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PubMed1993

Cauliflower mosaic virus gene I product (P1) forms tubular structures which extend from the surface of infected protoplasts.

Immunofluorescent staining of Chinese cabbage protoplasts infected with cauliflower mosaic virus (CaMV) showed different patterns of staining when antibodies raised against virus particles or antibodies specific for the CaMV movement protein (P1) were used. Anti-virion serum showed that particles accumulated as small discrete aggregates within the cytoplasm of infected protoplasts. In contrast, anti-P1 serum identified long thread-like structures extending from the protoplast surface. The same structures were seen when protoplasts were isolated from infected plants and held in culture. Electron microscopical examination after immunogold labeling and negative staining of protoplasts revealed some extracellular tubular structures which labeled with anti-P1 and which contained virus particles. Western blot analysis of isolated tubules showed them to be composed of the full-length P1 but not the smaller P1 products detected in infected tissues or protoplasts. The time course of P1 accumulation in infected protoplasts paralleled the accumulation of virus coat protein, as measured by immunofluorescence microscopy, and did not indicate that P1 was an early gene product as has been suggested for other virus movement proteins.

Blotting, Western↗
PubMed1983

Transport of nutrients in yeast protoplasts.

Protoplasts were prepared with snail-gut juice from Saccharomyces cerevisiae and Candida utilis. Transport of D-xylose (A), trehalose (B), 2-deoxy-D-glucose (C), L-leucine (D), L-proline (E), inorganic phosphate (F) and H+ ions (G) was studied with special emphasis on the first species. Transport of A which is not sensitive to glucose stimulation in its synthesis was not affected by protoplast formation. Similarly unaffected were transports of B, D, E and F in their "residual" form in starved cells. However, transport of these substances after glucose-stimulated synthesis was practically fully suppressed by protoplast formation. This may be connected with the virtually complete inhibition of the proton pump (G) in protoplasts with the implication that B, D, E and F are transported in conjunction with protons but only those that are immediately produced by the proton pump (glucose consumption, oxygen utilization and membrane potential were not substantially altered in protoplasts). Transport of C was stimulated nearly two-fold in protoplasts. Uranyl ions (0.1 mM) had a pronouncedly lower inhibitory effect on all the transports studied in protoplasts.

Amino Acids↗
PubMed1991

Isolation, structural and functional characterization of Staphylococcus aureus protoplasts obtained using lysoamidase.

The action of the lysoamidase bacteriolytic complex on Staphylococcus aureus VKM B-209P cells has been studied to obtain protoplasts. The cells in the midlogarithmic phase were the most sensitive to lysoamidase action. It led to local destruction of cell wall due to hydrolysis of the peptidoglycan. Protoplast formation occurred in two steps in the presence of 1 M sucrose. First, osmotically fragile spheroplasts were formed. Then, the protoplasts were released from the destructed cell wall. The protoplast yield was about 80%. The protoplasts preserved the intact ultrastructure and were able to synthesize peptidoglycan fibrillae. Mainly the spheroplasts that maintained the cell-wall residues reversed into bacterial forms. The protoplasts had respiratory activity similar to cells. Respiration of cells and protoplasts was stimulated by various substrates. High rates of oxygen consumption were observed with alpha-glycerophosphate and ethanol as substrates.

Cell Wall↗
PubMed1976

Mating reaction in yeast protoplasts.

Protoplasts prepared from complementary haploid strains of Saccharomyces cervisiae were studied with regard to their ability of conjugating. Neither fresh protoplasts nor the growing protoplasts possessing fibrillar walls exhibited sex specific agglutination or fusion. However, they were capable of inducing sexual activation in normal cells of opposite mating type. After completing the regeneration of cell walls the protoplasts could conjugate either with each other or with cells of opposite sex. The frequency of conjugations was low, about 1%, and was largely dependent on the degree of completition of the wall during regeneration. From the results the following conclusions may be drawn: 1. The initiation of mating is dependent on the integrity of the cell wall. 2. The sex specific morphogenetic changes do not occur in wall-less protoplasts but may happen after the protoplasts have regenerated their cell walls. 3. The lysis of cell walls does not occur until the walls come into close contact. 4. The fusion of plasma membranes in sex-activated protoplasts cannot be induced by arteficial agglutination.

Cell Wall↗
PubMed2003

Gating of Cl- currents in protoplasts from the marine alga Valonia utricularis depends on the transmembrane Cl- gradient and is affected by enzymatic cell wall degradation.

The electrical properties of protoplasts of the turgor pressure-regulating giant marine alga Valonia utricularis were investigated by using the patch-clamp technique. In the whole-cell configuration, large inward currents were elicited by negative-going voltage pulses. The time-dependent component was predominantly carried by Cl-, as revealed by 'tail current' analysis. When experiments were performed on protoplasts directly after mechanical release from the 'mother cell', small outward currents were additionally observed at membrane voltages more positive than ECl-. These outward currents disappeared to a large extent after treatment of the protoplasts with a mixture of cell wall-degrading enzymes. Plots of the chord conductance versus the clamped membrane voltage revealed that enzymatic treatment affected the gating properties. By fitting Boltzmann distributions to the data, a midpoint potential of + 5 +/- 5 mV (n = 7) was obtained in symmetrical Cl- solutions for mechanically released protoplasts. In contrast, protoplasts treated additionally with enzymes exhibited a midpoint potential of -13 +/- 5 mV (n = 8). By varying the external and internal Cl- concentration, gating was also shown to depend on the Cl- gradient across the plasmalemma both in enzymatically treated and untreated protoplasts. Plotting of the midpoint potential against the Nernst potential of Cl- rendered a slope less than 1 (0.70 and 0.64, respectively) indicating that gating did not strictly depend on the electrochemical Cl- gradient. The voltage- and Cl--dependence as well as inhibition experiments with 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) suggested that the Cl- conductance of the membrane is dominated by the Valonia Anion Channel 1 (VAC1) described by Heidecker, M., Wegner, L.H., Zimmermann, U. 1999: A patch-clamp study of ion channels in proto-plasts prepared from the marine alga Valonia utricularis. J. Membrane Biol. 172:235-247. The relevance of the findings for membrane potential control and turgor regulation in V. utricularis as well as the general implications of the data for electrophysiological work on protoplasts (that are usually obtained by enzymatic digestion of plant tissue) are discussed.

Cells, Cultured↗
PubMed2006

Isolation of cytoplasts from Satsuma mandarin (Citrus unshiu Marc.) and production of alloplasmic hybrid calluses via cytoplast-protoplast fusion.

Cytoplasm of Satsuma mandarin (Citrus unshiu Marc.) is known to influence seedlessness. Transfer of cytoplasm to a seedy cultivar could possibly lead to the production of seedless citrus fruits. In the present paper cytoplasts were isolated from cell suspension-derived protoplasts of Satsuma mandarin via ultra-centrifugation in a discontinuous gradient. No nucleus could be detected in the cytoplasts by DAPI (4', 6-diamidino-2-phenylindole) staining compared with normal protoplasts. The cytoplasts, with high viability and small size, did not divide during solid embedding culture. Cytoplasts of Satsuma mandarin were electrically fused with embryogenic protoplasts of Murcott tangor (C. reticulata x C. sinensis), which led to regeneration of several cell lines. Flow cytometry (FCM) indicated that the cell lines were diploids. Simple sequence repeats (SSR) and cleaved amplified polymorphism sequence (CAPS) showed that the cell lines got their nuclear DNA from the protoplast parent, whereas the cytoplast parent donated the mtDNA, confirming transfer of mtDNA from Satsuma mandarin into Murcott tangor via cytoplast-protoplast fusion though no polymorphism was detected in chloroplast DNA between the fusion partners. This is the first report on isolation and characterization of cytoplasts, together with cytoplast-protoplast fusion in Citrus, which has a potential for citrus cultivar improvement involving cytoplasm transfer via cytoplast-protoplast fusion.

Cell Fusion↗
PubMed2006

Osmotic water permeability of plasma and vacuolar membranes in protoplasts II: theoretical basis.

Water permeability of the plasma membrane (PM) and the vacuolar membrane (VM) is important for intracellular and transcellular water movement in plants, because mature plant cells have large central vacuoles. We have developed a new method for measuring the osmotic water permeability of the PM and VM (P ( f1) and P ( f2), respectively) in individual plant cells. Here, the theoretical basis and procedure of the method are discussed. Protoplasts isolated from higher plant tissues are used to measure P ( f1) and P ( f2). Because of the semi-permeability (selective permeability) of cellular membranes, protoplasts swell or shrink under hypotonic or hypertonic conditions. A theoretical three-compartment model is presented for simulating time-dependent volume changes in the vacuolar and cytoplasmic spaces in a protoplast during osmotic excursions. The model describes the theoretical relationships between P ( f1), P ( f2) and the bulk osmotic water permeability of protoplasts (P ( f(bulk))). The procedure for measuring the osmotic water permeability is: (1) P ( f(bulk)) is calculated from the time when half of the total change in protoplast volume is completed, by assuming that the protoplast has a single barrier to water movement across it (two-compartment model); (2) P ( f2) of vacuoles isolated from protoplasts is obtained in the same manner; and (3) P ( f1) is determined from P ( f(bulk)) and P ( f2) according to the three-compartment model. The theoretical relationship between P ( fl ) (m s(-1)) and L ( Pl ) (hydraulic conductivity, l=1, 2) (m s(-1) Pa(-1)) is also discussed.

Cell Membrane Permeability↗
PubMed1987

Measurement of cytoplasmic calcium in aleurone protoplasts using indo-1 and fura-2.

Previous attempts to measure cytoplasmic Ca2+ in plant cells using the new generation of fluorescent probes, indo-1 and fura-2, have been unsuccessful. We investigated the use of indo-1 and fura-2 to measure cytoplasmic Ca2+ in barley aleurone protoplasts and found that indo-1 could be successfully used when it was loaded into protoplasts in the Ca2+-sensitive form. The acetoxymethyl esters of both dyes accumulated in aleurone protoplasts, but fura-2 was sequestered in the vacuole and indo-1 was not adequately hydrolyzed. We developed a non-disruptive method for loading the Ca2+-sensitive form of indo-1 into aleurone protoplasts in mildly acidic solutions. Using this approach, protoplasts accumulate indo-1 in a pH-dependent manner. The accumulated dye is Ca2+-sensitive, it is not sequestered in vacuoles or the endomembrane system, and it is not rapidly secreted. Fluorescence from indo-1 in individual cells was quenched by Mn2+ in the presence of digitonin. We estimate the cytoplasmic Ca2+ concentration in aleurone protoplasts to be approximately 250 nM. The Ca2+ ionophore, ionomycin does not induce changes in the fluorescence of protoplasts loaded with indo-1, but fluorescence changes could be induced by changes in extracellular Ca2+ in the presence of digitonin. We conclude that the strategy of loading indo-1 at acidic pH provides a useful means of measuring cytoplasmic Ca2+ in the barley aleurone that may also be applicable to other types of plant cells.

Benzofurans↗
PubMed2005

Uptake of sodium in quince, sugar beet, and wheat protoplasts determined by the fluorescent sodium-binding dye benzofuran isophthalate.

The uptake of sodium into protoplasts of quince (Cydonia oblonga Mill, clone BA29), sugar beet (Beta vulgaris L. cv. Monohill), and wheat (Triticum aestivum L. cv. Kadett) was determined by use of the acetoxy methyl ester of the fluorescent sodium-binding benzofuran isopthalate (SBFI-AM). In the presence of 1 mM CaCl2, little sodium was taken up in the cytosol of quince mesophyll cells compared to cytosols of sugar beet and wheat. Upon addition of 40 mM NaCl, approximately the same amount of sodium was taken up in leaf and root protoplasts of wheat, but no sodium was taken up in quince. However, in calcium-free medium, obtained by addition of ethylene glycol tetra acetic acid (EGTA), quince protoplasts transiently took up sodium in the cytosol when 200-400 mM NaCl was added to the protoplast medium. Moreover, after cultivation of quince in the presence of 200 mM sodium for 4 weeks, the cytosol of isolated protoplasts did not take up any sodium at all from a calcium-free medium. The results show that protoplasts from salt tolerant quince only temporarily take up sodium in the cytosol and that they have a mechanism for fast extrusion of sodium from that compartment. These mechanisms are probably important for the high salt tolerance of quince. Calcium blocks the sodium uptake into the cytosol of both quince and wheat protoplasts.

