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PubMed1983

An ultrastructural study of the interaction of liposomes with plant protoplasts.

A one-step procedure using a mixture of glutaraldehyde and osmium tetroxide was devised to fix in situ large unilamellar liposomes of phosphatidylserine for transmission electron microscopy (TEM), since the conventional fixation method was found to be inadequate in this respect. The new fixation procedure enabled us to visualize the sequence of events in the interaction of liposomes with protoplasts from Vinca rosea suspension cultures in the presence of polyethylene glycol. Liposomes were thus found adhering to the surface of protoplasts, in association with invaginating plasmalemma, and within intracellular vesicles. These observations showed that liposomes enter plant protoplasts via endocytosis. Ultrastructural profiles indicating fusion of liposomes with protoplasts were not observed.

Cell Membrane↗

PubMed1991

High-yield isolation of protoplasts from microgram amounts of shoot meristematic tissues and rapid DNA content determination by flow cytometry.

This paper describes a novel approach to rapid cell-cycle analysis of shoot meristematic cells. The method involves fixation and disaggregation of meristems into protoplast suspension and flow-cytometric analysis of these protoplasts stained with fluorescent dyes. We have developed a procedure for a high-yield isolation of protoplasts allowing an accurate flow-cytometric analysis with a few micrograms of meristem tissues. We present here determinations of total DNA content of protoplasts stained with propidium iodide in the dicotyledon Sinapis alba, and the monocotyledon Lolium temulentum.

Cell Cycle↗

PubMed1984

Replication of peanut stunt virus and its associated RNA 5 in cowpea protoplasts.

Peanut stunt virus (PSV) RNA containing PSV-associated RNA 5 (PARNA 5) was used as the inoculum in the successful infection of cowpea protoplasts. Total nucleic acid extracts of protoplast samples at different times after inoculation were analyzed for the presence of PSV genomic RNAs and PARNA 5 using glyoxal denaturation, agarose gel electrophoresis, blotting to nitrocellulose, and hybridization to specific probes. It appears that (+)-stranded PSV genomic RNAs are synthesized up to 36 hr after inoculation after which their synthesis levels off, whereas PARNA 5 synthesis continues much later during infection. Oligomers of PARNA 5 were found in the infected protoplasts, in double-stranded RNA preparations from PSV-infected tissues, and in single-stranded PSV-RNA preparations isolated from purified virus. However, we were unable to demonstrate the presence of circular PARNA 5 molecules in infected protoplasts or tissues. These results leave open the question whether PARNA 5 is replicated via a rolling circle type replication mechanism, as proposed for viroids and tobacco ringspot virus satellite, or via a virus-like replication mechanism, as certain structural features of PARNA 5 would indicate. It is not impossible that both types of mechanisms are operative at different phases of PARNA 5 replication.

Arachis↗

PubMed1988

Subcellular localization of tobacco mosaic virus minus strand RNA in infected protoplasts.

Radioactive RNA probes were prepared which specifically hybridize with sequences complementary to 5' and 3' regions of tobacco mosaic virus (TMV) RNA. These probes were used in Northern hybridization to locate TMV-RNA minus strands in the subcellular fractions of infected tobacco protoplasts. When the protoplasts were lysed with Triton X-100, full-length minus strands were present in the cytoplasmic but not in the nuclear fraction. With mechanically broken protoplasts, the crude nuclear fraction (250 g pellet) contained small amount of minus strands which appeared to derive from unbroken protoplasts, but most of minus strands were recovered in a fraction sedimented between 250 and 2500 g, little if any being found in lighter fractions. The results indicate that TMV-RNA replicates in association with an extranuclear structure.

Blotting, Northern↗

PubMed1990

Defective-interfering RNAs and elevated temperatures inhibit replication of tomato bushy stunt virus in inoculated protoplasts.

