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PubMed1986

Subcellular localization of the 30K protein in TMV-inoculated tobacco protoplasts.

We investigated the intracellular localization of the 30K protein in TMV-inoculated tobacco protoplasts by means of pulse-labeling and pulse-chase experiments with [35S]methionine. Protoplasts were lysed with a nonionic detergent and the extracts were centrifuged to yield soluble and crude nuclear fractions. Most of the 30K protein was found in the crude nuclear fraction. The nuclear fraction was further purified by centrifugation in a step-wise Percoll gradient. Nuclei and the 30K protein were found in the same fractions. The results of pulse-chase experiments indicated that the 30K protein is synthesized in the soluble fraction and then translocated to the crude nuclear fraction. The 30K protein of Ls1, a temperature-sensitive (ts) mutant affecting the cell-to-cell viral transport function, was also found in the nuclei, even at a nonpermissive temperature. These results suggested that the 30K protein has to be localized in the nuclei to function, and that impaired translocation of the 30K protein to the nuclei is not responsible for the ts lesion of mutant Ls1.

Centrifugation, Density Gradient↗
PubMed1989

Expression of the 16K cistron of tobacco rattle virus in protoplasts.

An antiserum was raised against a synthetic peptide corresponding to the 18 C-terminal amino acids of a putative 16K protein encoded by the 3'-terminal open reading frame of tobacco rattle virus (TRV) RNA-1. This antiserum was used to demonstrate expression of the 16K cistron in vivo. TRV-infected tobacco protoplasts accumulated similar amounts of 16K protein and viral coat protein but in tobacco plants only the coat protein was detectable. Time course experiments revealed that in protoplasts the accumulation of 16K protein lagged somewhat behind that of coat protein. The 16K protein was incorporated in a high-molecular-weight cellular component that was resistant to treatment with nonionic detergents.

Cells, Cultured↗
PubMed1992

Reinvestigation of intracellular localization of the 30K protein in tobacco protoplasts infected with tobacco mosaic virus RNA.

It has been shown that the 30K protein of tobacco mosaic virus (TMV) is responsible for the cell-to-cell movement function of the virus. It is still obscure how the protein is involved in this function at the molecular level. We formerly found that the 30K protein is localized to the plasmodesmata of TMV-infected plants. We also reported that the 30K protein was detected in a nuclei-rich fraction of TMV-infected protoplasts after biochemical fractionation. To clarify the inconsistency, the 30K protein was immunocytologically localized in TMV-infected protoplasts using a newly prepared antibody against the 30K protein. On some sections, the 30K protein was found near the nucleus but not in or on the nucleus. At later stages of infection a novel electron-transparent structure was detected in the cytoplasm where the 30K proteins were localized. This structure might reflect an intermediate form between its synthesis in the cytoplasm and its targeting to the plasmodesmata in whole plants.

Amino Acid Sequence↗
PubMed1980

Giant protoplasts from Nitella cells.

The process converting an intact Nitella cell into a protoplast was recorded continuously by means of time-lapse cinematography. The mechnican properties of these enzymatically isolated protoplasts were examined by several methods.

Cell Wall↗
PubMed1981

Inhibition of RNA synthesis in yeast protoplasts by a peptide factor from Tetrahymena cells.

Protoplasts of Schizosaccharomyces pombe, grown on a rich nutrient medium, were treated with a peptide factor isolated from cultures of the protozoan Tetrahymena pyriformis. The peptide factor is known to inhibit RNA synthesis in Tetrahymena. It has now been shown that the peptide factor also inhibits RNA synthesis in yeast protoplasts without affecting protein synthesis.

Animals↗
PubMed2001

Fusicoccin- and light-induced activation and in vivo phosphorylation of phosphoenolpyruvate carboxylase in vicia guard cell protoplasts.

The in vivo regulation of phosphoenolpyruvate carboxylase (PEPCase; EC 4.1.1.31), was studied in purified guard cell protoplasts (GCPs) of Vicia faba L. Incubation of GCPs with fusicoccin (FC) led to the rapid activation of PEPCase and reduced its sensitivity towards the feedback-inhibitor malate. This was accompanied by an increase in the phosphorylation state of the enzyme. Additionally, PEPCase could be transiently activated by white light. Activation and phosphorylation of PEPCase upon illumination were dependent on the presence of potassium in the incubation medium. Treatment of GCPs with inhibitors of H(+)-ATPases, and with abscisic acid (ABA) suppressed the activation of PEPCase in a concentration-dependent manner. Treatment of protoplasts with butyrate also led to PEPCase activation, suggesting a role for the cytosolic pH (pH(cyt)) in the signal transduction process. The presented data indicate that guard cell PEPCase is regulated by reversible phosphorylation of at least one isoform and elucidate first components of the signaling pathway.

