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At least 685 records · Page 38Linked to original sources
PubMed1990

Inoculation of sugar beet protoplasts with beet necrotic yellow vein virus particles by mild sonication.

A novel procedure employing mild sonication for in vitro inoculation of plant protoplasts with virus particles has been established. Sugar beet (Beta vulgaris L.) protoplasts were briefly exposed to 20 kHz ultrasound in the presence of beet necrotic yellow vein virus (BNYVV) particles. The accumulation of BNYVV coat protein was analyzed by an enzyme-linked immunosorbent assay. The infection was detectable after 16 h and reached maximum 3-3 1/2 days after inoculation. Maximum levels of BNYVV coat protein in inoculated protoplasts were obtained by sonication for 500-600 ms at 45-60 W. This reduced the viability to 15-30%. The efficiency of the inoculation increased with the concentration of BNYVV particles up to 28 micrograms/ml. Infection was found to be optimal when the BNYVV particles were added just before sonication but low levels of infection could be obtained by addition of virus particles up to 60 min after sonication.

Capsid↗
PubMed1996

Infection and replication of a planthopper transmitted virus-rice stripe virus in rice protoplasts.

Rice stripe virus (RSV), a planthopper-transmitted virus, was inoculated into rice protoplasts, and a one-step growth curve was determined. The amount of virus in the protoplasts decreased following the inoculation, and then increased after 8 h. The replication of RSV reached its peak 20 h after inoculation. RSV replication in the inoculated protoplasts was further demonstrated by electron microscopy.

Animals↗
PubMed1990

Loading of quin2 into the oat protoplast and measurement of cytosolic calcium ion concentration changes by phytochrome action.

The loading of quin2 into oat protoplasts was carried out in an incubation medium (0.6 M sorbitol, 1 mM CaCl2, 5 mM Mes, 5 mM Tris, 0.05% BSA, 1 mM KCl, 1 mM MgSO4 (pH 6.8)), in which we found the best viability of the protoplast and the highest membrane permeability of quin2/AM, compared with the results obtained from any other incubation medium we had tried to use. 50 microns of quin2/AM was added in the suspension medium containing 5 x 10(5)/ml of oat protoplasts, and incubation at 4 degrees C was performed for 24 h. From atomic absorption data, we confirmed that quin2 loading was 1.78 mmol per liter of cells. Red-light (660 nm) irradiation for 5 min caused an increase of the cytosolic Ca2+ concentration from 30 to 193 nM. On the other hand, a subsequent irradiation with far-red light (730 nm) for 5 min decreased it by about 48 nM. Even when the extracellular Ca2+ was completely chelated with 1 mM EDTA, red light increased the cytosolic Ca2+ concentration by about 51 nM and far-red light decreased it to 3 nM. These results imply that the Pfr form of phytochrome functions not only in the process of influx of Ca2+, but also in the mobilization process of Ca2+ from the intracellular Ca2+ pools. The fact that the Pr form of phytochrome lowers the cytosolic Ca2+ concentration is also presented in this report.

Aminoquinolines↗
PubMed1994

Clinostation influence on regeneration of cell wall in Solanum tuberosum L. protoplasts.

Regeneration of cell walls in protoplasts was investigated using light- and electronmicroscopic methods. The protoplasts were isolated from mesophyll of Solanum tuberosum leaves and were cultivated on the horizontal low rotating clinostat (2 rpm) and in control for 10 days. Using a fluorescent method (with Calcofluor white) it was demonstrated that changes in vector gravity results in a regeneration inhibition of cell wall. With electron-microscopical and electro-cytochemical methods (staining with alcianum blue) dynamics of the regeneration of cell walls in protoplasts was studied; carbohydrate matrix of cell walls is deposited at the earliest stages of this process. The influence of microgravity on the cell wall regeneration is discussed in higher plants.

Cell Wall↗
PubMed1994

Development of plant protoplasts during the IML-1 mission.

During the 8 day IML-1 mission, regeneration of cell walls and cell divisions in rapeseed protoplasts were studied using the Biorack microscope onboard the Space Shuttle "Discovery". Samples from microgravity and 1g protoplast cultures were loaded on microscope slides. Visual microscopic observations were reported by the payload specialist Roberta Bondar, by down-link video transmission and by use of a microscope camera. Protoplasts grown under microgravity conditions do regenerate cell walls but to a lesser extent than under 1g. Cell divisions are delayed under microgravity. Few cell aggregates with maximum 4-6 cells per aggregate are formed under microgravity conditions, indicating that microgravity may have a profound influence on plant cell differentiation.

Brassica↗
PubMedDate not supplied

Use of a transient expression assay for the optimization of direct gene transfer into tobacco mesophyll protoplasts by electroporation.

Plasmid DNA was transfected into tobacco mesophyll protoplasts by electroporation. Transfection efficiency was estimated, using a transient expression assay based on the measurement of chloramphenicol transacetylase activity or by scoring colonies expressing resistance to paromomycin, an aminoglycoside related to kanamycin. Under conditions of cell survival superior to 50% after electroporation, transient expression signals and transformation efficiencies were found to be proportional. Factors affecting the efficiency of transformation were studied. A clear-cut optimum voltage (250-300 V/cm) was detected. Among various salts tested, potassium chloride was the best electrolyte. No improvement of electroporation efficiency was obtained by a heat-shock (45 degrees C/5 min) treatment prior to electroporation or by the presence of polyethylene glycol in the electroporation medium. The physiological state of plants used as the protoplast source significantly affected the transfection ability of the resulting protoplasts. These results are discussed and compared to previously published procedures.

Acetyltransferases↗
PubMed1995

Lentil root protoplasts: a transient expression system suitable for coelectroporation of monoclonal antibodies and plasmid molecules.

Protoplasts were isolated from lentil (Lens culinaris) roots and their suitability as a transient expression system was investigated. After transfecting the protoplasts with the beta-glucuronidase (GUS) gene by either electroporation or polyethylene glycol (PEG), the specific activity of the reporter enzyme and the cell viability were determined. Electroporation was more effective than PEG treatment as transfection procedure and its efficiency was affected by the plasmid length. The feasibility of electro-transferring at the same time (coelectroporation) inhibitory anti-lipoxygenase monoclonal antibodies and the GUS-carrying plasmid pBI 221 was investigated as well. The amount of transferred immunoglobulins was quantitated by ELISA and the inhibitory ability of monoclonal antibodies on the intracellular target enzyme was determined. Evidence is presented for the successful coelectroporation of immunoglobulins and plasmid DNA into lentil protoplasts, the two types of macromolecules acting independently of each other in the recipient cells.

Antibodies, Monoclonal↗
PubMed1992

Multiple transformation of Saccharomyces cerevisiae by protoplast fusion.

A technique for the multiple transformation of yeast by protoplast fusion is described. This involved the PEG-induced fusion of protoplasts from cells which had been treated with chromosome-fragmenting agents (in this case cupferron and hydroxylamine) with protoplasts of triply auxotrophic cells. The recovery of transformants was increased significantly if one of the amino acid requirements of the recipient strain was included in the selection medium. Transformants isolated on supplemented media remained auxotrophic for that requirement. Prototrophic, uninucleate transformants had a DNA content and cellular volume similar to that of the parental strains. Possible mechanisms of gene transfer are discussed. This technique offers the possibility of transferring desirable characteristics from one yeast strain to another without altering the ploidy level of the recipient strain.

Hydroxylamine↗
PubMed1992

Characterization of genes expressed in mesophyll protoplasts of Nicotiana sylvestris before the re-initiation of the DNA replicational activity.

To decipher the early events preceding the re-entry of somatic cells into the cell cycle, we constructed a cDNA library from 6-h-old protoplasts of Nicotiana sylvestris. We characterized three mRNAs, via their cDNAs, that accumulate at very high levels 6 h after the beginning of the culture. Two of them could be identified by comparison of the deduced amino acid sequence to databanks. 6P10 is a novel type I trypsin inhibitor, which has the peculiarity of being devoid of the pro-sequence peptide described to be essential for transport to the vacuole. 6P73 is a novel, moderately anionic peroxidase. 6P50 belongs to a gene family not yet identified. These genes are highly expressed in protoplasts at the beginning of the culture and moderately in roots, but are neither expressed in response to chemical treatment, heat shock, pathogen attacks nor during tumor induction. These findings suggest that the activation of these genes corresponds not only to a specific adaptation of protoplasts to the new environment but also, since their level of expression decreases at the onset of division, to a sequence of events connected with the establishment of the new program of gene expression of the dividing cell.

Agrobacterium tumefaciens↗
PubMed2006

Automatic positioning of a microinjector in mouse ES cells and rice protoplasts.

A 2-channel (2C) microinjector was prepared by pulling a glass capillary with a theta-shaped cross section. One channel was used as a potential measuring electrode (MeaE) and the other was used as an electrophoretic introduction electrode (IntE). The 2C microinjector was propelled by an oil pressure manipulator driven by a pulse motor, while the MeaE output was recorded continuously. When the 2C microinjector penetrated the cell membrane of a mouse ES cell or a rice protoplast, the output potential changed sharply. The differential of this potential change was used as a stop signal for the pulse motor. Thus, the microinjector was correctly positioned in the cell without losing cell viability. Its success rate was 73% and 84% for ES cells and rice protoplasts, respectively. After the positioning of the microinjector in the cell, Lucifer yellow (LY) was introduced via IntE. Under these conditions, the rate of viable cells was 16% and 62% for ES cells and rice protoplasts, respectively.

Animals↗
PubMed2005

Biotin-labeled abscisic acid as a probe for investigating abscisic acid binding sites on plasma membranes of barley aleurone protoplasts.

Plant hormone abscisic acid (ABA) plays important roles in dormancy and stress responses, but its binding sites have not yet been fully elucidated. In this report, we suggest the utility of biotin-labeled abscisic acid (bioABA) as a probe to investigate ABA-binding sites on the plasma membrane of barley aleurone protoplasts. BioABA was approximately 100 times less effective than ABA in inhibiting expression of gibberellin-inducible alpha-amylase and in inducing expression of a reporter gene fused to the dehydrin promoter. To ascertain that bioABA could bind to ABA-binding sites on the plasma membrane, we used fluorescence flow cytometry to measure the fluorescence intensity of aleurone protoplasts treated with a combination of bioABA and fluorescence-labeled streptavidin. Addition of bioABA increased the fluorescence of aleurone protoplasts in a concentration-dependent manner, but addition of non-active bioABA derivatives did not. Furthermore, the increase in fluorescence intensity observed upon addition of bioABA was eliminated by co-treatment with excess ABA, but it was not eliminated by co-treatment with other plant hormones. These results suggest that bioABA binds to ABA-binding sites, and that bioABA should be a valuable probe for investigating ABA-binding sites on the plasma membrane.

Abscisic Acid↗
PubMed2005

The nucleocytoplasmic microfilament network in protoplasts from cultured soybean cells is a plastic entity that pervades the cytoplasm except the central vacuole.

The microfilament network of cultured Glycine max cells (SB-1 line), and protoplasts was visualized with rhodamine-phalloidin under conditions that lysed the protoplast and changed the cell shape. The whole cell had the typical microfilament distribution of a "cage" around the nucleus, from which the large subcortical cables and transvacuolar strands radiated towards the cortex until it reached the cortical microfilament network. Upon cell wall removal, the network conserved its compartmentalization. Thus, the redistribution of the shape where the vacuole becomes a central entity, made the cytoplasm displace peripherally, but the network distribution was conserved. When protoplasts were lysed in a low osmotic medium, the vacuoles were gradually released intact. Under these conditions, the F-actin staining remained within the ghost of the cell, but none was detected in either emerging or almost completely released vacuoles. Most of the released F-actin was found in debris from the cell lysate in the form of microfilaments. When the ghosts were constrained in a coverslip with an air bubble, the shape of the ghost changed accordingly, but the microfilament network distribution remained constant. These results provide further evidence that the vacuole of plant cells does not have detectable associated F-actin. In addition, we demonstrate that the actin microfilament network is a moldable entity that can change its shape but keeps its distribution under constant conditions, in these cultured cells.

Actin Cytoskeleton↗
PubMed2004

Strong expression of the rice catalase gene CatB promoter in protoplasts and roots of both a monocot and dicots.

The rice (Oryza sativa L.) catalase (EC 1.11.1.6) gene CatB is expressed in roots and cultured cells. We examined the promoter activity of its 5'-flanking region in a monocot and in two dicots. Transient expression assays in rice Oc and tobacco BY-2 suspension cell protoplasts showed that CatB's 5'-flanking DNA fragments (nucleotides -1066 to +298) had about 20 and 3-4 times as much promoter activity, respectively, as the CaMV 35S promoter. Serial deletion analyses of the CatB promoter region revealed that the shortest fragment (-56 to +298) still had about 10 times as much promoter activity as the CaMV 35S promoter in rice protoplasts. In tobacco protoplasts, the activity of the fragment (-56 to +298) was about half of the CaMV 35S promoter. Transgenic rice and Arabidopsis plants carrying GUS genes driven by the 5'-truncated CatB promoters were generated and their GUS activity was examined. The region ranging from -329 to +298 showed preferential expression in the roots of rice and Arabidopsis, and in the shoot apical meristems of Arabidopsis. In situ hybridization revealed that CatB was highly expressed in branch root primordia and root apices of rice. Fusion of the GUS gene to the region (-329 to +298) conferred strong expression in these same areas, indicating that the presence of this region was sufficient to express CatB specifically in the roots. There may be new regulatory element(s) in this region, because it contained no previously known cis-regulatory elements specific for gene expression in roots.

Arabidopsis↗
PubMed1997

The correlation between adhesion of schizophyllan to yeast glucan and its effect on regeneration of yeast protoplast.

Schizophyllan, a water-soluble (1-->3)-beta-D-glucan with a triple-helical conformation, adheres to yeast glucan and curdlan gel. As the molecular weight of schizophyllan decreases, both its adhesion to the water-insoluble glucans and its ability to promote the regeneration of yeast protoplasts are reduced. Therefore, we hypothesize that schizophyllan can surround yeast protoplasts by adhering to a fragment of yeast glucan remaining or/and resynthesized on the cell surface and that this encapsulation allows regeneration of the protoplast cells to occur at very high frequency.

Carbohydrate Conformation↗
PubMed1997

Studies on ion channel antagonist-binding sites in sunflower protoplasts.

The cytological location of ion channel antagonist-binding sites was studied in sunflower protoplasts using the fluorescent probes DM-Bodipy-PAA and DM-Bodipy-DHP. The binding specificity of the probes was established by competition experiments with Bepridil, phenylalkylamine (Verapamil) and dihydropyridine (Nifedipine) which are known as calcium and potassium channel antagonists. Quantitative image analysis of the fluorescence emitted by the protoplasts showed the existence of interactions between PAA- and DHP-binding sites. Moreover, studies on the cytolocalization of the PAA receptors by confocal imaging showed that in freshly isolated protoplasts, DM-Bodipy-PAA binds exclusively at sites located in the cortical region of the cell.

Binding Sites↗
PubMed2003

A specific cis-hairpin ribozyme facilitates infection of a TMV-based DNA vector in tobacco protoplasts.

The effect of a specific cis-hairpin ribozyme on TMV-based vectors in the infection of tobacco protoplasts was studied. Vectors contained full-length TMV genome cDNA linked to a T7 promoter or a CaMV 35S promoter at the 5'-end and an NOS gene polyadenylation signal at the 3'-end. The coat protein (CP) gene was replaced with the green fluorescent protein (GFPuv) gene allowing quantification of protoplast infection. In plasmids pTMVGFPRIB (T7-driven) and pSTMVGFPRIB (CaMV 35S-driven), the cDNA fragment of the cis-hairpin ribozyme (designed to specifically cleave the transcripts immediately downstream of the 3'-terminus of TMV RNA) was inserted between the 3'-terminus of TMV genome and NOS sequence. The in vitro transcript TMVGFPRIB was three- to fivefold more infectious than the control TMVGFPNOS. Northern blot analysis indicated that the 3'-terminal non-viral sequence had been cleaved from the in vitro transcripts by the cis-hairpin ribozyme soon after in vitro transcription. pSTMVGFPRIB and pSTMVGFPNOS plasmid DNAs were, as expected, less infectious than their in vitro transcript counterparts. However, pSTMVGFPRIB was somewhat more infectious than pSTMVGFPNOS. Northern blot analysis indicated that pSTMVGFPRIB synthesized more genomic and sub-genomic RNAs in the protoplasts. The significant increase in infectivity and viral RNA synthesis is due to the specific activity of the cis-hairpin ribozyme in vivo. Therefore, the cis-hairpin ribozyme described here may improve TMV-based vectors in the expression of foreign protein in plants.

Base Sequence↗
PubMed1990

External electric fields stimulate the electrogenic calcium/sodium exchange in plant protoplasts.

External electric fields of low intensity stimulated calcium influx in protoplasts isolated from carrot cell suspension cultures in field intensity dependent and frequency-dependent ways. The field-induced calcium uptake involved a temperature-dependent system that was saturable by external calcium. The induction process appeared mainly cumulative as long as the morphology of the protoplasts did not change (up to 10 min). The stimulation elicited by the electric fields was effective even after switching the field off; the influx increased for 5 min and then slowed down to its initial value 15 min later. During electrostimulation, an additional amount of ATP was accumulated; on removal of the stimulatory field, the extra amount of ATP was consumed, whereas the plasma membrane was hyperpolarized and sodium ions were expelled from the protoplasts. Inhibition of either ATP accumulation or consumption results in the inhibition of both calcium influx and sodium efflux, demonstrating that these processes are coupled. From the data obtained in this work, it may be concluded that the electric field stimulates an ATP synthase like activity; the consumption of the ATP thus formed elicits an electric potential (probably due to the efflux of cations and more specifically sodium) that drives the influx of calcium.

Adenosine Triphosphate↗
PubMed1998

The effect of external Ca2+ and Ca(2+)-channel modulators on red-light-induced swelling of protoplasts of Phaseolus radiatus L.

Red-light-induced swelling of the protoplasts isolated from hypocotyl of etiolated mung bean (Phaseolus radiatus L.) was observed only when Ca2+ ions were present in the medium. The optimal CaCl2 concentration was 250 microM. Swelling response declined when Ca2+ was supplied into the medium after red light irradiation. The Ca(2+)-chelator EGTA eliminated the red-light-induced swelling and 45Ca2+ accumulation in the protoplasts. In contrast, A23187, a Ca(2+)-ionophore, could mimic the effect of red light in darkness. These results indicate that Ca2+ may play a role in light signal transduction. In addition, swelling response was prevented by TFP and CPZ (both are CaM antagonists), implying the involvement of CaM in red-light-induced and Ca(2+)-dependent protoplast swelling.

Calcimycin↗