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At least 991 records · Page 55Linked to original sources
PubMed1995

Multiple serine phosphorylation sites on the 30 kDa TMV cell-to-cell movement protein synthesized in tobacco protoplasts.

p30, the protein required for cell-to-cell movement of tobacco mosaic virus (TMV), has a slightly reduced mobility on SDS-polyacrylamide gels when isolated by immunoprecipitation from TMV-infected protoplasts compared with that of p30 translated from viral RNA in vitro. Further investigation established a probable cause for the difference in mobility between the two: protoplasts incorporate [32P]orthophosphate into p30 at multiple sites, predominantly as phosphoserine. Tryptic peptide mapping reveals at least five internal phosphopeptides in p30, besides the C-terminal tryptic phosphopeptide already reported, involving at least two distinct domains of the protein (at residues 61-114 and residues 212-231), which may be substrates for different protein kinases. These structural results are consistent with a three-domain model for the TMV movement protein with two regulatory domains similar to that recently proposed on genetic grounds for dianthovirus movement proteins.

Peptide Mapping↗
PubMed2000

Transient expression and heat-stress-induced co-aggregation of endogenous and heterologous small heat-stress proteins in tobacco protoplasts.

Heat-stress granules (HSG) are highly ordered, cytoplasmic chaperone complexes found in all heat-stressed plant cells. We have developed an experimental system involving expression of cytosolic class I and class II small heat-stress proteins (Hsps) of pea, Arabidopsis and tomato in tobacco protoplasts to study the structural prerequisites for the assembly of HSG or HSG-like complexes. Class I and class II small Hsps formed class-specific dodecamers of 210-280 kDa, which, upon heat stress, were incorporated into HSG complexes. Interestingly, class II dodecamers alone could form HSG-like complexes (auto-aggregation), whereas class I dodecamers could do so only in the presence of class II proteins (recruitment). By analysing C-terminal deletion forms of Hsp17 class II, we obtained evidence that the intact C-terminus is critical for the oligomerization state, for the heat-stress-induced auto-aggregation and for recruitment of class I proteins. The class-specific formation of dimers as a prerequisite for oligomerization was analysed by the yeast two-hybrid system. In the presence of the endogenous (tobacco) set of heat-stress-induced proteins, all heterologous class I and class II proteins were incorporated into HSG complexes, whose ultrastructure was different from that of complexes formed by class I and class II proteins alone. Although other, more distantly related, members of the Hsp20 family, i.e. the plastidic pea Hsp21, the Drosophila Hsp23 and the mouse Hsp25, were well expressed in tobacco protoplasts and formed homo-oligomers of 200-700 kDa, none of them could be recruited to HSG complexes.

Amino Acid Sequence↗
PubMed2003

A novel procedure for the localization of viral RNAs in protoplasts and whole plants.

Analysis of virus spread using co-expressed reporter proteins has provided important details on cell-to-cell and long-distance movement of viruses in plants. However, most viruses cannot tolerate insertion of large non-viral segments or loss of any open-reading frames, procedures required to detect viruses non-evasively. A technique used to localize mRNAs intracellularly in yeast has been modified for detection of viral RNAs in whole plants. The technique makes use of the binding of the coat protein of MS2 bacteriophage (CPMS2) to a 19 base hairpin (hp). A fusion protein, consisting of the CPMS2, green fluorescent protein (GFP), and a nuclear localization signal (NLS), was nuclear-localized upon transient expression in protoplasts. However, addition of the hp to the 3' untranslated region of Turnip crinkle virus (TCV-hp) and co-transfection of the virus and fusion protein construct into protoplasts resulted in the re-location of GFP to the cytoplasm. Neither the insertion of the hp nor the interaction with the fusion protein impaired any viral functions. Transgenic plants expressing the GFP-NLS-CPMS2 fusion protein were generated, and GFP was detected in nuclei of young plant cells. Foci of GFP cytoplasmic fluorescence were detected in TCV-hp-inoculated leaves at 2 days post-inoculation. Later, GFP was detected in young leaves near the midvein and in the base (support) cells of trichomes in the vicinity of secondary and tertiary veins. In older leaves, cytoplasmic GFP could be visualized throughout many of the leaves. This technique should be amenable for detection of any virus with a transformable plant (or animal) host and may also prove useful for localizing properly engineered host RNAs.

3' Untranslated Regions↗
PubMed2002

The number of K(+) channels in the plasma membrane of guard cell protoplasts changes in parallel with the surface area.

The activity of the two dominant K(+) channels in the plasma membrane of Vicia faba guard cell protoplasts was examined during pressure-driven swelling. For this purpose, the K(+) currents and the membrane capacitance (C(m)) of guard cell protoplasts were recorded in parallel. A rise in C(m), reflecting an increase of the membrane surface area, was coupled to a proportional rise in conductance of both the K(+) inward and K(+) outward rectifier. The activation kinetics of the K(+) channels were not affected during this process. The quantitative and temporal coupling of C(m) and K(+) conductance can hence be interpreted as the result of the addition of active inward and outward rectifier K(+) channels to the plasma membrane during an increase in surface area.

Cell Membrane↗
PubMed1980

Biparental products of bacterial protoplast fusion showing unequal parental chromosome expression.