Tomato bushy stunt virus (TBSV) genomic RNA and one of its defective interfering (DI) RNAs were inoculated in various combinations to protoplasts of Nicotiana benthamiana. Ethidium bromide staining of electrophoretically separated RNAs from infected protoplasts, incorporation of [3H]uridine into TBSV and DI RNAs, and Northern hybridization at different times after inoculation clearly demonstrated reduced accumulation of genomic RNA in the presence of DI RNA. Accumulation of genomic RNA was very rapid between 3 and 9 hr postinfection. The presence of equimolar amounts of genomic and DI RNA in the inoculum resulted in a 65% suppression of genomic RNA accumulation. Suppression of genomic RNA was mediated by a reduction in the rate at which genomic RNA accumulated. Analysis of protoplasts inoculated with increasing ratios of DI:genomic RNA suggested that DI RNA-mediated suppression of genomic RNA synthesis results from competition for factors essential for viral replication. Incubation of protoplasts at different temperatures also had a profound effect on replication of both genomic and DI RNAs. Both replicated well at 27 degrees but were barely detectable at 32 degrees. Suppression of genomic RNA synthesis by DI RNA was similar at all temperatures tested. Thus, this study suggests that DI suppression of TBSV symptoms in whole plants and symptom attenuation at elevated temperatures are primarily the result of reduced viral replication.

Blotting, Northern↗

PubMed1994

Transgenic Indica rice (Oryza sativa L.) plants obtained by direct gene transfer to protoplasts.

We have established a reproducible procedure for transformation of protoplasts and regeneration of transgenic plants for an improved Indica rice cultivar IR43. Mature embryo-derived calli were placed in liquid culture medium containing maltose to establish meristematically active, embryogenic cell suspension lines. In order to obtain transgenic plants, a chimeric hygromycin phosphotransferase hph gene under the control of the cauliflower mosaic virus CaMV 35S promoter was introduced into protoplasts from these cell suspension lines using polyethylene glycol. Protoplasts were cultured in maltose-containing medium. Hygromycin B selection was applied to 14-day-old dividing cell colonies. Resistant calli were readily obtained after 3 weeks of selection. Seventy-three plantlets were regenerated from resistant calli from several independent experiments, and a few of the 29 plants grown in the greenhouse reached maturity. Stable integration of the transgene in the genome of these plants was confirmed by Southern blot analysis and the expression of the transgene in plants by hygromycin phosphotransferase assay. The procedure described yielded 5 to 18 resistant colonies and approximately four transgenic plantlets per million treated protoplasts.

Base Sequence↗

PubMed1993

Cymbidium mosaic virus RNA synthesis in isolated orchid protoplasts.

An in vitro protoplast host system has been developed to facilitate the study of cymbidium mosaic virus (CyMV) replication mechanisms. CyMV, a member of the potexvirus group of plant viruses, is among the most significant pathogens of cultivated orchid plants, but a convenient system for studying its molecular genetics has not been available previously. A cDNA probe representing a segment of the CyMV genome was used to detect viral RNA following inoculation of Vanda floral protoplasts with virus particles. Detection was carried out by RNA-DNA hybridization methods (RNA slot blots). Newly synthesized viral RNA was detectable by 6-9 h postinfection and appeared to reach a maximum around 12 h. These results are consistent with the time course of viral RNA synthesis in protoplasts infected with other RNA viruses. Immunoblot detection methods revealed the presence of newly synthesized viral protein indicating that viral gene expression occurs in the protoplasts in addition to genome replication.

Blotting, Western↗

PubMed1992

The effect of microgravity on the development of plant protoplasts flown on Biokosmos 9.

