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PubMed1992

Intergeneric protoplast fusion between Gluconobacter oxydans and Corynebacterium species.

Intergeneric protoplast fusion between 2,5-diketo-gluconic acid producing Gluconobacter oxydans (ATCC 9937) and a mutant strain of Corynebacterium species (ATCC 31090), capable of reducing 2,5-diketo-gluconic acid to 2-keto-L-gulonic acid, a penultimate step in vitamin C production) resulted in viable recombinants. Some of the fusion products exhibited the capacity to convert D-glucose to 2-keto-L-gulonic acid, but the conversion rate is low.

Acetobacteraceae↗
PubMed1991

Compartmented metabolite pools in protoplasts from the green alga Chlamydomonas reinhardtii: changes after transition from aerobiosis to anaerobiosis in the dark.

A rapid fractionation method for determination of metabolite levels in the chloroplast and the extrachloroplast compartment of Chlamydomonas reinhardtii has been developed. Protoplasts containing one large chloroplast were fractionated by passing them through a multilayer gradient containing digitonin, polyacrylamide, and a mixture of silicone oil and bromodecane. Lysis of the plasma membrane and the separation of the chloroplasts from most of the extrachloroplast material was achieved within less than 5 s. The chloroplast enriched fraction was contaminated with 3% fumarase (mitochondria) and 13% phosphoenol pyruvate carboxylase (cytosol). Metabolites of the upper glycolytic chain were detected mainly in the chloroplasts, whereas 2-phosphoglycerate was found only in the extrachloroplast compartment. Analysis of changes in metabolite concentrations after transition to anaerobic conditions in the dark pointed to a regulation of carbohydrate catabolism by chloroplast phosphofructokinase and by cytosolic pyruvatekinase.

Aerobiosis↗
PubMed1996

Regulation of the supply of oxaloacetate for mitochondrial metabolism via phosphoenolpyruvate carboxylase in barley leaf protoplasts. II. Effects of metabolites on PEPC activity at different activation states of the protein.

The regulation of the supply of oxaloacetate (OAA) for mitochondrial metabolism via phosphoenolpyruvate carboxylase (PEPC) by metabolites is studied in barley (Hordeum vulgare L.) leaf protoplasts in light or darkness as well as under photorespiratory or non-photorespiratory conditions. Measurements on PEPC activity were performed on samples quickly frozen in liquid nitrogen to break the cell and stop metabolism and thus preserve the in vivo activation state. Glycine, serine, pyruvate, acetyl-CoA, glycolate, fructose 1,6-bisphosphate, fructose 2,6-bisphosphate and ADP had no significant effect on PEPC activity. Malate, aspartate and glutamate were strong inhibitors of PEPC activity decreasing the activity more in light versus darkness. However, at the physiological cytosolic concentration of these metabolites under the respective conditions, inhibition of PEPC activity was about the same with the exception of aspartate which inhibits more under non-photorespiratory than under photorespiratory conditions. 2-Oxoglutarate and glyoxylate decreased PEPC activity by 20 to 40% in the range of its physiological cytosolic concentration. Inhibition by physiological cytosolic concentrations of glutamine was limited. Glucose 6-phosphate, fructose 6-phosphate, 3-phosphoglycerate, dihydroxyacetonphosphate and P(i) stimulated PEPC activity significantly in their physiological cytosolic concentration range. Physiological cytosolic concentrations of glucose 6-phosphate and fructose 6-phosphate activated PEPC activity to about the same extent under all conditions applied, while 3-phosphoglycerate and dihydroxyacetonphosphate stimulating stronger under non-photorespiratory versus photorespiratory conditions. Moreover, dihydroxyacetonphosphate stimulated PEPC activity more in light versus darkness under non-photorespiratory conditions. P(i) activation of PEPC activity decreases in light versus darkness under non-photorespiratory conditions. Stimulation of PEPC activity by citrate in its physiological concentration range is limited. Glucose 1-phosphate and AMP activated PEPC activity only at concentrations higher than their physiological levels in the cytosol. Determinations of PEPC activity in the presence of different malate/glucose 6-phosphate ratios revealed that glucose 6-phosphate totally relieved the inhibitory effect of malate. The regulatory properties of PEPC activity will be discussed in relation to its functions in C3 plants.

Amino Acids↗
PubMed1992

Intraspecific hybridisation of Trichoderma pseudokoningii by anastomosis and by protoplast fusion.