Benzofurans↗
PubMed2003

Production of cell wall accumulative enzymes using immobilized protoplasts of Catharanthus roseus in agarose gel.

Catharanthus roseus cells and protoplasts were used for production of peroxidase and alpha-galactosidase which are accumulated in the cell wall. Only 4% (0.026 U ml(-1)) of the total peroxidase was secreted into the broth by cultured cells while in cultured protoplasts, 45% (0.12 U ml(-1)) was secreted. Protoplasts were protected against the physical and osmotic stresses by immobilizing them in 3% agarose gel (high mass transfer, non-electric charge, low gelation temperature). In order to increase peroxidase production, the immobilized protoplasts were cultivated in shake cultures at low osmotic pressure (12.3 to 16.4 atm) without disruption. During batch peroxidase production, the total activities obtained with free cells at 4.9 atm, free protoplasts at 19.3 atm, and immobilized protoplasts at 12.3 atm were 0.17, 2.54, and 5.16 U, respectively. When four repeated-batch cultures of the immobilized protoplasts were performed at 16.4 atm, 11.8 U of peroxidase was obtained. This system was also useful for the production of alpha-galactosidase.

Catharanthus↗
PubMed2001

Cell death of barley aleurone protoplasts is mediated by reactive oxygen species.

The barley aleurone layer is a terminally differentiated secretory tissue whose activity is hormonally controlled. The plant hormone gibberellic acid (GA) stimulates the secretion of hydrolytic enzymes and triggers the onset of programmed cell death (PCD). Abscisic acid (ABA) antagonizes the effects of GA and inhibits enzyme secretion and PCD. Reactive oxygen species (ROS) are key players in many types of PCD, and data presented here implicate ROS in hormonally regulated death of barley aleurone cells. Incubation of aleurone layers or protoplasts in H(2)O(2)-containing media results in death of GA-treated but not ABA-treated aleurone cells. Cells that are programmed to die are therefore less able to withstand ROS than cells that are programmed to remain alive. Illumination of barley aleurone protoplasts with blue or UV-A light results in a rapid increase in intracellular H(2)O(2) production. GA-treated protoplasts die rapidly in response to this increase in intracellular H(2)O(2) production, but ABA-treated protoplasts do not die. The rate of light-induced death could be slowed by antioxidants, and incubating protoplasts in the dark with the antioxidant butylated hydroxy toluene reduces the rate of hormonally induced death. Taken together, these data demonstrate that GA-treated aleurone protoplasts are less able than ABA-treated protoplasts to tolerate internally generated or exogenously applied H(2)O(2), and strongly suggest that ROS are components of the hormonally regulated cell death pathway in barley aleurone cells.

Abscisic Acid↗
PubMed2003

Sugar uptake and proton release by protoplasts from the infected zone of Vicia faba L. nodules: evidence against apoplastic sugar supply of infected cells.

Symbiotic dinitrogen fixation of legume nodules is fuelled by phloem-imported carbohydrates. These have to pass several cell layers to reach cells infected with Rhizobium bacteroids. It is unclear whether apoplastic steps are involved in carbohyd-rate translocation within the nodule. Protoplasts were isolated from the infected and uninfected cells of the central tissue of Vicia faba nodules using a recently developed protocol. These protoplasts were used to elucidate pathways for sugar transport in this tissue. Both types of protoplasts released protons into the medium. Acidification was inhibited by vanadate and erythrosin B. However, it was stimulated by fusicoccin only in uninfected cells. A symport of sugars with protons can therefore be energized in both cell types. Uptake of 14C-labelled sugars was determined using a phthalate centrifugation technique. Uninfected protoplasts accumulated glucose through high-affinity H+/glucose-symport that was not competitively inhibited by fructose or sucrose. Uninfected protoplasts also absorbed sucrose with biphasic kinetics. At 0.1, 1, and 10 mM sucrose, uptake was inhibited by CCCP. Fusicoccin did not stimulate the linear phase of sucrose uptake. Glucose inhibited sucrose uptake nearly completely. This was not related to sucrose cleavage in the medium because sucrose was absorbed at a much higher rate than glucose, and glucose concentration did not increase in sucrose-containing protoplast suspensions. By contrast with uninfected protoplasts, infected cells did not show transporter-mediated glucose or sucrose uptake. The findings underline a role of uninfected cells in sugar translocation. Infected cells are not apoplastically supplied with sugars and possibly depend on uninfected cells for carbon supply.

Carbohydrate Metabolism↗
PubMed1975

Ribosomal RNA metabolism in cucumber leaf mesophyll protoplasts.

Aspects of the metabolism of RNA have been studied in enzymatically isolated protoplasts from cotyledon and first leaf mesophyll tissue of two cultivars of cucumber. The first leaf mesophyll protoplasts incorporated (3H)-uridine into ribosomal RNA at a constant rate for up to 25 hr in a simple salts medium and for up to 45 hr in a growth medium. Pulse-chase labelling experiments on such preparations showed a rapid dilution of the intracellular (3H)-uridine pool(s) and a high metabolic rate in the cells in one cultivar but not in another. Gel electrophoretic analysis of the RNA from both cotyledon and first leaf protoplasts showed that both protoplast types incorporated either (14C)- or (3H)-uridine into ribosomal RNA species. Incorporation of (3H)-uridine into chloroplasts RNA was minimal in cotyledon protoplasts, but significant in leaf protoplasts. Greater incorporation into the chloroplast RNA species could be achieved by longer pulses. Synthesis of all of the ribosomal RNA species was sensitive to actinomycin D at 10 and 25 mug/ml concentrations in all protoplasts tested.

Cells, Cultured↗
PubMed2001

Effects of movement protein mutations on the formation of tubules in plant protoplasts expressing a fusion between the green fluorescent protein and Cauliflower mosaic virus movement protein.

Fusions between the green fluorescent protein (GFP) and the Cauliflower mosaic virus (CaMV) movement protein (MP) induce the formation of fluorescent foci and surface tubules in Arabidopsis thaliana leaf mesophyll protoplasts. Tubules elongate coordinately and progressively in an assembly process approximately 6 to 12 h following transfection of protoplasts with GFP-MP constructs. Tubules are not formed in protoplasts transfected by GFP-MP(ER2A), a MP mutation that renders CaMV noninfectious. A small number of short tubules are formed on protoplasts transfected by GFP-MP(N6) and GFP-MP(N13), two second-site revertants of ER2A that partially restore infectivity. Protoplasts cotransfected with cyan fluorescent protein (CFP)-MP(WT) and GFP-MP(ER2A) form tubules containing both MP fusions, indicating that although the GFP-MP(ER2A) cannot induce tubule formation, GFP-MP(ER2A) can coassemble or colocalize with CFP-MP(WT) in tubules. Thus, CaMV MP-induced tubule formation in protoplasts correlates closely with the infectivity of mutation ER2A and its revertants, suggesting that tubule-forming capacity in plant protoplasts reflects a process required for virus infection or movement.

Arabidopsis↗
PubMed1983

Genetic interactions in Streptomyces rimosus mediated by conjugation and by protoplast fusion.

The development of a protoplast fusion technique for oxytetracycline-producing Streptomyces rimosus strains, and its evaluation for the application for a breeding programme, has been described. Treatment of S. rimosus protoplasts with 40% (w/v) PEG 1550 for 30 min gave optimal numbers of recombinants ranging from 1 to 10% of the total progeny. Therefore, by comparison with conjugation, protoplast fusion increased the frequency of recombination by two to three orders of magnitude. The proportion of multiple crossover classes amongst recombinants was higher, by a factor of ten, after protoplast fusion (13.3%) than after conjugation (1.5%). Participation of less frequent complementary genotype doubled from 9.0% in conjugation to 17.9% in protoplast fusion. Overall, this suggested that the opportunities for crossing over in a fusion of S. rimosus protoplasts were spatially and/or temporally extended leading to a loosening of linkage with a near-random assortment of genotypes in a cross. However, by minimizing the multiple crossover classes and calculating allele frequency gradients, it was shown that the protoplast fusion technique allows arrangement of genetic markers on the S. rimosus chromosome. These are ideal characteristics for the recombination of divergent lines in a strain improvement programme.

Conjugation, Genetic↗
PubMed1994

Alterations of auxin perception in rolB-transformed tobacco protoplasts. Time course of rolB mRNA expression and increase in auxin sensitivity reveal multiple control by auxin.