An experiment using plant protoplasts has been accepted for the IML-1 Space Shuttle mission scheduled for 1991. Preparatory experiments have been performed using both fast and slow rotating clinostats and in orbit to study the effect of simulated and real weightlessness on protoplast regeneration. Late access to the space vehicles before launch has required special attention since it is important to delay cell wall regeneration until the samples are in orbit. On a flight on Biokosmos 9 ("Kosmos-2044") in September 1989 some preliminary results were obtained. Compared to the ground control, the growth of both carrot and rapeseed protoplasts was decreased by 18% and 44% respectively, after 14 days in orbit. The results also indicated that there is less cell wall regeneration under micro-g conditions. Compared to the ground controls the production of cellulose in rapeseed and carrot flight samples was only 46% and 29% respectively. The production of hemicellulose in the flight samples was 63% and 67% respectively of that of the ground controls. In both cases all samples reached the stage of callus development. The peroxidase activity was also found to be lower in the flight samples than in the ground controls, and the number of different isoenzymes was decreased in the flight samples. In general, the regeneration processes were retarded in the flight samples with respect to the ground controls. From a simulation experiment for IML-1 performed in January 1990 at ESTEC, Holland, regenerated plants have been obtained. These results are discussed and compared to the results obtained on Biokosmos 9. Protoplast regeneration did not develop beyond the callus stage in either the flight or the ground control samples from the Biokosmos 9 experiment.

Brassica↗

PubMed1991

F-actin contractile rings in protoplasts of the yeast Schizosaccharomyces.

By rhodamine-phalloidin fluorescence, distinct continuous F-actin rings were visualized in 18-20% of the protoplasts of Schizosaccharomyces pombe and S. japonicus var. versatilis, in addition to randomly distributed F-actin dots. Whereas the reversion of ring-lacking protoplasts coincided with the polarization of the dotted F-actin pattern, the ring-containing protoplasts became furrowed as the F-actin rings constricted. The furrowing was more conspicuous in S. japonicus var. versatilis than in S. pombe protoplasts and it was blocked when the reversion was inhibited by Novozyme 234 indicating that the cell wall formation is essential for the F-actin ring constriction.

Actins↗

PubMedDate not supplied

Expression and maintenance of plasmid resistance in regenerating protoplasts of Bacillus subtilis.

Expression of plasmid-encoded resistances in regenerating protoplasts of Bacillus subtilis occurs only after wall synthesis has been resumed. This is observed for protoplasts obtained from cells already containing plasmids (pC194, pT127, pK545) or for plasmid-bearing cells coming from a PEG-mediated transformation. Recovery of expression needs a 2-h incubation of protoplasts, previously washed to get rid of their lysozyme content, in rich hypertonic medium (SMMP). A longer incubation (24-h) results in the obtention of regenerants; however, most of them have lost their resistant phenotype in contrast to those obtained from the usual solid regeneration plates. This finding suggests either a high curing effect or some kind of gene inactivation phenomenon. Discussion is focused on the critical points that have to be considered when polyethylenglycol-mediated transformation of protoplasts is applied to recombinant DNA technology.

Bacillus subtilis↗

PubMed2005

Internalisation of cell-penetrating peptides into tobacco protoplasts.

Cells are protected from the surrounding environment by plasma membrane which is impenetrable for most hydrophilic molecules. In the last 10 years cell-penetrating peptides (CPPs) have been discovered and developed. CPPs enter mammalian cells and carry cargo molecules over the plasma membrane with a molecular weight several times their own. Known transformation methods for plant cells have relatively low efficiency and require improvement. The possibility to use CPPs as potential delivery vectors for internalisation in plant cells has been studied in the present work. We analyse and compare the uptake of the fluorescein-labeled CPPs, transportan, TP10, penetratin and pVEC in Bowes human melanoma cells and Nicotiana tabacum cultivar (cv.) SR-1 protoplasts (plant cells without cell wall). We study the internalisation efficiency of CPPs with fluorescence microscopy, spectrofluorometry and fluorescence-activated cell sorter (FACS). All methods indicate, for the first time, that these CPPs can internalise into N. tabacum cv. SR-1 protoplasts. Transportan has the highest uptake efficacy among the studied peptides, both in mammalian cells and plant protoplast. The internalisation of CPPs by plant protoplasts may open up a new effective method for transfection in plants.

Carrier Proteins↗

PubMed2006

Dielectric spectroscopy of Anabaena 7120 protoplast suspensions.

The dielectric spectroscopy of Anabaena 7120 protoplast suspensions has been investigated over the frequency range of 40 Hz-110 MHz. The protoplast suspensions showed a complicated dielectric dispersion consisting of at least four distinct sub-dispersions with the increasing frequencies due to the Maxwell-Wagner interfacial polarization. The double-shell model, in which an equivalent shell of thylakoid was assumed inside the cytoplasm, was adopted to describe the special morphology of the protoplast. Under the assumption that the conductivity of plasmalemma was negligible and the conductivity of the equivalent shell was 0.1 microS/cm, we attempted to estimate the dielectric properties of various protoplast components by fitting theoretical curve to experimental data. The relative permittivity of the plasmalemma epsilon(mem) was estimated to be 6.5+/-0.5, and the permittivity of the equivalent shell of thylakoid epsilon(thy) was estimated to be about 3.2+/-0.2. The permittivity epsilon(cyt) and conductivity kappa(cyt) of the cytoplasm were estimated to be 60 and 0.88+/-0.11 mS/cm, respectively. The permittivity epsilon(nuc) and conductivity kappa(nuc) of the nucleoplasmic region were determined to be 100 and 0.13+/-0.02 mS/cm, respectively.

Anabaena↗

PubMed2005

Self-fusion of protoplasts enhances chitinase production and biocontrol activity in Trichoderma harzianum.

Protoplasts were isolated from Trichoderma harzianum strain PTh18 using lysing enzymes and self-fusion of T. harzianum protoplasts was carried out using polyethylene glycol in STC buffer. The fused protoplasts of T. harzianum were regenerated and 15 self-fusants were selected to study the chitinase production and biocontrol activity. High chitinase activity was measured in the culture filtrates of most of the self-fusants (87%) than the parent. Among the fusants, the strain SFTh8 produced maximum chitinase with a two-fold increase as compared to the parent strain. All the self-fusants exhibited increased antagonistic activity against Rhizoctonia solani than the parent. The crude chitinase preparation of SFTh8 lysed the mycelia of T. harzianum, Trichoderma viride and Trichoderma reesei and released the protoplasts in higher number than the crude chitinase preparation of parent strain PTh18.

Chitinases↗

PubMed2005

Light-enhanced dark respiration in leaves, isolated cells and protoplasts of various types of C4 plants.

The rate of respiratory CO2 evolution from the leaves of Zea mays, Panicum miliaceum, and Panicum maximum, representing NADP-ME, NAD-ME, and PEP-CK types of C4 plants, respectively, was increased by approximately two to four times after a period of photosynthesis. This light-enhanced dark respiration (LEDR) was a function of net photosynthetic rate specific to plant species, and was depressed by 1% O2. When malate, aspartate, oxaloacetate or glycine solution at 50 mM concentration was introduced into the leaves instead of water, the rate of LEDR was enhanced, far less in Z. mays (by 10-25%) than in P. miliaceum (by 25-35%) or P. maximum (by 40-75%). The enhancement of LEDR under glycine was relatively stable over a period of 1 h, whereas the remaining metabolites caused its decrease following a transient increase. The metabolites reduced the net photosynthesis rate in the two Panicum species, but not in Z. mays, where this process was stimulated by glycine. The bundle sheath cells from P. miliaceum exhibited a higher rate of LEDR than those of Z. mays and P. maximum. Glycine had no effect on the respiration rate of the cells, but malate increased in cells of Z. mays and P. miliaceum by about 50% and 30%, respectively. With the exception of aspartate, which stimulated both the O2 evolution and O2 uptake in P. maximum, the remaining metabolites reduced photosynthetic O2 evolution from bundle sheath cells in Panicun species. The net O2 exchange in illuminated cells of Z. mays did not respond to CO2 or metabolites. Leaf mesophyll protoplasts of Z. mays and P. miliaceum, and bundle sheath protoplasts of Z. mays, which are unable to fix CO2 photosynthetically, also produced LEDR, but the mesophyll protoplasts, compared with bundle sheath protoplasts, required twice the time of illumination to obtain the maximal rate. The results suggest that the substrates for LEDR in C4 plants are generated during a period of illumination not only via the Calvin cycle reactions, but also by the conversion of endogenous compounds present in leaf cells. The stimulation of LEDR under glycine is discussed in relation to its direct or indirect effect on mitochondrial respiration.