Double auxotrophic and morphological mutants of Trichoderma pseudokoningii Rifai were fused by anastomosis and by protoplast fusion. The recovery of recombinants from heterokaryons on different selective media and from heterokaryotic colonies indicated the occurrence of parasexual events. Prototrophic colonies growing on minimal medium produced binucleate spores, green in colour, revealing a non-autonomous system for conidial pigmentation. Recombinants were obtained from these dikaryotic colonies suggesting the occurrence of a highly unstable diploid phase.

Cellulase↗
PubMed1990

Effect of CpG methylation on gene expression in transfected plant protoplasts.

Activity of the cat gene driven by the cauliflower mosaic virus 35S promoter has been assayed by transfecting petunia protoplasts with the pUC8CaMVCAT plasmid. In vitro methylation of this plasmid with M.HpaII (methylates C in CCGG sites) and M.HhaI (methylates GCGC sites) did not affect bacterial chloramphenicol acetyltransferase (CAT) activity. It should be noted, however, that no HpaII or HhaI sites are present in the promoter sequence. In contrast, in vitro methylation of the plasmid with the spiroplasma methylase M.SssI, which methylates all CpG sites, resulted in complete inhibition of CAT activity. The promoter sequence contains 16 CpG sites and 13 CpNpG sites that are known to be methylation sites in plant DNA. In the light of this fact, and considering the results of the experiments presented here, we conclude that methylation at all CpG sites leaving CpNpG sites unmethylated is sufficient to block gene activity in a plant cell. Methylation of CpNpG sites in plant cells may, therefore, play a role other than gene silencing.

Blotting, Southern↗
PubMed1991

The extensin signal peptide allows secretion of a heterologous protein from protoplasts.

Extensins are hydroxyproline-rich glycoproteins which are amongst the most abundant proteins present in the cell wall of higher plants. Here, we describe the structural analysis of an extensin-encoding gene from Nicotiana plumbaginifolia. The encoded protein (46 kDa) has a highly repetitive structure and contains 37% proline, 18.1% tyrosine, 13.4% lysine, 8.1% serine and 7.1% histidine. The extensin-encoding sequence contains a typical signal peptide for translocation of the protein to the endoplasmic reticulum. By using chimeric genes consisting of different 5' parts of the extensin-encoding gene and the neomycin phosphotransferase II-encoding gene (nptII) as reporter gene, we show that the N-terminal part of extensin can mediate the secretion of NPTII from electroporated N. tabacum protoplasts.

Amino Acid Sequence↗
PubMed2006

Identification and characterization of genes associated with the induction of embryogenic competence in leaf-protoplast-derived alfalfa cells.

Alfalfa leaf protoplast-derived cells can develop into somatic embryos depending on the concentration of 2,4-dichlorophenoxyacetic acid (2,4-D) in the initial culture medium. In order to reveal gene expression changes during the establishment of embryogenic competence, we compared the cell types developed in the presence of 1 and 10 microM 2,4-D, respectively, at the time of their first cell divisions (fourth day of culture) using a PCR-based cDNA subtraction approach. Although the subtraction efficiency was relatively low, applying an additional differential screening step allowed the identification of 38 10 microM 2,4-D up-regulated transcripts. The corresponding genes/proteins were annotated and representatives of various functional groups were selected for more detailed gene expression analysis. Real-time quantitative PCR (RT-QPCR) analysis was used to determine relative expression of the selected genes in 2,4-D-treated leaves as well as during the whole process of somatic embryogenesis. Gene expression patterns confirmed 2,4-D inducibility for all but one of the 11 investigated genes as well as for the positive control leafy cotyledon1 (MsLEC1) gene. The characterized genes exhibited differential expression patterns during the early induction phase and the late embryo differentiation phase of somatic embryogenesis. Genes coding for a GST-transferase, a PR10 pathogenesis-related protein, a cell division-related ribosomal (S3a) protein, an ARF-type small GTPase and the nucleosome assembly factor family SET protein exhibited higher relative expression not only during the induction of somatic embryogenesis but at the time of somatic embryo differentiation as well. This may indicate that the expression of these genes is associated with developmental transitions (differentiation as well as de-differentiation) during the process of somatic embryogenesis.

2,4-Dichlorophenoxyacetic Acid↗
PubMed2004

Hyperosmotically induced accumulation of a phosphorylated p38-like MAPK involved in protoplast volume regulation of plasmolyzed wheat root cells.

A 46 kDa protein resembling immunochemically to the mammalian dually phosphorylated p38-MAPK was detected in wheat root cells under hyperosmotic conditions, using Western blot analysis. This protein accumulated in a time- and dose-dependent fashion and exhibited pharmacological sensitivity similar to the activated p38-MAPK. The application of a highly specific p38-MAPK inhibitor revealed that the p38-like MAPK is probably implicated in hyperosmotically induced tubulin cytoskeleton reorganization as well as in protoplast volume regulation and osmotic tolerance of wheat root cells. As far as we know, the p38-MAPK has not been previously reported in higher plants.

