in the secreted enzyme has been estimated by pulse-chase labelling of secreting protoplasts, followed by fractionation and counting radioactivity in the enzyme band in polyacrylamide gels after electrophoresis at pH 5.0. A precursor-product relationship between the Triton X-100-extractable fraction of the protoplast-bound acid phosphatase and the secreted enzyme is apparent. 3. The solubilized acid phosphatase is essentially indistinguishable from the secreted enzyme with regard to a number of enzymatic properties and its stability towards pH and temperature. Both enzymes also behave alike on polyacrylamide-gel electrophoresis, producing a single acid phosphatase band with glycoprotein character and comparable mobility. 4. A striking difference is seen in isopycnic equilibrium sedimentation in CsCl: the secreted acid phosphatase is homogeneous, with a buoyant density of p equals 1.47 g/cm3, while the Triton X-100-extractable part of the protoplast-bound acid phosphatase is heterogeneous; besides heavier material a major component with buoyant density of p equals 1.37 g/cm3 is always visible.

Acid Phosphatase↗