Expression and physiological effects of the root-inducing rolB gene of Agrobacterium rhizogenes T-DNA were studied simultaneously in tobacco (Nicotiana tabacum) mesophyll protoplasts. The kinetic study of the expression of rolB mRNA following exogenous auxin application showed that auxin transiently stimulated rolB expression, with mRNA levels starting to accumulate 6 to 9 h after auxin was supplied and increasing 300-fold after 12 to 18 h. The parallel study of the auxin sensitivity of rolB-transformed protoplasts, as assayed by their electrical response to the hormone, showed that the auxin treatment generated an increase in sensitivity by a factor of up to 100,000, whereas in untransformed protoplasts the same auxin treatment induced an increase in auxin sensitivity that never exceeded 30- to 50-fold. This reflects a strong cooperative effect of auxin and rolB in transformed protoplasts. Surprisingly, the maximal increase in sensitivity was observed several hours before the maximal accumulation of rolB mRNA, suggesting that the dramatic control of auxin sensitivity by auxin in rolB-transformed protoplasts requires only low levels of rolB expression. Antibodies directed against ZmER-abp1, the major auxin-binding protein from maize, differentially altered the auxin sensitivity of the electrical response of rolB-transformed and normal protoplasts. This suggests that alterations of the auxin reception-transduction pathway at the plasma membrane of rolB-transformed protoplasts may account for their increased auxin sensitivity.

Agrobacterium tumefaciens↗
PubMed1994

The regulation of gene expression in transformed maize aleurone and endosperm protoplasts. Analysis of promoter activity, intron enhancement, and mRNA untranslated regions on expression.

Gene expression in the aleurone and endosperm is highly regulated during both seed development and germination. Studies of alpha-amylase expression in the aleurone of barley (Hordeum vulgare) have generated the current paradigm for hormonal control of gene expression in germinating cereal grain. Gene expression studies in both the aleurone and endosperm tissues of maize (Zea mays) seed have been hampered because of a lack of an efficient transformation system. We report here the rapid isolation of protoplasts from maize aleurone and endosperm tissue, their transformation using polyethylene glycol or electroporation, and the regulation of gene expression in these cells. Adh1 promoter activity was reduced relative to the 35S promoter in aleurone and endosperm protoplasts compared to Black Mexican Sweet suspension cells in which it was nearly as strong as the 35S promoter. Intron-mediated stimulation of expression was substantially higher in transformed aleurone or endosperm protoplasts than in cell-suspension culture protoplasts, and the data suggest that the effect of an intron may be affected by cell type. To examine cytoplasmic regulation, the 5' and 3' untranslated regions from a barley alpha-amylase were fused to the firefly luciferase-coding region, and their effect on translation and mRNA stability was examined following the delivery of in vitro synthesized mRNA to aleurone and endosperm protoplasts. The alpha-amylase untranslated regions regulated translational efficiency in a tissue-specific manner, increasing translation in aleurone or endosperm protoplasts but not in maize or carrot cell-suspension protoplasts, in animal cells, or in in vitro translation lysates.

Animals↗
PubMed2000

Iron deficiency decreases the Fe(III)-chelate reducing activity of leaf protoplasts.

The ferric-chelate reductase (FC-R) activity of mesophyll protoplasts isolated from Fe-sufficient (control) and Fe-deficient sugar beet (Beta vulgaris L.) leaves has been characterized. Measurements were made in an ionic environment similar to that in the apoplastic space of the sugar beet mesophyll cells. The FC-R activity of Fe-sufficient and Fe-deficient protoplasts was dependent on light. Fe deficiency decreased markedly the FC-R activity per protoplast surface unit. The optimal pH for the activity of the FC-R in mesophyll protoplasts was in the range 5.5 to 6.0, typical of the apoplastic space. Beyond pH 6.0, the activity of the FC-R in mesophyll protoplasts decreased markedly in both Fe-sufficient and Fe-deficient protoplasts. These data suggest that both the intrinsic decrease in FC-R activity per protoplast surface and a possible shift in the pH of the apoplastic space could lead to the accumulation of physiologically inactive Fe pools in chlorotic leaves.

FMN Reductase↗