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At least 199 records · Page 11Linked to original sources
PubMed2000

Invertase production by Saccharomyces cerevisiae protoplasts immobilized in strontium alginate gel beads.

The invertase productivity of Saccharomyces cerevisiae IFO 0309 protoplasts in a static culture was 35 times (extracellular) and 9 times (extracellular + intracellular) higher than those of cells. When S. cerevisiae protoplasts were immobilized in 1.0% strontium alginate gel as an artificial cell wall, the protoplasts could be cultivated in a shake flask without disruption, and invertase was secreted into the broth. However, cell wall regeneration in the immobilized protoplasts was detected at about 24 h of cultivation. This implies that prevention of cell wall regeneration is a prerequisite for long term process with protoplasts. When 0.5 microg/ml aculeacin A (inhibitor of beta-1,3 glucan synthesis) was added to the broth, active protoplasts were maintained without cell wall regeneration for more than 24 h and invertase was produced extracellularly. Immobilized S. cerevisiae T7 protoplasts (this strain produces twice as much invertase as S. cerevisiae IFO 0309) were used for invertase production in a bubble column reactor, and a high and stable level of invertase (45 U/ml) was maintained for 72 h.

Journal Article↗
PubMed1988

Transformation of the cryobehavior of rye protoplasts by modification of the plasma membrane lipid composition.

The freezing tolerance of protoplasts isolated from nonacclimated rye leaves (Secale cereale L. cv Puma) was significantly altered by using a pH-induced protoplastliposome fusion technique to modify the lipid composition of the plasma membrane. The increase in freezing tolerance was elicited by fusion with liposomes composed of either the total phospholipid fraction isolated from the plasma membrane of cold-acclimated leaves or single mono- or diunsaturated species of phosphatidylcholine (PtdCho). Of the PtdCho species tested, dilinoleoylphosphatidylcholine ([Lin(2)]PtdCho) and dilinolenoylphosphatidylcholine ([Lnn(2)]PtdCho) liposomes were the most effective; 1-palmitoyl-2-oleoylphosphatidylcholine, 1-palmitoyl-2-linoleoylphosphatidylcholine, or dioleoylphosphatidylcholine liposomes were somewhat less effective; dimyristoylphosphatidylcholine or dipalmitoylphosphatidylcholine liposomes had no effect. The increased freezing tolerance was the result of a transformation in the cryobehavior of the plasma membrane during freeze-induced osmotic contraction. In control nonacclimated protoplasts, osmotic contraction resulted in endocytotic vesiculation of the plasma membrane which was irreversible and resulted in lysis during osmotic expansion after melting of the suspending medium. In nonacclimated protoplasts fused with mono- or diunsaturated species of PtdCho, osmotic contraction resulted in the reversible formation of exocytotic extrusions of the plasma membrane-as normally occurs in protoplasts isolated from cold-acclimated leaves (acclimated protoplasts). In scanning electron micrographs, the morphology of the extrusions of nonacclimated protoplasts fused with [Lin(2)]PtdCho was virtually indistinguishable from that of the extrusions formed in acclimated protoplasts. These studies provide direct evidence that changes in the lipid composition of the plasma membrane are causally related to one facet of the cold-acclimation process.

Journal Article↗
PubMed1996

The Complexity of Enzymic Control of Hydrogen Peroxide Concentration May Affect the Regeneration Potential of Plant Protoplasts.

Total peroxidase, NADH-peroxidase, ascorbate peroxidase, superoxide dismutase, and catalase activities were measured in tobacco (Nicotiana tabacum) leaves and in regenerating and nonregenerating protoplasts isolated from the same tissue and cultured for 2 weeks. The specific ranges of H2O2 concentration at which the enzymes scavenging the active forms of oxygen may efficiently operate and the activities of those enzymes were determined in an extract from tobacco leaves and in dividing and nondividing tobacco mesophyll protoplasts. The overall H2O2-scavenging enzyme activities were similar in both protoplast populations during the 2 to 3 d of culture. After 3 d, the regenerating protoplasts started to divide and both the antioxidant enzyme activities and the total peroxidase activity increased; in contrast, the viability and the H2O2-scavenging enzyme activities in nonregenerating protoplasts dramatically decreased. Surprisingly, the regenerative potentiality in dividing protoplasts was specifically correlated with a higher NADH-peroxidase activity, which resulted in a net H2O2 accumulation in the cells. Light, which causes the accumulation of active forms of oxygen in photosynthetic organelles, also stimulated catalase and ascorbate peroxidase activities in dividing protoplasts. We suggest that the localization of H2O2 rather than its absolute concentration might be responsible for oxidative stress and that controlled amounts of H2O2 are necessary to allow proper cell-wall reconstitution and the consequent cell division.

Journal Article↗
PubMed1981

Parameters influencing the liposome-mediated insertion of fluorescein diacetate into plant protoplasts.

Maximum uptake of liposome-encapsulated fluorescein diacetate by Daucus carota protoplasts was observed when 6 x 10(6) protoplasts per milliliter were incubated with 2.4 x 10(7) liposomes per milliliter for 1 hour. In the case of Nicotiana glutinosa protoplasts, optimum ratio of protoplasts to liposomes was 1:10, where 2.3 x 10(5) protoplasts per milliliter were provided. Neutral and positive liposomes were found to be efficient vehicles to transfer their contents into plant protoplasts. When protoplasts treated with liposomes were cultured in a synthetic medium for 1 week, 20% resumed cell divisions.

Journal Article↗
PubMed1982

Isolation of NADH Oxidation System from the Plasmalemma of Corn Root Protoplasts.

A plasmalemma-bound NADH oxidation system (Lin 1982 Proc Natl Acad Sci USA 79: 3773-3776) in corn root protoplasts was isolated by a mild treatment of intact protoplasts with trypsin. The majority of NADH stimulated O(2) consumption activity of the protoplasts could be recovered in the supernatant isolated from the intact protoplasts which have been treated with trypsin. The activation energy of NADH oxidation in the supernatant is similar to that of the intact protoplasts (8.7 versus 9.4 kilocalories per mole per degree). Unlike that of the intact protoplasts, an Arrhenius plot of the temperature response (from 5 to 25 degrees C) of the activity in the supernatant shows no transition suggestive of a dissociation of the enzyme from the membrane. Trypsin treatment did not affect K(+) uptake into cell volume of the protoplast. However, the NADH-stimulated K(+) uptake and the increase of cell volume were greatly reduced. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of trichloroacetic acid-precipitated protein from the supernatant showed one extra peptide band with approximately 42 kilodalton molecular weight.

Journal Article↗
PubMed1985

A simple method for estimating intactness of spinach leaf protoplasts by glycolate oxidase assay.

A method was developed for the quantitative analysis of intactness of spinach leaf protoplasts using glycolate oxidase activity as an index. Since glycolate does not penetrate into protoplasts at neutral pH, the increase of O(2) consumption by the addition of glycolate to protoplast suspension was due to the glycolate oxidase activity released from damaged protoplasts. The proportion of damaged protoplasts in the whole preparation was calculated from the ratio of released and total glycolate oxidase activity. Freshly prepared spinach leaf protoplasts were found to be 80 to 90% intact as estimated by the method. The effect of osmolarity on the respiratory activities of spinach leaf protoplasts was also examined by applying the same principle.

Journal Article↗
PubMed1985

Intracellular calcium and calmodulin involvement in protoplast fusion.