Efficiently regenerated single colonies from mixed multiply auxotrophic Bacillus subtilis protoplasts, fused with polyethylene glycol, reveal colonies carrying each of the parent types (biparentals) and recombinant colonies. The latter appear in high yields (up to 1% of certain recombinant classes); the yield of biparentals may be as large as 10%, in the range of the indicated physical fusion events. Many of the biparentals are diploids although, contrary to expectation, they are not complementing prototrophs, but show precisely the phenotype of one (either one) of the parent strains. Extensive pedigree analysis and subcloning of diploid lines show that they can propagate with varying stability on the appropriate parental selective medium to reproduce diploid progeny, parental segregants, and late-appearing recombinants, including some prototrophs. Thus, the principal product of intertype protoplast fusion is a diploid carrying two chromosomes, only one of which is expressed in each particular clone. The extinction of one parental genome is especially well demonstrated when it includes suppression of a normally dominant antibiotic sensitivity marker. In transformation experiments, DNA made from a selected diploid clone was able to transfer several of the unexpressed genes. The structural or topological character of DNA associated with the chromosome extinction remains unexplained.

Bacillus subtilis↗
PubMed1990

Expression of photosynthesis-related gene fusions is restricted by cell type in transgenic plants and in transfected protoplasts.

We have analyzed the expression of chimeric genes in populations of protoplasts isolated from the photosynthetic and nonphotosynthetic tissues within leaves of transgenic tobacco plants and separated by fluorescence-activated cell sorting. Expression of transcriptional gene fusions controlled by promoters from photosynthesis-associated genes showed a striking dependence on cell type. These patterns of expression were preserved when the gene fusions were transfected into normal (nontransgenic) tobacco leaf protoplasts.

Base Sequence↗
PubMed1990

Location of the cis-acting auxin-responsive region in the promoter of the par gene from tobacco mesophyll protoplasts.

We have isolated a genomic clone of an auxin-regulated par gene, which is expressed during the transition from G0 phase to S phase in the early stage of tobacco mesophyll protoplasts cultured in vitro, from a tobacco genomic library using the par cDNA as a probe. When a chimeric gene, in which a reporter gene for bacterial beta-glucuronidase (GUS) was placed downstream of the 5' flanking sequences of the par gene, was introduced into tobacco mesophyll protoplasts by electroporation, the chimeric gene elicited auxin-regulated expression of GUS activity. Because deletion of a 111-base-pair (bp) direct repeat in the 5' flanking sequences of the par gene abolished the auxin-induced GUS activity, it is deduced that in the 111-bp direct repeat of the par gene promoter is localized an auxin-responsive region, which regulates auxin-mediated activation of transcription.

Amino Acid Sequence↗
PubMed1992

Gibberellic acid and abscisic acid coordinately regulate cytoplasmic calcium and secretory activity in barley aleurone protoplasts.

The effects of gibberellic acid (GA3) and abscisic acid (ABA) on the temporal and spatial dynamics of cytosolic calcium concentration ([Ca2+]i) in aleurone protoplasts of barley (Hordeum vulgare L. cv. Himalaya) were measured by using fluorescence ratio analysis and confocal microscopy. After 4-6 h of treatment, GA3 induced a sustained increase in [Ca2+]i from 50 to 150 nM in aleurone protoplasts. The increase in [Ca2+]i preceded the GA3-induced increase in alpha-amylase synthesis and secretion by 4 h. The elevation of [Ca2+]i was highest in the peripheral cytoplasm and may play a role in coordinating the secretory events there. Reducing Ca2+ levels in the incubation medium to below 500 microM inhibited the increase in [Ca2+]i and the GA3-induced stimulation of alpha-amylase synthesis and secretion. These data suggest that GA3 may increase [Ca2+]i by increasing the influx of Ca2+ at the plasma membrane. ABA reversed the effect of GA3 on [Ca2+]i within 3 h, 2 h before its effect on alpha-amylase production could be detected. Thus changes in [Ca2+]i may play a role in mediating the effects of GA3 and ABA on the synthesis and secretion of alpha-amylase in the aleurone cell.

Abscisic Acid↗
PubMed2005

The thylakoid delta pH/delta psi are not required for the initial stages of Tat-dependent protein transport in tobacco protoplasts.

The twin-arginine translocation (Tat) system transports folded proteins across the chloroplast thylakoid membrane and bacterial plasma membrane. In vitro import assays have pointed to a key role for the thylakoid delta pH in the initial assembly of the full translocon from two subcomplexes; more generally, the delta pH is believed to provide the overall driving force for translocation. Here, we have studied the role of the delta pH in vivo by analyzing the translocation of Tat substrates in transfected tobacco protoplasts. We show that the complete maturation of the precursor of the 23-kDa lumenal protein (pre-23K) and of a fusion of the 23K presequence linked to green fluorescent protein (pre-GFP) are unaffected by dissipation of the delta pH. High level expression of Tat substrates in protoplasts has recently been shown to result in "translocation reversal" in that a large proportion of a given substrate is partially translocated across the thylakoid membrane, processed to the mature size, and returned to the stroma. However, the efficiency of translocation of pre-23K is undiminished in the absence of the delta pH and/or delta psi, and the rate and extent of maturation of both pre-23K and pre-GFP by the lumen-facing processing peptidase is similarly unaffected. These data demonstrate that the proton motive force is not required for the functional assembly of the Tat translocon and the initial stages of translocation in higher plant chloroplasts in vivo. We conclude that unknown factors play an influential role in both the mechanism and energetics of this system under in vivo conditions.

Bacteria↗
PubMed1990

The use of flow cytometry to monitor chitin synthesis in regenerating protoplasts of Candida albicans.

Flow cytometry was used to monitor chitin synthesis in regenerating protoplasts of the yeast Candida albicans. Comparisons of cells stained with Calcofluor White, a fluorochrome with known affinity for chitin, and cells incubated in the presence of N-[3H]-acetylglucosamine, the precursor substrate for chitin, showed a linear relationship between fluorescence and incorporation of label over time. