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At least 703 records · Page 39Linked to original sources
PubMed1980

Purine metabolism in mesophyll protoplasts of tobacco (Nicotiana tabacum) leaves.

The overall metabolism of purines was studied in tobacco (Nicotiana tabacum) mesophyll protoplasts. Metabolic pathways were studied by measuring the conversion of radioactive adenine, adenosine, hypoxanthine and guanine into purine ribonucleotides, ribonucleosides, bases and nucleic acid constituents. Adenine was extensively deaminated to hypoxanthine, whereupon it was also converted into AMP and incorporated into nucleic acids. Adenosine was mainly hydrolysed to adenine. Inosinate formed from hypoxanthine was converted into AMP and GMP, which were then catabolized to adenine and guanosine respectively. Guanine was mainly deaminated to xanthine and also incorporated into nucleic acids via GTP. Increased RNA synthesis in the protoplasts resulted in enhanced incorporation of adenine and guanine, but not of hypoxanthine and adenosine, into the nucleic acid fraction. The overall pattern of purine-nucleotide metabolic pathways in protoplasts of tobacco leaf mesophyll is proposed.

Adenine↗
PubMed1982

Observations on the phosphate status and intracellular pH of intact cells, protoplasts and chloroplasts from photosynthetic tissue using phosphorus-31 nuclear magnetic resonance.

Individual pools of intracellular inorganic phosphate (Pi) can be observed in the dark in intact cells, protoplasts and chloroplasts from photosynthetic tissue by using 31P nuclear magnetic resonance (n.m.r.). Estimates for the pH of vacuolar and extravacuolar compartments are reported although it is shown that intracellular pH is determined by the pH of the suspending medium. Mannose treatment of asparagus (Asparagus officinalis) cells and spinach (Spinacia oleracea) protoplasts results in the inhibition of photosynthesis. The mechanism of mannose phosphate sequestration of free Pi is supported by the 31P n.m.r. spectra of mannose-treated tissue. There is a fundamental difference in 31 P n.m.r. spectra of mannose-treated spinach protoplasts and asparagus cells, reflecting a difference in the availability of vacuolar Pi for cellular metabolism in these species. The 31P n.m.r. spectrum of intact spinach chloroplasts is reported.

Chloroplasts↗
PubMed1982

Isolation of plasma membrane from protoplasts of Lolium multiflorum (ryegrass) endosperm cells.

Plasma membranes have been isolated from protoplasts of suspension-cultured ryegrass (Lolium multiflorum) endosperm cells. The protoplast membrane is coated before cell disruption with murine myeloma protein J539, a galactose-binding immunoglobulin A. The plasma membrane is labelled with 125I by using chemically or enzymically catalysed iodination techniques, or, more conveniently, by using 125I-labelled myeloma protein J539, which enables the membrane to be simultaneously coated and labelled. Protoplast lysis is effected by gentle mechanical means after swelling in hypo-osmotic medium. The plasma-membrane fraction is recovered at low centrifugal forces by fractionation of cell lysates on a discontinuous sucrose/sorbitol gradient. The plasma-membrane fraction is enriched 96-fold on a protein basis with respect to the specific radioactivity of 125I-labeled myeloma protein J539 in the homogenate. Electron microscopy showed long membrane profiles often associated with one another.

Carrier Proteins↗
PubMed2003

Direct delivery of bacterial avirulence proteins into resistant Arabidopsis protoplasts leads to hypersensitive cell death.

Many bacterial avirulence (Avr) proteins, including the Pseudomonas syringae proteins, AvrRpt2 and AvrB, appear to be recognized inside the host plant cell by resistance mechanisms mediated by the cognate resistance (R) genes. It is thought that Avr proteins are either delivered directly into the host cell via the bacterial type III secretion system (TTSS) or taken up by the plant cell following secretion into the apoplast through the TTSS. Recently, it was shown that the Xanthomonas campestris AvrBs2 protein can be delivered directly into the host plant cell by the TTSS. However, it is not known whether other type III effectors of phytopathogens behave similarly. Here, using a novel protein transfection method, we demonstrate that AvrRpt2 and AvrB must enter the plant cell to be recognized by R gene-mediated mechanisms. First, we established a hypersensitive cell death assay for protoplasts using the membrane-impermeable, nuclear-staining dye, YO-PRO-1, and transgenic Arabidopsis plants that carry an inducible avrRpt2 gene. Second, we transfected E. coli-produced AvrRpt2 or AvrB proteins into Arabidopsis protoplasts using a protein transfection kit based on the carrier peptide Pep-1, and demonstrated that hypersensitive cell death occurs in a gene-for-gene-specific manner. In contrast, these Avr proteins failed to elicit hypersensitive cell death when they were applied to protoplasts without the carrier peptide. We conclude that our preparations of E. coli-produced AvrRpt2 and AvrB are active, that AvrRpt2 and AvrB must be delivered into the plant cell to be recognized, and that a method based on a carrier peptide can be used to introduce proteins into plant cells.