Cell Size↗
PubMed2006

Transformation of undomesticated strains of Bacillus subtilis by protoplast electroporation.

A rapid method combining the use of protoplasts and electroporation was developed to transform recalcitrant wild strains of Bacillus subtilis. The method described here allows transformation with both replicative and integrative plasmids, as well as with chromosomal DNA, and provides a valuable tool for molecular genetic analysis of interesting Bacillus strains, which are hard to transform by conventional methods.

Bacillus subtilis↗
PubMed2006

Bemisia tabaci transmission of specific Lettuce infectious yellows virus genotypes derived from in vitro synthesized transcript-inoculated protoplasts.

Bemisia tabaci transmission was demonstrated for virions derived from cloned infectious cDNAs of Lettuce infectious yellows virus (LIYV). RNA transcripts synthesized from the cDNA clone of LIYV RNA 1 and for clones of seven genotypes (pR6, p1-1a, p1-2b, p1-2f, p1-4h, p1-5b, and p1-5d) of LIYV RNA 2 produced virions or virion-like particles (VLPs) when inoculated to tobacco protoplasts. pR6, p1-1a, and p1-2f virions were transmissible to plants by B. tabaci and transmission frequencies ranged from 15 to 80%, depending on the virion concentration. In contrast, no transmission was observed for p1-5b VLPs even though their concentrations were comparable to that of transmissible virions. p1-5b VLPs also differed from transmissible virions by the absence of an intact CPm, and this correlated with an out-of-frame stop codon mutation in the CPm gene. This is the first report of the vector transmissibility of infectious cDNAs-derived virions for a virus in the genus Crinivirus.

Amino Acid Sequence↗
PubMed1997

Microviscosity of cucumber (Cucumis sativus L.) fruit protoplast membranes is altered by triacontanol and abscisic acid.

Cucumber (Cucumis sativus L.) fruit protoplast membranes were probed with diphenylhexatriene (DPH) and pyrene, and also with two different plant growth regulators, triacontanol (TRIA) and abscisic acid (ABA). Fluorescence anisotropies of DPH and pyrene were measured after incorporating them into the membranes. The fluorescence lifetime of membrane-bound pyrene was also measured by using neodymium-doped yttrium aluminium garnet (Nd:YAG) laser of 35 ps pulses. The microviscosities of the membranes were calculated using the values of fluorescence anisotropy and lifetime. In the presence of TRIA and ABA, there was a sharp decrease in the fluorescence lifetime of pyrene. Similarly, there was also a decrease in the microviscosities of the membranes and increase in the rate of rotation of membrane-bound fluorophore, induced by the plant growth regulators. Furthermore, TRIA or TRIA + ABA could reduce the fluorescence anisotropy of both the fluorophores whereas, ABA decreased the anisotropy of only pyrene. This property of ABA may be due to its confinement to a specific spacial facet in the membrane. Fatty acid analysis indicated that membrane microviscosity fluctuations were not due to altered fatty acid composition alone as it is known that change in lipid-protein interaction would also alter the physical status of the membrane.

Abscisic Acid↗
PubMed2001

P34(cdc2) kinase is associated with cortical microtubules from higher plant protoplasts.

The cell cycle regulatory enzyme p34(cdc2) kinase is known to be localized to the preprophase band, the spindle and the phragmoplast, but not to interphase cortical microtubules. This was investigated further by mechanically cleaving substrate-attached protoplasts to leave plasma membrane disks bearing microtubules freed of nuclear and cytosolic signal. Antibodies to PSTAIRE and to specific C-terminal peptides of cdc2a, were used in immunofluorescence, protein blotting and immunogold electron microscopy to demonstrate that antigen is located on the cortical microtubules of carrot, tobacco BY-2 and Arabidopsis cells.

Animals↗
PubMed1997

Detection of glycosyl-phosphatidylinositol-anchored proteins on the surface of Nicotiana tabacum protoplasts.

Glycosyl-phosphatidylinositol (GPI)-anchored plasma membrane proteins have been found to be widespread in eukaryotes and protozoa but have not been reported in higher terrestrial plants. A sensitive biotin-based assay has been used to detect the presence of GPI-anchored proteins on the outer surface of cultured Nicotiana tabacum cells. Six proteins with molecular weights of 92, 84, 60.5, 54.5, 39.5 and 37 kDa were found to move from a Triton X-114 detergent-rich phase to an aqueous phase following incubation with phosphatidylinositol-specific phospholipase C (PtdIns-PLC). The behaviour of these proteins is consistent with the presence of a GPI-anchor. Seven GPI-anchored proteins were also detected on the surface of tobacco leaf protoplasts with molecular weights of 67.5, 62, 39, 33.5, 27, 23 and 15.6 kDa. These data demonstrate the presence of multiple GPI-anchored proteins on the plasma membrane of higher plant cells.