Aspartic Acid↗

PubMed2005

Salt stress-induced programmed cell death in tobacco protoplasts is mediated by reactive oxygen species and mitochondrial permeability transition pore status.

The status of mitochondrial permeability transition pore (PTP) and levels of reactive oxygen species (ROS) play key roles in regulating apoptosis in animal cells. To investigate if the PTP and cellular oxidation-reduction state are also involved in salt stress-induced programmed cell death (PCD) in tobacco (Nicotiana tabacum, cultivar BY-2) protoplasts, flow cytometry was used to simultaneously monitor ROS levels, PTP status and PCD. Increased ROS and decreased mitochondrial membrane potential (delta psi(m)) were observed before the appearance of PCD. Pre-treatment with an inhibitor of the PTP opening, cyclosporin A (CsA), effectively retarded the onset of PCD, the delta psi(m) decrease and the ROS content increase. Addition of ascorbic acid (AsA) during the salt stress significantly decreased the percentage of protoplasts undergoing PCD and ROS levels but increased delta psi(m). Hydrogen peroxide effectively induced the appearance of PCD and caused an increase in ROS and a decrease in delta psi(m). Pre-treatment of protoplasts with CsA weakened the effects of H2O2. All these results suggest that the open state of PTP and ROS are necessary elements for salt stress-induced PCD in tobacco protoplasts. The open states of PTP and ROS could promote each other suggesting that ROS could lead to a self-amplifying process. This positive feedback loop may act as an all-or-nothing switch, which is in good accordance with the hypothesis that PTP is an important coordinator and executioner of PCD in both animals and plants.

Apoptosis↗

PubMed2000

An improved protocol for the preparation of protoplasts from an established Arabidopsis thaliana cell suspension culture and infection with RNA of turnip yellow mosaic tymovirus: a simple and reliable method.

An improved method for preparation of protoplasts of Arabidopsis thaliana cells grown in suspension culture is presented. This method is fast, reliable and can be used for the production of virtually an unlimited number of protoplasts at any time. These protoplasts can be transformed efficiently with RNA from turnip yellow mosaic tymovirus (TYMV) by polyethyleneglycol-mediated transfection. The simple transfection procedure has been optimized at various steps. Replication of TYMV can be monitored routinely by detection of the coat protein in as few as 2 x 10(4) infected protoplasts.

Arabidopsis↗

PubMed1998

Effects of clinorotation on the polysaccharide content of resynthesized walls of protoplasts.

Changes in cellulose and callose content during cell wall regeneration in Brassica oleracea protoplasts have been examined by cytofluorimetry following their exposure to the conditions of the horizontal clinostat (2 r.p.m.) for 10 days. In comparison with controls, cellulose content decreased 4-fold and 28% of the protoplasts failed to resynthesize a wall in the clinorotated sample. The callose content was almost doubled in clinostated cells. Callose synthesis fluctuated in both control and clinorotated protoplasts. The results support the idea that inhibition of cellulose synthesis in protoplasts grown on the clinostat is caused by a change of plasmalemma fluidity and functioning, and also by a disturbance to the state of cytoplasmic calcium under conditions of simulated microgravity.

Brassica↗

PubMedDate not supplied

Initial steps of wall protoplast regeneration in Candida albicans.

Cell wall regeneration of individual Candida albicans yeast and mycelial protoplasts was studied with confocal and electron microscopy using polyclonal antibodies and lectins. Quantitative measurements of the fluorescence emitted by individual protoplasts during the process of regeneration indicate that chitin is the first polymer to be laid down, whereas beta (1,3)- and beta (1,6)glucan are incorporated at a later stage. Mannoproteins were found on the surface of fresh protoplasts and those newly synthesized were then deposited with time. During the first steps of wall regeneration, the proteins that interacted covalently with chitin or glucan were different, but the same species were found linked to each polymer in yeast and mycelial regenerating forms. The aggregates formed by regenerating protoplasts were shown to be due to the chitin and mannoprotein network initially laid.

Blotting, Western↗