Journal Article↗
PubMed2004

Efficient selection of hybrids by protoplast fusion using drug resistance markers and reporter genes in Saccharomyces cerevisiae.

We have developed a selection system for hybrids by protoplast fusion using dominant selective drug resistance markers, Tn601(903) against geneticin and AUR1-C against aureobasidin A, and reporter genes, ADH1p-PHO5-ADH1t and CLN2p-CYC1-lacZ, in Saccharomyces cerevisiae. To examine the effectiveness of this system, plasmids with each marker and reporter gene were introduced into auxotrophic sake yeasts. From the resulting transformants, eight colonies were screened by protoplast fusion in combination with the drug resistance markers and the reporter genes. Among them, seven strains were judged as hybrids between parental strains by analysis of growth on a minimal medium. This selection system was applied to wine yeasts having no genetic markers. Six strains were regarded as hybrids between parental strains by polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) analysis of the MET2 gene and by karyotype analysis using a contour-clamped homogeneous electric field (CHEF). We propose that the plotoplast fusion using dominant selective geneticin- and aureobasidin A-resistance markers and reporter genes is useful for the selection of hybrids from wine yeasts, which are homothallic and have low sporulation ability.

Journal Article↗
PubMed1991

Characterization of the auxin-regulated par gene from tobacco mesophyll protoplasts.

The auxin-regulated par gene from tobacco mesophyll protoplasts was characterized in detail to deduce its possible function. An homology search of the par gene in the NBRF databases revealed that the par gene has homology to the stringent starvation protein (ssp) gene of Escherichia coli, which is induced under starved conditions and binds in an equimolar ratio to a holoenzyme of RNA polymerase. Hence, it is supposed that the par gene product could play a similar role to that of ssp. Although sequence homology of the par gene to the Gmhsp 26-A gene from soybean was observed, both genes were shown to respond differently to plant hormones and stresses. Gmhsp 26-A is induced by heat shock, 2,4-dichlorophenoxyacetic acid (2,4-D), cytokinin and abscisic acid (ABA), whereas the par gene was induced only by auxins. Furthermore, cycloheximide treatment prevents 2,4-D-mediated accumulation of Gmhsp 26-A mRNA, but not that of par mRNA. Both par and Gmhsp 26-A respond to CdCl2, but splicing of the par pre-mRNA proceeded in a normal way, whereas splicing off the Gmhsp 26-A pre-mRNA was inhibited. Hence, the par and Gmhsp 26-A genes should have a common ancestor, but have evolved in different directions. Detailed time-course experiments confirmed that the par gene was induced immediately after the addition of auxin and expressed upon the initiation of meristematic activity in tobacco mesophyll protoplasts. As the par gene was induced by the sole treatment of cycloheximide, it was proposed that the par gene belongs to a category of 'superinduction' genes.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2004

Organelle inheritance in plant cell division: the actin cytoskeleton is required for unbiased inheritance of chloroplasts, mitochondria and endoplasmic reticulum in dividing protoplasts.

Nuclear inheritance is highly ordered, ensuring stringent, unbiased partitioning of chromosomes before cell division. In plants, however, little is known about the analogous cellular processes that might ensure unbiased inheritance of non-nuclear organelles, either in meristematic cell divisions or those induced during the acquisition of totipotency. We have investigated organelle redistribution and inheritance mechanisms during cell division in cultured tobacco mesophyll protoplasts. Quantitative analysis of organelle repositioning observed by autofluorescence of chloroplasts or green fluorescent protein (GFP), targeted to mitochondria or endoplasmic reticulum (ER), demonstrated that these organelles redistribute in an ordered manner before division. Treating protoplasts with cytoskeleton-disrupting drugs showed that redistribution depended on actin filaments (AFs), but not on microtubules (MTs), and furthermore, that an intact actin cytoskeleton was required to achieve unbiased organelle inheritance. Labelling the actin cytoskeleton with a novel GFP-fusion protein revealed a highly dynamic actin network, with local reorganisation of this network itself, appearing to contribute substantially to repositioning of chloroplasts and mitochondria. Our observations show that each organelle exploits a different strategy of redistribution to ensure unbiased partitioning. We conclude that inheritance of chloroplasts, mitochondria and ER in totipotent plant cells is an ordered process, requiring complex interactions with the actin cytoskeleton.