(45)Ca(2+) uptake was compared between fusogenic and nonfusogenic Daucus carota L. protoplasts. Fusogenic protoplasts took 10 minutes to reach calcium equilibrium compared to 5 minutes in the nonfusogenic protoplasts. Intracellular stores of calcium were manipulated by isolating protoplasts in different calcium regimes. Lowering of intracellular calcium lowered fusion potential, while raising intracellular stores of calcium enhanced fusion potential. Regardless of the amount of calcium sequestered in a store, mobilization with A23187 increased fusion levels within 10 minutes. Calmodulin antagonists were potent inhibitors of protoplast fusion. This inhibition was obtained by treating cells with the calmodulin antagonists during protoplast isolation. A23187, however, only allowed a partial recovery from this inhibition, indicating that calcium flux alone was not sufficient for maximum fusion potential. On the basis of the evidence presented, we propose that calcium fluxes during protoplast isolation activate a calmodulin-mediated biochemical process that is necessary for the formation or maintenance of a fusion permissive state.

Journal Article↗
PubMed1986

Hormonal Regulation of alpha-Amylase Gene Transcription in Wild Oat (Avena fatua L.) Aleurone Protoplasts.

The time of appearance and relative amounts of alpha-amylase mRNA in wild oat (Avena fatua L.) aleurone protoplasts incubated with 1 micromolar gibberellin A(4) (GA(4)) were closely correlated with the amounts of alpha-amylase enzyme secreted by the protoplasts. In the absence of GA(4), or when protoplasts were incubated with 25 micromolar abscisic acid (ABA) together with 1 micromolar GA(4) no alpha-amylase mRNA was detected and only very low levels of alpha-amylase were secreted. Nuclei were isolated in high yields (65-71%) from aleurone protoplasts and in an in vitro transcription system displayed characteristics of a faithful DNA-dependent RNA synthesizing system. The time course of incorporation of [(3)H]-UTP suggested that the RNA synthesized was mainly ;run off' transcription and therefore that the transcripts produced in vitro were those being synthesized in the protoplasts at the times when the nuclei were isolated. By hybridizing in vitro synthesized [(32)P]RNA to barley alpha-amylase cDNA and control filters we have estimated that 90 +/- 10 ppm of the transcripts synthesized by nuclei isolated from GA(4) treated protoplasts can be attributed to alpha-amylase sequences and that statistically insignificant amounts of these transcripts are obtained from control and GA(4) plus ABA treatments. The results suggest that GA(4) and ABA influence the transcription of alpha-amylase genes in aleurone protoplasts of wild oat.

Journal Article↗
PubMed1986

Ca-stimulated secretion of alpha-amylase during development in barley aleurone protoplasts.

The effects of gibberellic acid (GA(3)) and Ca(2+) on the synthesis and secretion of alpha-amylase from protoplasts of barley (Hordeum vulgare L. cv Himalaya) aleurone were studied. Protoplasts undergo dramatic morphological changes whether or not the incubation medium contains GA(3), CaCl(2), or both. Incubation of protoplasts in medium containing both GA(3) and Ca(2+), however, causes an increase in the alpha-amylase activity of both incubation medium and tissue extract relative to controls incubated in GA(3) or Ca(2+) alone. Isoelectric focusing shows that adding Ca(2+) to incubation media containing GA(3) increases the levels of alpha-amylase isozymes having high isoelectric points (pI). In the presence of GA(3) alone, only isozymes with low pIs accumulate. The increase in alpha-amylase activity in the incubation medium begins after 36 hours of incubation, and secretion is complete after about 72 hours. Protoplasts require continuous exposure to Ca(2+) to maintain elevated levels of alpha-amylase release. Immunoelectrophoresis shows that Ca(2+) stimulates the release of low-pI alpha-amylase isozymes by 3-fold and high-pI isozymes by 30-fold over controls incubated in GA(3) alone. Immunochemical data also show that the half-maximum concentration for this response is between 5 and 10 millimolar CaCl(2). The response is not specific for Ca(2+) since Sr(2+) can substitute, although less effectively than Ca(2+). Pulse-labeling experiments show that alpha-amylase isozymes produced by aleurone protoplasts in response to GA(3) and Ca(2+) are newly synthesized. The effects of Ca(2+) on the process of enzyme synthesis and secretion is not mediated via an effect of this ion on alpha-amylase stability or on protoplast viability. We conclude that Ca(2+) directly affects the process of enzyme synthesis and transport. Experiments with protoplasts also argue against the direct involvement of the cell wall in Ca(2+)-stimulated enzyme release.