Arabidopsis↗
PubMed1994

Transposition of a Ds element from a plasmid into the plant genome in Nicotiana plumbaginifolia protoplast-derived cells.

Nicotiana plumbaginifolia haploid protoplasts were co-transformed with two plasmids, one with a NPT-II/Ds element and one with a gene encoding an amino-terminal truncated Ac transposase. It is shown that Ds can efficiently transpose from extrachromosomal DNA to N. plumbaginifolia chromosomes when the Ac transposase gene is present in trans. Ds has been shown to have transposed into the plant genome in a limited number of copies (1.9 copies per genome), for 21/32 transgenic lines tested. The flanking sequences present in the original plasmid are missing in these 21 plants. In only two of 21 plants was part of the transposase construct integrated. By segregation analysis of transgenic progeny, Ds was shown to be present in the heterozygous state in 10 lines even though haploid protoplasts had been originally transformed. This observation could indicate that integration occurred after or during DNA replication that leads to protoplast diploidization.

Base Sequence↗
PubMed2000

Early activation of lipoxygenase in lentil (Lens culinaris) root protoplasts by oxidative stress induces programmed cell death.

Oxidative stress caused by hydrogen peroxide (H2O2) triggers the hypersensitive response of plants to pathogens. Here, short pulses of H2O2 are shown to cause death of lentil (Lens culinaris) root protoplasts. Dead cells showed DNA fragmentation and ladder formation, typical hallmarks of apoptosis (programmed cell death). DNA damage was evident 12 h after the H2O2 pulse and reached a maximum 12 h later. The commitment of cells to apoptosis caused by H2O2 was characterized by an early increase of lipoxygenase activity, of ultraweak luminescence and of membrane lipid peroxidation, which reached 720, 350 and 300% of controls, respectively, at 6 h after H2O2 treatment. Increased lipoxygenase activity was paralleled by an increase of its protein and mRNA level. Lipoxygenase inhibitors nordihydroguaiaretic acid, eicosatetraynoic acid and plamitoyl ascorbate prevented H2O2-induced DNA fragmentation and ultraweak luminescence, only when added together with H2O2, but not when added 8 h afterwards. Inhibitory anti-lipoxygenase monoclonal antibodies, introduced into the protoplasts by electroporation, protected cells against H2O2-induced apoptosis. On the other hand, lentil lipoxygenase products 9- and 13-hydroperoxy-octadecadienoic acids and their reduced alcohol derivatives were able to force the protoplasts into apoptosis. Altogether, these findings suggest that early activation of lipoxygenase is a key element in the execution of apoptosis induced by oxidative stress in plant cells, in a way surprisingly similar to that observed in animal cells.

Apoptosis↗
PubMed1994

Light induces rapid changes of the phosphorylation pattern in the cytosol of evacuolated parsley protoplasts.

The fractionation of cells of a parsley suspension culture [Petroselinum crispum (Mill.) A. Hill] by protoplasting and subsequent removal of the vacuoles led to physiologically intact evacuolated protoplasts retaining light inducibility of chalcone synthase expression. Lysis of the evacuolated protoplasts permitted the isolation of a pure, highly concentrated cytosolic fraction containing major cytosolic membranes but only minor contamination by proplastids, mitochondria, and nuclei. Short-time irradiations of the cytosol with red or UV-containing white light resulted in very fast changes of the phosphorylation pattern of 18-, 40-, 48-, 55- to 70-, and 120-kDa proteins. Major differences were observed between the phosphorylation patterns obtained by red or UV-containing white light treatment, indicating a different primary action of the excited photoreceptors in vitro. Separation of the microsomal fraction from the cytosolic matrix established the localization of these proteins. Chase and photoreversibility experiments revealed that phytochrome in vitro regulates the phosphorylation of the 40-kDa protein by modifying a soluble cytosolic kinase/phosphatase system.

Cell Fractionation↗
PubMed1997

T-strand integration in maize protoplasts after codelivery of a T-DNA substrate and virulence genes.

We describe a plant protoplast transformation method that provides transformants with a simple pattern of integration of a foreign gene. The approach is to deliver into plant protoplasts by direct gene transfer the Agrobacterium virulence genes virD1 and virD2 with or without virE2, together with a target plasmid containing a gene of interest flanked by Agrobacterium T-DNA border repeat sequences of 25 bp. We present evidence of T-DNA formation in maize protoplasts and its integration into the maize genome. The frequency of VirD1-VirD2-mediated integration events was about 20-35% of the total number of transformants. The addition of virE2 doubled the transformation efficiency. The method described here is of sufficient efficiency and simplicity to be useful for the production of transgenic plants with single-copy well-defined transgenic inserts.

Bacterial Proteins↗
PubMed2002

Isolation and regeneration of protoplasts from the yeast and mycelial form of the dimorphic zygomycete Benjaminiella poitrasii: role of chitin metabolism for morphogenesis during regeneration.

Experimental parameters for isolation and regeneration of protoplasts from the mycelial and yeast form cells of the dimorphic zygomycete Benjamininiella poitrasii are reported. Using a chitosanase containing preparation from Streptomyces sp. MCl we obtained protoplasts after 5 h incubation with a yield of 2+/-0.3 x 10(6) ml(-1) and 3+/-0.4 x 10(7) ml(-1) for the mycelial and yeast form, respectively. During regeneration under conditions triggering dimorphism the two morphological forms were observed after 36 h. Initially, for 10-12 h only an irregular mass was formed as a result of deregulated cell wall synthesis. Among the tested inhibitors influencing cell wall metabolism, chitin metabolism inhibitors showed distinctive effects on the regeneration of protoplasts suggesting that the respective enzymes significantly contribute to determining the morphogenesis of the dimorphic fungus B. poitrasii.

Acetylglucosaminidase↗
PubMed1986

Monoclonal antibodies directed against protoplasts of soybean cells: analysis of the lateral mobility of plasma membrane-bound antibody MVS-1.

A monoclonal antibody (MVS-1) was used to monitor the lateral mobility of a defined component (Mr approximately 400,000) of the plasma membrane of soybean protoplasts prepared from suspension cultures of Glycine max (SB-1 cell line). The diffusion coefficient (D) of antibody MVS-1 bound to its target was determined (D = 3.2 X 10(-10) cm2/s) by fluorescence redistribution after photobleaching. Pretreatment of the protoplasts with soybean agglutinin (SBA) resulted in a 10-fold reduction of the lateral mobility of antibody MVS-1 (D = 4.1 X 10(-11) cm2/s). This lectin-induced modulation could be partially reversed by prior treatment of the protoplasts with either colchicine or cytochalasin B. When used together, these drugs completely reversed the modulation effect induced by SBA. These results have refined our previous analysis of the effect of SBA on receptor mobility to the level of a defined receptor and suggest that the binding of SBA to the plasma membrane results in alterations in the plasma membrane such that the lateral diffusion of other receptors is restricted. These effects are most likely mediated by the cytoskeletal components of the plant cell.

Antibodies, Monoclonal↗
PubMed1959

Studies of L forms and protoplasts of group A streptococci. I. Isolation, growth, and bacteriologic characteristics.

L forms of Group A streptococci have been isolated by the use of penicillin gradient agar plates. Osmotically fragile protoplasts of Group A streptococci have been obtained by the use of Group C phage-associated lysin which lyses Group A streptococci and their isolated cell walls. Membranes surrounding these enzymatically derived protoplasts have been isolated, and chemical and immunological studies indicate that they are free of cell wall carbohydrate and M protein. The streptococcal protoplasts reproduce as colonies which are morphologically indistinguishable from streptococcal L forms. Evidence is presented to show that these two streptococcal derivatives are serologically and physiologically related to each other as well as to the parent streptococcal strain from which they were isolated.

Bacteriophages↗
PubMed1997

A plant cation-chloride co-transporter promoting auxin-independent tobacco protoplast division.