Actins↗
PubMed1986

Lateral diffusion of phospholipids in the plasma membrane of soybean protoplasts: Evidence for membrane lipid domains.

Fluorescent lipid and phospholipid probes were incorporated at 4 degrees C into soybean protoplasts prepared from cultured soybean (SB-1) cells. Fluorescence microscopy showed that the plasma membrane as well as the nucleus were labeled. Fluorescence redistribution after photobleaching (FRAP) analysis was performed on these cells at 18 degrees C to monitor the lateral mobility of the incorporated probes. After labeling at low concentrations (40 mug/ml) of phosphatidyl-N-(4-nitrobenzo-2-oxa-1,3-diazolyl)ethanolamine (NBD-PtdEtn), a single mobile component was observed with a diffusion coefficient (D) of approximately 3 x 10(-9) cm(2)/sec. After labeling at higher probe concentrations (>/=100 mug/ml), two diffusing species were observed, with diffusion coefficients of approximately 3 x 10(-9) cm(2)/sec ("fast") and approximately 5 x 10(-10) cm(2)/sec ("slow"). Similar results were observed with fluorescent derivatives of phosphatidylcholine and fatty acids. In contrast to these results, parallel analysis of 3T3 fibroblasts, using the same probes and conditions, yielded only a single diffusion component. These results suggest that the soybean plasma membrane may contain two distinct lipid domains in terms of lipid mobility. Consistent with this idea, experiments with soybean protoplasts yielded a single diffusion component under the following conditions: (i) labeling with NBD-PtdEtn (100 mug/ml), FRAP analysis at 37 degrees C (D = 1.1 x 10(-8) cm(2)/sec); (ii) labeling with NBD-PtdEtn (100 mug/ml), FRAP analysis at 18 degrees C in the presence of 2 mM EGTA (D = 4.2 x 10(-9) cm(2)/sec); (iii) labeling with 5-(N-dodecanoyl)aminofluorescein (a short-chain lipid probe), FRAP analysis at 18 degrees C or 37 degrees C (D = 2.5 x 10(-8) cm(2)/sec). These results suggest that the plasma membrane of soybean cells may contain stable immiscible domains of fluid and gel-like lipids.

Journal Article↗
PubMed1987

Voltage dependence of K channels in guard-cell protoplasts.

Stomatal pores in leaves enable plants to regulate the exchange of gases with their environment. Variations of the pore aperture are mediated by controlled changes of potassium salt concentrations in the surrounding guard cells. The voltage-dependent gating of K(+)-selective channels in the plasma membrane (plasmalemma) of cell-wall-free guard cells (protoplasts) was studied at the molecular level in order to investigate the regulation of K(+) fluxes during stomatal movements. Inward and outward K(+) currents across the plasmalemma of guard cells were identified by using the whole-cell configuration of the patch-clamp technique. Depolarizations of the membrane potential from a holding potential of -60 mV to values more positive than -40 mV produced outward currents that were shown to be carried by K(+). Hyperpolarizations elicited inward K(+) currents. Inward and outward currents were selective for K(+) over Na(+) and could be partially blocked by exposure to extracellular Ba(2+). In cell-attached and excised membrane patches, previously identified K(+)-selective single channels in guard cells were studied. Averaging of single-channel currents during voltage pulses resulted in activation and deactivation kinetics that were similar to corresponding kinetics of inward and outward currents in whole cells, showing that K(+)-selective channels were the molecular pathways for the K(+) currents recorded across the plasmalemma of single guard-cell protoplasts. Estimates demonstrate that K(+) currents through the voltage-gated K(+) channels recorded in whole guard cells can account for physiological K(+) fluxes reported to occur during stomatal movements in leaves.

Journal Article↗
PubMed1988

Binding of the phytotoxin zinniol stimulates the entry of calcium into plant protoplasts.