Journal Article↗
PubMed1988

Effects of la on surface charges, dielectrophoresis, and electrofusion of barley protoplasts.

When dielectrophoresis and electrofusion of barley (Hordeum vulgare var Moor) leaf protoplasts were assayed in the presence of 0.1 to 1 millimolar lanthanum ion (La(3+)) in the basal medium (0.7 molar mannitol, 1 millimolar piperazine-N, N-bis[2-ethanesulfonic acid]-Na [pH 6.7], 0.1 millimolar CaCl(2)), dielectrophoresis and induction of electrofusion were strongly inhibited. The latter remained inhibited and the former recovered by about 60% after washing the La(3+) -treated protoplasts without EDTA. These inhibitions were almost completely abolished by washing the La(3+) -treated protoplasts with 1 millimolar EDTA. Inductively coupled plasma atomic emission spectroscopic analysis revealed that protoplasts retained a considerable amount of La(3+) after washing without EDTA and released most of the bound La(3+) by washing with 1 millimolar EDTA. This tightly bound La(3+) seemed responsible for the inhibition of electrofusion and dielectrophoresis that was observed in the La(3+) -treated protoplasts after washing. zeta-potentials of protoplasts were -39.0+/-3.2 millivolts, -16.7 +/- 2.6 millivolts, and virtually zero in media containing 0, 0.1, and 0.3 millimolar La(3+) (I = 7.2 millimolar), respectively, and had a positive value (+ 14.2 +/- 2.2 millivolts) in the presence of 1 millimolar La(3+). These effects of La(3+) on zeta-potentials were easily abolished by washing without EDTA. This indicates that charged species located at the surface of plasma membrane of protoplasts cannot account for the sites at which La(3+) exerts its inhibition of dielectrophoresis and electrofusion. In contrast, the promotion of spherical fusion and the reduction of broken fusion products observed in the presence of La(3+) were almost completely abolished by washing without EDTA. Our results also indicate that the initial induction and development of electrofusion can be studied independently.

Journal Article↗
PubMed1991

In Plant Protoplasts, the Spontaneous Expression of Defense Reactions and the Responsiveness to Exogenous Elicitors Are under Auxin Control.

When auxin was omitted during either the preparation or the culture of tobacco mesophyll protoplasts, as well as during both periods, synthesis of beta-glucanase was spontaneously induced. In contrast, when protoplasts were prepared and cultured in the presence of 16 micromolar 1-naphthaleneacetic acid (optimal concentration for protoplast division), the expression of beta-glucanase was maintained close to the minimal level observed in tobacco leaves. This inhibitory effect was only promoted by active auxins (1-naphthaleneacetic acid, 2,4-dichlorophenoxyacetic acid, 2,4,5-trichlorophenoxyacetic acid, and 3-indoleacetic acid) but not by inactive auxin analogs. Tobacco protoplasts responded to exogenous elicitors from the cell wall of Phytophthora megasperma glycinea (Pmg) by accumulating beta-glucanase in the presence of 16 micromolar 1-naphthaleneacetic acid. At higher auxin concentrations, the elicitor-induced beta-glucanase synthesis was inhibited. Naphthaleneacetic acid concentration (3 x 10(-5) molar) required to inhibit by 50% the expression of this defense reaction triggered by a near-optimal elicitor concentration was about 100 times higher than that sufficient to inhibit by 50% the spontaneous expression in nonelicited protoplasts. This is the first demonstration of an auxin-fungal elicitor interaction in the control of a defined defense reaction. The above observations were extended to soybean cell protoplasts. The Pmg elicitor-induced stimulation of the synthesis of pathogenesis related P17 polypeptides and of a 39-kilodalton peptide immunologically related to tobacco beta-glucanase was only observed when the spontaneous accumulation of these proteins was inhibited in auxin-treated protoplasts.

Journal Article↗
PubMed1992

Dark Respiration Protects Photosynthesis Against Photoinhibition in Mesophyll Protoplasts of Pea (Pisum sativum).

The optimal light intensity required for photosynthesis by mesophyll protoplasts of pea (Pisum sativum) is about 1250 microeinsteins per square meter per second. On exposure to supra-optimal light intensity (2500 microeinsteins per square meter per second) for 10 min, the protoplasts lost 30 to 40% of their photosynthetic capacity. Illumination with normal light intensity (1250 microeinsteins per square meter per second) for 10 min enhanced the rate of dark respiration in protoplasts. On the other hand, when protoplasts were exposed to photoinhibitory light, their dark respiration also was markedly reduced along with photosynthesis. The extent of photoinhibition was increased when protoplasts were incubated with even low concentrations of classic respiratory inhibitors: 1 micromolar antimycin A, 1 micromolar sodium azide, and 1 microgram per milliliter oligomycin. At these concentrations, the test inhibitors had very little or no effect directly on the process of photosynthetic oxygen evolution. The promotion of photoinhibition by inhibitors of oxidative electron transport (antimycin A, sodium azide) and phosphorylation (oligomycin) was much more pronounced than that by inhibitors of glycolysis and tricarboxylic acid cycle (sodium fluoride and sodium malonate, respectively). We suggest that the oxidative electron transport and phosphorylation in mitochondria play an important role in protecting the protoplasts against photoinhibition of photosynthesis. Our results also demonstrate that protoplasts offer an additional experimental system for studies on photoinhibition.

Journal Article↗
PubMed1994

Regeneration of Fertile Barley Plants from Mechanically Isolated Protoplasts of the Fertilized Egg Cell.

A simple procedure is described for the mechanical isolation of protoplasts of unfertilized and fertilized barley egg cells from dissected ovules. Viable protoplasts were isolated from ~75% of the dissected ovules. Unfertilized protoplasts did not divide, whereas almost all fertilized protoplasts developed into microcalli. These degenerated when grown in medium only. When cocultivated with barley microspores undergoing microspore embryogenesis, the protoplasts of the fertilized egg cells developed into embryo-like structures that gave rise to fully fertile plants. On average, 75% of cocultivated protoplasts of fertilized egg cells developed into embryo-like structures. Fully fertile plants were regenerated from ~50% of the embryo-like structures. The isolation-regeneration techniques may be largely genotype independent, because similar frequencies were obtained in two different barley varieties with very different performance in anther and microspore culture. Protoplasts of unfertilized and fertilized eggs of wheat were isolated by the same procedure, and a fully fertile wheat plant was regenerated by cocultivation with barley microspores.

Journal Article↗
PubMed1985

Efficient plasmid transformation of Streptomyces ambofaciens and Streptomyces fradiae protoplasts.

A procedure for efficient transformation of Streptomyces ambofaciens and Streptomyces fradiae protoplasts with plasmid DNA was developed. Transformation frequencies with S. fradiae protoplasts were strongly influenced by the temperatures for cell growth, protoplast formation, and protoplast regeneration. Transformation frequencies for both species were also influenced by the culture age before protoplast formation, the source and concentration of polyethylene glycol, the transformation-inducing agent, the concentration of protoplasts used in the transformation procedure, and the number of protoplasts added to regeneration plates. Transformation frequencies were substantially higher for both species when calf thymus DNA and protamine sulfate were added to the transformation mix. With S. fradiae, transformation frequencies were much lower with plasmid DNA prepared from other species than with the same plasmids prepared from S. fradiae, suggesting that S. fradiae expresses restriction and modification. With the modified transformation procedures using DNA prepared from homologous hosts, S. ambofaciens and S. fradiae are now transformed routinely at frequencies of 10(6) to 10(7) transformants per micrograms of plasmid DNA.