Although auxin plays a central role in plant development, little is known about the signal transduction pathways triggered by auxin regulating cell elongation, division and differentiation. We describe the molecular analysis of the mutant tobacco line axi 4/1, which was regenerated from an auxin-independent callus created by activation T-DNA tagging. Transcriptional enhancer-mediated deregulated expression of the tagged plant gene axi 4 uncouples division of axi 4/1 protoplasts from external auxin stimuli, whereas in untransformed protoplasts expression of axi 4 and cell division are auxin dependent. axi 4 encodes a 109 kDa protein with significant homology to a family of electroneutral cation-chloride co-transporters (CCCs). We show that overexpression of AXI 4 or another member of the CCC family, the Na+/K+/2Cl--co-transporter from shark, triggers auxin-independent growth of tobacco protoplasts. We suggest that Na+/K+/2Cl--co-transporters may play a role in signalling cell division and that this function is highly conserved between AXI 4 and the shark Na+/K+/2Cl--co-transporter. We also demonstrate that a C-terminal fragment of AXI 4 is sufficient to promote auxin-independent cell division, showing that the C-termini of CCCs are functional subunits triggering cell division. This may allow a molecular dissection of this process not only in plants but also in animal cells.

Amino Acid Sequence↗
PubMed1993

The activity of daptomycin on Enterococcus faecium protoplasts: indirect evidence supporting a novel mode of action on lipoteichoic acid synthesis.

The effect of daptomycin, an acidic lipopeptide antibiotic active against Gram-positives, was studied in Enterococcus faecium protoplasts. This antibiotic killed 99% of the protoplasts within 60 minutes of treatment, while vancomycin was ineffective, thus excluding peptidoglycan synthesis as the only target of the action of daptomycin. As previously seen with whole cells, in protoplasts lipoteichoic acid synthesis was the earliest and most strongly inhibited among types of macro-molecular synthesis. Radioactive daptomycin tightly bound only to the cytoplasmic membrane, in which the enzymes involved in lipoteichoic acid synthesis are located. These conclusions strongly support our previous proposal that daptomycin, though active against peptidoglycan synthesis, primarily inhibits lipoteichoic acid synthesis.

Anti-Bacterial Agents↗
PubMed1992

Gene rescue in plants by direct gene transfer of total genomic DNA into protoplasts.

To study the possibility of gene rescue in plants by direct gene transfer we chose the Arabidopsis mutant GH50 as a source of donor DNA. GH50 is tolerant of chlorsulfuron, a herbicide of the sulfonylurea class. Tobacco protoplasts were cotransfected with genomic DNA and the plasmid pHP23 which confers kanamycin resistance. A high frequency of cointegration of the plasmid and the genomic DNA was expected, which would allow the tagging of the plant selectable trait with the plasmid DNA. After transfection by electroporation the protoplasts were cultivated on regeneration medium supplemented with either chlorsulfuron or kanamycin as a selective agent. Selection on kanamycin yielded resistant calluses at an absolute transformation frequency (ATF) of 0.8 x 10(-3). Selection on chlorsulfuron yielded resistant calluses at an ATF of 4.7 x 10(-6). When a selection on chlorsulfuron was subsequently applied to the kanamycin resistant calluses, 8% of them showed resistance to this herbicide. Southern analysis carried out on the herbicide resistant transformants detected the presence of the herbicide resistance gene of Arabidopsis into the genome of the transformed tobacco. Segregation analysis showed the presence of the resistance gene and the marker gene in the progeny of the five analysed transformants. 3 transformants showed evidence of genetic linkage between the two genes. In addition we show that using the same technique a kanamycin resistance gene from a transgenic tobacco could be transferred into sugar beet protoplasts at a frequency of 0.17% of the transformants.

Blotting, Southern↗
PubMed1993

Activity of U-snRNA genes with modified placement of promoter elements in transfected protoplasts and stably transformed tobacco.