Zinniol [1,2-bis(hydroxymethyl)-3-methoxy-4-methyl-5-(3-methyl-2-butenyloxy)benzene], a toxin produced by fungi of the Alternaria group, causes symptoms in plants that resemble those induced by the fungi. The phytotoxin binds to carrot protoplasts and isolated membranes in a saturable and reversible manner. Receptor occupancy stimulates entry of calcium into protoplasts. Zinniol can partially reverse the effects and binding of the calcium-channel blockers desmethoxyverapamil and bepridil. Selected cell lines that are insensitive to zinniol lose part of their binding capacity and sensitivity to the action of the agonist-like compound but are still able to bind calcium-channel blockers. We conclude that zinniol acts on calcium entry but that the targets of the toxin and of calcium-channel blockers are dissimilar, suggesting the occurrence of sites affected both by zinniol and by channel blockers and of sites affected only by zinniol.

Journal Article↗
PubMed1992

Control of excision frequency of maize transposable element Ds in Petunia protoplasts.

The complete coding region of maize transposable element Ac and truncated but active derivatives of it were placed under the control of promoters of different strength and tested for the ability to excise transposable element Ds from a beta-glucuronidase reporter gene in a cotransfection assay in Petunia protoplasts. The highest excision values (5% of the protoplasts able to express the beta-glucuronidase gene in a control experiment) were observed with a truncated version of the Ac coding region under the control of the 2' promoter. The weak Ac promoter is sufficient to give rise to excision values not much lower than those found with much stronger promoters such as the 2' and nos promoters. A decrease in excision frequency was observed when translation of the Ac coding region was hindered by out-of-frame ATG codons in addition to the use of weak promoters. Increasing the level of Ac transposase thus does not seem to be sufficient to raise the level of Ds excision observed in this system and possibly also in maize. Therefore another factor limits the excision of Ds elements. Previously, it was reported that in tobacco cells the deletion of Ds sequence between base pairs 186 and 245 led to a decrease of the Ds excision frequency by the full-length but not by the truncated Ac product. In the Petunia assay system, however, deletion of these sequences decreased the excision rate with both the full length and the truncated Ac coding region. A cDNA construct was found similarly active as the corresponding genomic DNA.

Journal Article↗
PubMed1993

Cytokinin stimulates dihydropyridine-sensitive calcium uptake in moss protoplasts.

Ca2+ influx through dihydropyridine (DHP)-sensitive Ca2+ channels is thought to be an early event in cytokinin-induced bud formation in moss protonema because DHP antagonists inhibit bud formation in the presence of cytokinin and DHP agonists stimulate bud formation in the absence of cytokinin [Conrad, P. A. & Helper, P. K. (1988) Plant Physiol. 86, 684-687]. In the present study, we established the presence of a DHP-sensitive Ca2+ transport system by measuring 45Ca2+ influx into moss protoplasts. Ca2+ influx was stimulated by external KCl (up to 5 mM), indicating that transport is voltage-dependent. K(+)-induced Ca2+ influx was DHP-sensitive with > 50% inhibition at 500 nM nifedipine. Ca2+ influx was stimulated by increasing concentrations of the DHP Ca2+ channel agonist Bay K8644 with half-maximal effects at 25 nM; this stimulation was seen only in the absence of K+, suggesting that the agonist works preferentially on polarized membranes. Ca2+ influx was also inhibited by phenylalkylamines (verapamil) and benzothiazepines (diltiazem). The phytohormone 6-benzylaminopurine consistently stimulated Ca2+ influx with a Km value of 1 nM, whereas adenine, indoleacetic acid, and gibberellic acid had no effect on Ca2+ transport. The cytokinins kinetin and trans-zeatin caused a greater stimulation of Ca2+ influx and induced more bud formation than did 6-benzylaminopurine. These results indicate that Ca2+ is taken up into moss protoplasts through voltage-dependent DHP-sensitive Ca2+ channels on the plasma membrane and that one of the cytokinin effects in the induction of bud formation is regulation of this plasma membrane Ca2+ channel.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1995

Auxins induce clustering of the auxin-binding protein at the surface of maize coleoptile protoplasts.

The predominant localization of the major auxin-binding protein (ABP1) of maize is within the lumen of the endoplasmic reticulum. Nevertheless, all the electrophysiological evidence supporting a receptor role for ABP1 implies that a functionally important fraction of the protein must reside at the outer face of the plasma membrane. Using methods of protoplast preparation designed to minimize proteolysis, we report the detection of ABP at the surface of maize coleoptile protoplasts by the technique of silver-enhanced immunogold viewed by epipolarization microscopy. We also show that ABP clusters following auxin treatment and that this response is temperature-dependent and auxin-specific.

