Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Protoplasts”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 145 records · Page 8Linked to original sources
PubMed1981

Multiple septation in multinuclear protoplasts of Schizosaccharomyces pombe.

The dependence of septation on karyokinesis was studied in protoplasts of the fission yeast Schizosaccharomyces pombe. The mononuclear protoplast produces a single centrally located septum. In multinuclear reverting protoplasts, on the other hand, the formation of a single septum was a rare event. In each protoplast instead of one, two to six septa were formed. These findings suggest some closer relations between the formation of septa and the number of nuclei present in the protoplast. Our results obtained with protoplasts also imply that the initiation of septum formation is possible only in the presence of a complete cell wall. In reverting multinuclear protoplasts undergoing more than one mitosis, no septation is initiated until the cell wall has been completed. Only the complete cell wall can induce the formation of one or several septa in mono- and multinuclear protoplasts, respectively.

Ascomycota↗
PubMed1996

Gypsy Moth Immune Defenses in Response to Hyphal Bodies and Natural Protoplasts of Entomophthoralean Fungi

Gypsy moth hemocytes phagocytosed and/or encapsulated walled entomophthoralean cells and protoplasts that were regenerating cell walls but rarely recognized the healthy protoplasts that lack cell walls. Experiments using lectin conjugates demonstrated different sugars localized at the surface of protoplasts, regenerating protoplasts, and walled cells. Protoplasts had few sugars, in small amounts, on their surfaces and these fungal cells evoked minimal cellular responses. Walled fungal cells had greater quantities of a variety of sugars in the cell walls and evoked the strongest defense response. Protoplasts of Entomophaga grylli, an orthopteran pathogen that cannot successfully develop in the gypsy moth, Lymantria dispar, evoked a stronger hemocytic response in larvae than protoplasts of the lepidopteran pathogen Entomophaga maimaiga that is able to successfully develop in L. dispar. Sugars detected at the surface of E. grylli and E. maimaiga protoplasts were similar, so it is likely that the surface sugars we tested do not determine the differences in nonpathogen recognition between these fungal species.

Journal Article↗
PubMed1989

Growth and soluble proteins of cell cultures derived from explants and protoplasts of Pinus pinaster cotyledons.

Pinus pinaster Ait. cell suspension cultures were derived from chopped cotyledons and from cotyledon protoplasts. When transferred after 12 weeks in culture, growth of both cell types showed a lag of 5 days followed by an exponential phase of 14 days for the protoplast-derived cells and 23 days for the organ-derived cells. During the exponential growth phase, packed cell volume of protoplast-derived cultures increased 7-fold and that of organ-derived cultures 13-fold. During the stationary phase, the diameters of protoplast-derived cells averaged 80 microm and those of organ-derived cells 100 microm. After 30 days, the media containing protoplast-derived and organ-derived cells decreased in osmolarity by 50 and 120 mOs per kg water, respectively, and in pH by 1.4 and 2.0 units, respectively. Throughout the growth cycle, protein content per unit of packed cell volume was always at least 35% higher in the protoplast-derived cultures than in the organ-derived cultures. Two-dimensional electrophoretic separation of soluble proteins revealed three peptides present in protoplast-derived cells that were absent from organ-derived cells and two peptides present in organ-derived cells that were absent from protoplast-derived cells. Other peptides differed quantitatively between the cell types.

Journal Article↗
PubMed1994

Perception of Gibberellin and Abscisic Acid at the External Face of the Plasma Membrane of Barley (Hordeum vulgare L.) Aleurone Protoplasts.

The response of protoplasts isolated from aleurone layers of barley (Hordeum vulgare L. cv Himalaya) to internally and externally applied hormone was analyzed to localize the site of perception of the hormonal signal. Protoplasts responded to externally applied gibberellic acid (GA3) with increased synthesis and secretion of [alpha]-amylase, transient expression of the glucuronidase reporter gene fused to the hormone-responsive elements of the [alpha]-amylase promoter, and the vacuolation typical of GA3-treated aleurone cells. When up to 250 [mu]M GA3 was microinjected into the protoplast cytoplasm, none of these responses were observed. This did not reflect damage to the protoplasts during the microinjection procedure, since microinjected protoplasts remained responsive to externally applied hormone. Nor did it reflect loss of microinjected GA3 from the protoplast, since 50% of microinjected [3H]GA20 was retained by protoplasts for at least 24 h. Externally applied abscisic acid (ABA) could reverse the stimulation of [alpha]-amylase synthesis and secretion, whereas microinjecting up to 250 [mu]M ABA was ineffective at antagonizing the stimulatory effect of GA3. These results suggest that the site of perception of GA3 and ABA in the barley aleurone protoplast is on the external face of the plasma membrane.

Journal Article↗
PubMed1978

Membrane transport of sugars and amino acids in isolated protoplasts.

A method has been developed for observing membrane transport in isolated protoplasts. Transport of sugars and amino acids has been studied in protoplasts isolated from the mesophyll of Pisum sativum L. That uptake was not due to passive diffusion through damaged membranes was demonstrated by supplying simultaneously two sugar stereoisomers, the one (3)H-labeled and the other (14)C-labeled. The protoplast membranes were sufficiently functional to discriminate strongly between these stereoisomers.To characterize transport the nonmetabolized glucose analogue 3-O-methyl glucose (MeG) and amino acid analogue alpha-aminoisobutyric acid (AIB) were employed. When uptake was compared per unit of protein as between leaf strips and protoplasts prepared from the same tissue, it was estimated that the protoplasts had retained approximately 40 to 50% of the uptake ability of the whole cells. Uptake of neither MeG nor AIB by protoplasts was linear with time, but the tendency to flatten was more marked for AIB. Addition of Mg-ATP to buffered medium significantly promoted AIB uptake, an effect not ascribable to either chelation or pH. Transport of both MeG and AIB was markedly pH-dependent, uptake falling with rise in pH.The stimulatory effect of Mg-ATP and the pH dependence confirm that uptake was not due to a diffusional inward "leak" but involved membrane function.This work demonstrates the feasibility of using isolated protoplasts for membrane transport studies. The potential advantages of using protoplasts for such studies are pointed out.

Journal Article↗
PubMed1981

Vacuole/Extravacuole distribution of soluble protease in hippeastrum petal and triticum leaf protoplasts.

The subcellular distribution of soluble protease in anthesis-stage, anthocyanin-containing Hippeastrum cv. Dutch Red Hybrid petal protoplasts has been reevaluated and that of Triticum aestivum L. var. Red Coat leaf protoplasts determined using (125)I-fibrin as a protease substrate and improved methods for protoplast and vacuole volume estimation. Results indicate that about 20% of the Hippeastrum petal-soluble protease and about 90% of the wheat leaf-soluble protease can be assigned to the vacuole. Protoplast isolation enzyme labeled with (125)I has been used to assess the efficiency of removing isolation enzyme from protoplasts by repeated washing and by separation of protoplasts from debris using density centrifugation. Results of these studies suggest that protoplasts prepared by both methods retain low levels of isolation enzyme. However, when protoplasts prepared by either method were lysed with washing medium lacking osmoticum, little isolation enzyme contaminated the lysates.

Journal Article↗
PubMed1982

Vacuolar localization of proteases and degradation of chloroplasts in mesophyll protoplasts from senescing primary wheat leaves.

Mesophyll protoplasts isolated from primary leaves of wheat seedlings were used to follow the localization of proteases and the breakdown of chloroplasts during dark-induced senescence. Protoplasts were readily obtained from leaf tissue, even after 80% of the chlorophyll and protein had been lost. Intact chloroplasts and vacuoles could be isolated from the protoplasts at all stages of senescence. All the proteolytic activity associated with the degradation of ribulose bisphosphate carboxylase in the protoplasts could be accounted for by that localized within the vacuole. Moreover, this localization was retained late into senescence. Protoplasts isolated during leaf senescence first showed a decline in photosynthesis, then a decline in ribulose bisphosphate carboxylase activity, followed by a decline in chloroplast number. There was a close correlation between the decline in chloroplast number and the loss of chlorophyll and soluble protein per protoplast, suggesting a sequential degradation of chloroplasts during senescence. Ultrastructural studies indicated a movement of chloroplasts in toward the center of the protoplasts during senescence. Thus, within senescing protoplasts, chloroplasts appeared either to move into invaginations of the vacuole or to be taken up into the vacuole.

Journal Article↗
PubMed1982

Light and Dark Controls of Nitrate Reduction in Wheat (Triticum aestivum L.) Protoplasts.

Protoplasts were isolated from the leaves of nitrate-cultured wheat (Triticum aestivum L. var. Frederick) seedlings. When incubated in the dark, protoplasts accumulated nitrite under anaerobic, but not under aerobic, conditions. The assimilation of [(15)N]nitrite by protoplasts was strictly light-dependent, and no loss of nitrite from the assay medium was observed under dark aerobic conditions. Therefore, the absence of nitrite accumulation under dark aerobic conditions was the result of an O(2) inhibition of nitrate reduction and not a stimulation of nitrite reduction. In the presence of antimycin A, protoplasts accumulated nitrite under dark aerobic conditions. The oxygen inhibition of nitrate reduction was apparently due to a competition between nitrate reduction and dark respiration for cytoplasmic-reducing equivalents.Oxygen control of nitrate reduction was not observed in the light, since protoplasts assimilated [(15)N]nitrate to amino-N under light aerobic conditions. It has been proposed that the increase of the ATP-to-ADP ratio in the light inhibits dark respiration and allows nitrate reduction to occur under aerobic conditions. To test this hypothesis, protoplast N and C assimilation was assayed in the presence of nigericin, an uncoupler of photophosphorylation. The dark to light increase of the protoplast energy charge was not observed in the presence of nigericin, and CO(2) fixation was completely inhibited by the uncoupler. In contrast, rates of in vivo nitrate reduction (N(2) and air) and nitrite reduction were relatively unaffected by nigericin, and light-driven nitrate assimilation was inhibited by only 20%. Nigericin had no effect on the dark-to-light increase of protoplast NADH and NADPH levels. It is proposed that the light-induced increase of cytoplasmic-reducing equivalents suppresses the competition between nitrate reduction and dark respiration and allows nitrate reduction to occur under aerobic conditions. Dark-to-light changes of the ATP-to-ADP ratio apparently are not critical to the regulation of nitrate reduction.

Journal Article↗
PubMed1982

Reversible light-activation of ribulose bisphosphate carboxylase/oxygenase in isolated barley protoplasts and chloroplasts.

The enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase displayed near-maximal activity in isolated, intact barley (Hordeum vulgare L. cv. Pennrad) mesophyll protoplasts. The carboxylase deactivated 40 to 50% in situ when protoplasts were dark-incubated 20 minutes in air-equilibrated solutions. Enzyme activity was fully restored after 1 to 2 minutes of light. Addition of 5 millimolar NaHCO(3) to the incubation medium prevented dark-inactivation of the carboxylase. There was no permanent CO(2)-dependent activation of the protoplast carboxylase either in light or dark. Activation of the carboxylase from ruptured protoplasts was not increased significantly by in vitro preincubation with CO(2) and Mg(2+). In contrast to the enzyme in protoplasts, the carboxylase in intact barley chloroplasts was not fully reactivated by light at atmospheric CO(2) levels. The lag phase in carbon assimilation was not lengthened by dark-adapting protoplasts to low CO(2) demonstrating that light-activation of the carboxylase was not involved in photosynthetic induction. Irradiance response curves for reactivation of the the carboxylase and for CO(2) fixation by isolated barley protoplasts were similar. The above results show that there was a fully reversible light-activation of the carboxylase in isolated barley protoplasts at physiologically significant CO(2) levels.