Cells, Cultured↗
PubMed1999

Flow cytometry and surface plasmon resonance analyses demonstrate that the monoclonal antibody JIM19 interacts with a rice cell surface component involved in abscisic acid signalling in protoplasts.

Abscisic acid (ABA) is a plant hormone involved in many developmental and physiological processes, but as yet, no ABA receptor has been identified. Flow cytometry of rice protoplasts and immunoblotting of purified plasma membranes (PMs) have been used to demonstrate that the monoclonal antibody JIM19 recognizes carbohydrate epitopes of cell surface glycoproteins. Using surface plasmon resonance technology specific binding of PMs to JIM19 was observed. Such interaction was antagonized significantly by ABA, but not by the biologically inactive ABA catabolite phaseic acid. These in vitro interactions were correlated with the biological activities of JIM19, ABA and phaseic acid on activation of the ABA-inducible Em promoter using two different transient reporter gene assays, beta-glucuronidase/luciferase and quantitative flow cytometry of Aequoria green fluorescent protein. Pre-treatment with JIM19 resulted in significant inhibition of ABA-inducible gene expression. Taken together, these data suggest that JIM19 interacts with a functional PM complex involved in ABA signalling.

Abscisic Acid↗
PubMed1999

Unitary exocytotic and endocytotic events in guard-cell protoplasts during osmotically driven volume changes.

Osmotically driven swelling and shrinking of guard-cell protoplasts (GCPs) requires adjustment of surface area which is achieved by addition and removal of plasma membrane material. To investigate the mechanism for adaptation of surface area we have used patch-clamp capacitance measurements. The recorded membrane capacitance (C(m)) trace of swelling and shrinking GCPs occasionally revealed discrete upward and downward deflecting capacitance steps, respectively, with a median value of about 2 fF. The observed capacitance steps resulted from the fusion and fission of single vesicles with a diameter of around 300 nm. We conclude that exo- and endocytosis of these vesicles accommodate for osmotically driven surface area changes in GCPs.

Cell Membrane↗
PubMed1981

[Isolation of yeast protoplasts using various preparations of the hepato-pancreatic juice of Helix pomatia].

Conversion of large amounts of Saccharomyces cerevisiae cells to protoplasts is studied using various preparations extracted from Helix pomatia hepato-pancreatic juice. The most favourable yield in two hours incubations (88 per cent) is obtained with 20 ml cytohelicase, a chitinase and glucanase enriched extract, per 400 g of yeast cells, harvested at the end of the logarithmic growth phase and preincubated in presence of 2-mercaptoethanol.

Animals↗
PubMed1999

Evidence of multiple sugar uptake across the plasma membrane of laticifer protoplasts from Hevea.

In Hevea, rubber synthesis is confined to the cytosol of the highly differentiated laticifer cells. Agronomic and biochemical studies showed that this process uses high amounts of sugars that are efficiently imported into the laticifer. A H(+)-sugar symport system located in the plasma membrane is involved in sugar uptake into laticifers. Laticifer protoplasts were prepared and used in electrophysiological and labeling experiments to test the capacity of this system to transport a variety of sugars such as oligosaccharides from the raffinose family, trace compounds in rubber. Translocation of sugars known to be transported with different efficiency across the plasma membrane of plant cells was also tested. A 1 mM sucrose affinity was found for the symport. All the sugars tested, except palatinose induce membrane depolarization indicating that they were actively absorbed by the laticifer network. This reveals the wide capacity of this peculiar sink for the uptake of sugars.

Carbohydrate Metabolism↗
PubMed1994

In vitro fertilisation of maize by single egg and sperm cell protoplast fusion mediated by high calcium and high pH.

We present evidence for the fusion of isolated single maize egg and sperm cell protoplasts in a mannitol solution (400-430 mosmol/kg H2O) containing 0.05 M CaCl2 at pH 11.0, followed by cell division of the fusion products. These findings allow the performance of in vitro fertilisation of higher plants by combining single gametes as in lower plant and animal systems. Further, our findings open new avenues for investigating the basic mechanisms of adhesion and fusion of higher plant gametes and eventually for examining processes that inhibit polyspermy in higher plants.

Botany↗