In higher plants the promoter elements of pol II- and pol III-transcribed U-snRNA genes are identical, comprising a -30 TATA box and an upstream sequence element, USE. The USE and TATA are centred approximately four and three helical DNA turns apart in pol II and pol III genes, respectively, and it is this difference in the element spacing that determines the RNA polymerase specificity of the gene. In this study we have analyzed the effect of spacing mutations on activity of Arabidopsis U2 and U6 genes in transfected protoplasts of Nicotiana plumbaginifolia and in stably transformed tobacco. In the pol III-transcribed U6 gene the insertions and deletions of either odd or even numbers of half helical turns completely inactivate transcription in transfected protoplasts, consistent with the high conservation of the element spacing found in all plant U-snRNA genes. Surprisingly, while insertions of 50 base pairs (bp) or more into the spacer of the pol II-specific U2 gene inactivate transcription, a deletion of 5 bp or insertions of as much as 20 bp decrease transcription by only 40 to 70%. This relaxed requirement for the conserved element spacing is only seen in transfected protoplasts since the same mutant U2 genes are not transcribed in stably transformed tobacco when transcription takes place from the chromosome. The results provide some clues about possible factor interactions at the promoters of plant U-snRNA genes and also offer an example of major differences in transcription between transiently and stably transformed cells.

Arabidopsis↗
PubMed1979

Entrapment of plasmid DNA by liposomes and their interactions with plant protoplasts.

Lecithin and lecithin/cholesterol liposomes formed in aqueous solutions of DNA entrap covalently closed circular, open circular and linear DNA molecules of size up to at least 13 kilobases. The sequestered DNA molecules are efficiently protected against exogenous deoxyribonuclease action although nicking and linearization of circular DNA can be observed. The size of these liposomes ranges from approximately 0.5 to 7.5 mu with an average of 2.5--4 mu. DNA filled liposomes strongly interact with plant protoplasts under conditions inducing protoplast fusion. Results suggest that sequestered plasmid DNA can be transferred to protoplast nuclei.

Cholesterol↗
PubMed2000

Abnormal formation of the glucan network from regenerating protoplasts in Schizosaccharomyces pombe cps8 actin point mutant.

To study the close relationship between the actin cytoskeleton and cell wall formation, the process of cell wall formation in reverting protoplasts of the fission yeast, Schizosaccharomyces pombe, cps8 actin point mutant was investigated by ultra-high-resolution low-voltage scanning electron microscopy (UHR-LVSEM) and transmission electron microscopy (TEM). The protoplast of the cps8 mutant began to form a glucan network in a unipolar manner and to secrete alpha-galactomannan. The site of cell wall formation grew in a cylindrical shape in the wild-type protoplast. The alpha-galactomannan did not fill in the intrafibrillar spaces completely, however, and the fibrils were exposed on the cell surface. UHR-LVSEM images indicated that the glucan fibrils were thin and rope-shaped, forming a looser network than the wild-type. TEM images indicated the finest fibrils were approximately 1.5 nm in diameter, the same diameter as the wild-type. These results suggest that the cps8 mutant was insufficient in developing cross-linkage with the glucan fibrils up to the wide ribbon shape as found in the wild-type [Osumi M et al. (1989) J. Electron Microsc. 38: 457-468; Osumi M (1998) Micron 29: 207-233]. These findings appear to indicate that the actin cytoskeleton controls formation of the glucan network and secretion of beta-1,6-glucan, and confirm the close relationship of the actin cytoskeleton and glucan formation.

Actins↗
PubMed1999

Identification and characterization of the inward K+ channel in the plasma membrane of Brassica pollen protoplasts.

Patch clamp techniques have been used to identify and characterize the whole-cell currents carried by inward K+ channels in isolated matured pollen protoplasts of Brassica chinensis var. chinensis. The whole-cell inward currents in the isolated pollen protoplasts were activated at hyperpolarized membrane potentials more negative than -100 mV. The magnitudes of the whole-cell inward currents were strongly dependent on the external K+ concentration, and were highly selective for K+ over other monovalent cations. The inward currents were not observed when external K+ was replaced with the same concentration of Cs+ or Na+. The addition of 1 mM or 10 mM Ba2+ in external solutions resulted in 30% or 80% inhibition of the inward currents at -180 mV, respectively. These results demonstrated that the inward K+ currents mainly account for the recorded whole-cell inward currents in Brassica pollen protoplasts. Increase of cytoplasmic Ca2+ concentrations from 10 nM to 30 microM or even 5 mM did not affect the inward K+ currents. Decrease of external Ca2+ concentrations from 10 mM to 1 mM inhibited the inward K+ currents by 25%, while the increase of external Ca2+ from 10 mM to 50 mM almost completely blocked the inward K+ currents. Physiological importance of K+ transport into pollen and its possible regulatory mechanisms are also discussed.

Brassica↗