Journal Article↗
PubMed1989

Selection for salt and drought tolerance in protoplast- and explant-derived tissue cultures of Colt cherry (Prunus avium x pseudocerasus).

Colt cherry (Prunus avium x pseudocerasus) callus cultures were derived from leaf protoplasts, protoplasts of root cell suspension cultures, or by direct culture of leaf and root tissues. Survival of calli cultured on basal proliferation medium containing 25, 50, 100 or 200 mN (millinormal) NaCl, Na(2)SO(4) or KCl, or iso-osmotic (with NaCl) concentrations of mannitol ranged from 1 to 15%. After six transfers on the same medium, surviving cell lines were subjected to three cycles of direct recurrent selection; i.e., in each cycle, they were cultured alternately on basal proliferation medium, and on basal proliferation medium supplemented with NaCl, KCl, Na(2)SO(4) or mannitol. Salt- or mannitol-tolerant cell lines selected in this way had smaller cells than unselected cell lines, and they grew more rapidly and had higher callus and cell survival rates than unselected cell lines when cultured in the presence of salt or mannitol. Cells lines selected for tolerance to one agent (sodium salt, potassium salt or mannitol) showed minimal tolerance to another agent. However, when plants were regenerated from salt- or mannitol-tolerant callus and new cultures derived from them, the new cultures showed tolerance to all of the salts and mannitol. Plant regeneration from the new cultures was not achieved under the conditions that led to the regeneration of the parent plants from callus.

Journal Article↗
PubMed1995

Subcellular Compartmentation of the 4-Aminobutyrate Shunt in Protoplasts from Developing Soybean Cotyledons.

The subcellular localization of enzymes involved in the 4-ami-nobutyrate shunt was investigated in protoplasts prepared from developing soybean [Glycine max (L.) Merrill cv Maple Arrow] cotyledons. Protoplast lysate was fractionated by differential and continuous Percoll-gradient centrifugation to separate organelle fractions. Glutamate decarboxylase (EC 4.1.1.15) was found exclusively in the cytosol, whereas 4-aminobutyrate:pyruvate transami-nase (EC 2.6.1.19) and succinic semialdehyde dehydrogenase (EC 1.2.1.16) were associated exclusively with the mitochondrial fractions. Mitochondrial fractions also catabolized [U-14C]4-aminobu-tyrate to labeled succinate.

Journal Article↗
PubMed1995

Properties of Proton Pumping in Response to Blue Light and Fusicoccin in Guard Cell Protoplasts Isolated from Adaxial Epidermis of Vicia Leaves.

Guard cell protoplasts (GCPs) were isolated from the adaxial epidermis of Vicia leaves. The properties of isolated adaxial GCPs (ad GCPs) were compared with those of abaxial GCPs (ab GCPs) with respect to H+-pumping activity. A saturating pulse of blue light (200 [mu]mol m-2 s-1, 30 s) induced H+ pumping in both ad GCPs and ab GCPs under red light. The maximum rate of blue-light-dependent H+ pumping was slightly higher in ad GCPs than in ab GCPs, but the magnitude of H+ pumping in ad GCPs was 68% of that in ab GCPs. H+ pumping was responsive to the second pulse, and the rate and magnitude of the pumping increased with the time between two pulses. The periods required to achieve 50% of the maximum rate were 12 and 22 min for ad GCPs and ab GCPs, respectively. The rates of blue-light-dependent H+ pumping were saturable, with half-saturation at 630 [mu]mol m-2 (21 [mu]mol m-2 s-1, 30 s) for ad GCPs and 105 [mu]mol m-2 (3.5 [mu]mol m-2 s-1, 30s) for ab GCPs. In contrast, fusicoccin, an activator of the plasma membrane H+- ATPase, induced H+ pumping with a slightly higher rate in ad GCPs than in ab GCPs. Both types of protoplast swelled similarly in response to fusicoccin. These results suggest that ad GCPs have almost the same activity for H+ pumping as ab GCPs, whereas ad GCPs require a larger number of photons to activate the H+ pump than ab GCPs.

Journal Article↗