DNA↗
PubMed1977

Yeast protoplasts from stationary and starved cells: preparation, ultrastructure and vacuolar development.

The conversion of stationary and starved yeast cells into protoplasts is described. The method is rapid, simple and can be applied to a variety of stationary yeast cells. Preincubation of yeast cells in the presence of pronase was essential for effective conversion into protoplasts. Baker's yeast and seven defined yeast strains, including one "petite", were studied. All of them were efficiently transformed into protoplasts in 60 to 90 min, depending on the strain culture conditions and the age of the culture. Protoplasts may be obtained even from late-stationary cells which contain spores. Saccharomyces cerevisiae cells subjected to complete starvation conditions in water, could also be completely transformed into protoplasts, even after 48 h of starvation. Electron microscope examination of stationary protoplasts from three different yeast strains showed no evidence of a remaining cell-wall. S. cerevisiae stationary cells show a very developed vacuolar system, a number of "lipid granules" and a few altered mitochondria. Endomycopsis fibuligera and Candida tropicalis stationary protoplasts show a similar fine structure, but "lipid granules" were completely absent.

Culture Media↗
PubMed1994

Auxin inducibility and developmental expression of axi 1: a gene directing auxin independent growth in tobacco protoplasts.

We describe the characterization of axi 1, a tobacco gene isolated by activation T-DNA tagging which apparently plays a role in auxin action. Upon deregulated expression, axi 1 confers on protoplasts the ability to grow in culture not only in the absence of auxin but also in high auxin concentrations where maximal frequencies of cell division are not observed in wild-type protoplasts. In wild-type plants axi 1 is transcribed principally in root tissue. In the tagged plant line, axi 159, axi 1 RNA can be detected in all tissues tested. Freshly isolated wild-type protoplasts require auxin for the accumulation of detectable levels of axi 1 transcript and this precedes maximal levels of cell division. In contrast, axi 1 RNA appears in protoplasts isolated from axi 159 plants in the absence of auxin. axi 1 was localized to 6.2 kb of plant genomic DNA flanking the right T-DNA border sequence. axi 1 is interrupted by nine introns and in tobacco it is a member of a small gene family. Database searching reveals no similarity within the coding region with other genes. Sequences within the fourth intron are similar to those located in the non-coding regions of other plant genes, some of which are known to be auxin inducible. A DNA fragment containing the conserved sequence acts as an auxin responsive element in transient expression assays in wild-type protoplasts and this response is higher in axi 159 protoplasts. This suggests that auxin induced axi 1 expression may be mediated by a region contained within an intron sequence and that the axi 1 product might play a role in this induction.

Base Sequence↗
PubMed1996

Electroporation of small RNAs into plant protoplasts: mitochondrial uptake of transfer RNAs.

To study tRNA import into plant mitochondria, we have set up a system to follow the fate in vivo of tRNA transcripts introduced into plant protoplasts by electroporation. Conditions were optimized for maximum tRNA uptake into potato protoplasts. We have shown that in vitro synthesized tRNA transcripts are poor substrates due to rapid degradation leading to low efficiencies of transfer and short life in protoplasts. Labelled natural tRNAs were more efficiently electroporated into protoplasts and they remained stable during protoplast culture. We have observed import into mitochondria of total and purified cytosolic tRNAs in protoplasts but the process was not specific for the tRNA species which are normally imported.

Cells, Cultured↗
PubMed1983

Advances in protoplast fusion and transformation in Streptomyces.

Rapid advances have been made in recent years on protoplast research in the economically important Streptomyces. The use of protoplasts has facilitated the development of efficient techniques for intra- and interspecific genetic recombination by fusion and by gene cloning. This report summarizes current protoplast methodologies as they relate to both protoplast fusion and genetic transformation, points out some genetic instabilities associated with protoplast techniques, and speculates on future directions to broaden the applications of protoplasts for heterospecific gene recombination and cloning in Streptomyces.

Cloning, Molecular↗