Journal Article↗
PubMed1982

Ploidy Effects in Isogenic Populations of Alfalfa : II. Photosynthesis, Chloroplast Number, Ribulose-1,5-Bisphosphate Carboxylase, Chlorophyll, and DNA in Protoplasts.

Photosynthetically-active protoplasts isolated from isogenic sets of diploid-tetraploid and tetraploid-octoploid alfalfa (Medicago sativa L.) leaves were used to investigate the consequences of polyploidization on several aspects related to photosynthesis at the cellular level. Protoplasts from the tetraploid population contained twice the amount of DNA, ribulose-1,5-bisphosphate carboxylase (RuBPCase), chlorophyll (Chl), and chloroplasts per cell compared to protoplasts from the diploid population. Although protoplasts from the octoploid population contained nearly twice the number of chloroplasts and amount of Chl per cell as tetraploid protoplasts, the amount of DNA and RuBPCase per octoploid cell was only 50% higher than in protoplasts from the tetraploid population. The rate of CO(2)-dependent O(2) evolution in protoplasts nearly doubled with an increase in ploidy from the diploid to tetraploid level, but increased only 67% with an increase in ploidy from the tetraploid to octoploid level. Whereas leaves and protoplasts had similar increases in RuBPCase, DNA, and Chl with increase in ploidy level, it was concluded that increased cell volume rather than increased cell number per leaf is responsible for the increase in leaf size with ploidy.

Journal Article↗
PubMed1984

Extensor and flexor protoplasts from samanea pulvini : I. Isolation and initial characterization.

Protoplasts were isolated from extensor and flexor regions of open pulvini of the nyctinastic tree Samanea saman. Both types of protoplasts undergo many changes during isolation. Extensor protoplasts are univacuolate in vivo, but some become multivacuolate. All flexor protoplasts are univacuolate. In an open pulvinus, extensor cells have a higher osmotic pressure than flexor cells. However, both types of protoplasts can be isolated with optimal yield using the same osmoticum (0.5 molar sorbitol) in the digestion medium. This suggests that some leakage of osmoticum occurs during harvest or digestion, especially from extensor tissue. Despite these changes, both types of protoplasts extrude protons in response to 10 micromolar fusicoccin (1.6-1.8 nanoequivalent/10(6) protoplasts/minute), demonstrating that the protoplasts are metabolically active and that proton transport mechanisms must be at least partially functional. The changes in vacuolar structure and osmotic pressure are what one might expect if the protoplasts, which are isolated from open pulvini, take on characteristics of cells in a closed pulvinus.

Journal Article↗
PubMed1984

Extensor and Flexor Protoplasts from Samanea Pulvini : II. X-Ray Analysis of Potassium, Chlorine, Sulfur, Phosphorus, and Calcium.

Concentrations of K, Cl, P, S, and Ca in extensor and flexor protoplasts from open pulvini of the nyctinastic tree Samanea saman were estimated using x-ray microanalysis. This technique is particularly suitable when absolute numbers of protoplasts are low, because less than 100 protoplasts are required to obtain statistically significant data. Flexor protoplasts contain similar concentrations of P and S but almost twice as much K and Cl as extensor protoplasts. Low levels of total measurable osmoticum suggest that extensive leakage has occurred during protoplast isolation. Both extensor and flexor protoplasts appear to contain some unidentified osmoticum not detectable by x-ray analysis. Extensor protoplasts must have more unidentified osmoticum to compensate for their lower levels of K and Cl.

Journal Article↗
PubMed1986

Behavior and viability of tobacco protoplasts in response to electrofusion parameters.

This investigation examines responses of protoplasts in a systematic and quantitative way to the various electrical treatments used to achieve electrofusion and their individual and cumulative effect on protoplast viability. Mesophyll and cell suspension protoplasts from two species of the same genera, Nicotiana tabacum and N. rustica var brasilia were used in these experiments. Optimal frequencies for alignment of tobacco protoplasts were between 500 kilohertz and 2 megahertz at 100 volts per centimeter. Variations in frequency and voltage of the alternating current (AC) field caused predictable movements of protoplasts within an electrofusion chamber. AC frequencies below 10 hertz or above 5 megahertz significantly decreased the viability of protoplasts in the fusion chamber as estimated by fluorescein diacetate staining 1 hour after treatment. Although the direct current (DC) pulse appeared to have a slight detrimental effect on protoplast viability, this effect was not significantly different from untreated control preparations.Protoplasts from both leaf mesophyll cells and suspension cells were induced to fuse with one or more 10 to 30 microseconds DC square wave pulses of approximately 1 kilovolt per centimeter after the protoplasts had been closely appressed with an AC field.

Journal Article↗
PubMed1988

Transport and subcellular localization of polyamines in carrot protoplasts and vacuoles.

Putrescine and spermidine uptake in carrot (Daucus carota L., cv "Tip top") protoplasts and isolated vacuoles was studied. Protoplasts and vacuoles accumulated polyamines very quickly, with maximum absorption within 1 to 2 minutes. The insertion of a washing layer containing 100 millimolar unlabeled putrescine or spermidine did not change this pattern, but strongly reduced the uptake of putrescine and spermidine in protoplasts and in vacuoles. The dependence of spermidine uptake on the external concentration was linear up to the highest concentrations tested in protoplasts, while that in vacuoles showed saturation kinetics below 1 millimolar (K(m) = 61.8 micromolar) and a linear component from 1 to 50 millimolar. Spermidine uptake in protoplasts increased linearly between pH 5.5 and 7.0, while there was a distinct optimum at pH 7.0 for vacuoles. Preincubation of protoplasts with 1 millimolar Ca(2+) affected only surface binding but not transport into the cells. Nonpermeant polycations such as La(3+) and polylysine inhibited spermidine uptake into protoplasts. Compartmentation studies showed that putrescine and spermidine were partly vacuolar in location and that exogenously applied spermidine could be recovered inside the cells. The characteristics of the protoplast and vacuolar uptake system induce us to put forward the hypothesis of a passive influx of polyamines through the plasmalemma and of the presence of a carrier-mediated transport system localized in the tonoplast.

Journal Article↗
PubMed1989

Ribulosebisphosphate carboxylase activity and photosynthetic o(2) evolution rate in vicia guard-cell protoplasts.

Activities of ribulose-1,5-bisphosphate carboxylase and rates of photosynthetic O(2) evolution were measured in guard-cell and mesophyll protoplasts from Vicia faba. The ribulose-1,5-bisphosphate carboxylase activity of guard-cell protoplasts was 30% of that of mesophyll protoplasts; however, the O(2) evolution rate was 3 times higher in guard-cell protoplasts than in mesophyll protoplasts on a chlorophyll basis. When the dark-adapted, guard-cell protoplasts were illuminated by red light, O(2) was evolved with an induction period, which became shorter when the protoplasts were reilluminated. High activity of irreversible NADP-glyceraldehyde-3-phosphate dehyrogenase was found in guard-cell protoplasts. Several lines of evidence revealed that there was virtually no contamination by mesophyll cells in guard-cell preparations. These results indicate that guard cells fix CO(2) photosynthetically and imply that the cells utilize a considerable proportion of reducing equivalents from water for reactions other than CO(2) fixation.

Journal Article↗
PubMed1990

Stress Responses in Alfalfa (Medicago sativa L.): VI. Differential Responsiveness of Chalcone Synthase Induction to Fungal Elicitor or Glutathione in Electroporated Protoplasts.

Protoplasts derived from cell suspensions of alfalfa (Medicago sativa L.) responded to treatment with fungal elicitor (FE) by an increase in endogenous chalcone synthase (CHS) activity but were unresponsive to reduced glutathione (GSH). Preexposure of protoplasts to polyethylene glycol and electroporation resulted in strong responsiveness to GSH but little change in responsiveness to FE. Protoplasts from suspension cultures which had been subcultured more than 12 times lost responsiveness to GSH, but not FE, as assessed by measuring expression of a chimeric gene containing a bean CHS promoter linked to a bacterial chloramphenicol acetyltransferase (CAT) reporter gene. In protoplasts in which putative cis-acting CHS promoter sequences had been coelectroporated in trans with the intact CHS promoter-CAT construct, the extent of CAT expression depended upon the elicitor used (FE or GSH), the age (number of times subcultured) of the cells from which the protoplasts were isolated, and the nature of the coelectroporated CHS promoter sequence. For example, a region of the CHS promoter from -326 to -141 behaved as a trans-activator when coelectroporated with the CAT construct into unelicited protoplasts isolated from newly initiated cell suspensions, but the same region acted as a trans-silencer in the same protoplasts in the presence of FE. This silencer activity was much reduced in GSH-treated protoplasts. The results suggest that there are differences in the signal transduction pathways for elicitation of CHS transcription by FE and GSH, which involve previously indentified cis-elements in the CHS promoter.

Journal Article↗
PubMed1990

Transfer of Isolated Nuclei into Protoplasts of Trichoderma harzianum.

Protoplasts released from young hyphae of Trichoderma harzianum contained 0 to 10 nuclei per protoplast, and most (about 80%) contained from 4 to 6 nuclei. Most protoplasts were larger than 3 mum in diameter. Nuclei were isolated from protoplasts of an auxotrophic mutant of T. harzianum and transferred into protoplasts obtained from another auxotroph of the same strain. This intrastrain nuclear transfer gave rise to numerous progeny which were stable, prototrophic, and heterokaryotic. Interstrain transfers in which nuclei from a wild-type prototroph of one strain were transferred into protoplasts from a lysine-deficient auxotroph of a second strain were also done. Heterokaryotic progeny were recovered from these interstrain transfers when the regenerating protoplasts were provided with a low concentration of lysine 48 h after the initial plating. Heterokaryotic progeny contained 11 to 17% of donor-type nuclei. Progeny homokaryotic for donor-type nuclei were obtained as single-spore isolates. These homokaryotic isolates expressed the isozyme pattern and colony morphology phenotype of the nuclear donor. When regenerating protoplasts were provided with lysine 10 days after the initial plating, only a single progeny was obtained. However, single-spore subprogeny of this nuclear transfer were prototrophic and exhibited a wide range of unstable morphological phenotypes.

Journal Article↗
PubMed1964

STABILIZATION OF STREPTOCOCCUS FAECALIS PROTOPLASTS BY SPERMINE.

Harold, F. M. (National Jewish Hospital, Denver, Colo.). Stabilization of Streptococcus faecalis protoplasts by spermine. J. Bacteriol. 88:1416-1420. 1964.-Lysis of protoplasts of Streptococcus faecalis subjected to osmotic shock was prevented by the presence of 10(-3)m spermine and other divalent cations. Protein and nucleic acids were largely retained, but compounds of low molecular weight were discharged into the medium and the capacity for glycolysis was lost. Under these conditions, spermine was bound to the protoplasts. It could not be removed by washing with water or nonelectrolytes, but was displaced by salts, polyanions, and polycations. Removal of the spermine restored the osmotic fragility of the protoplasts, which could once again be protected from lysis by impermeant solutes. Protoplasts were also stabilized, in the absence of osmotic shock, by prolonged incubation with cations in 0.5 m sucrose. By either procedure, the protoplasts became resistant not only to osmotic lysis but also to sonic oscillation. It is concluded that the stabilization of protoplasts resulted from ionic binding of the cation to acidic sites on the external surface of the plasma membrane. This conferred upon the membrane additional mechanical strength, perhaps by the cross-linking of subunits, but did not alter its permeability to extracellular solutes.

Bacteriolysis↗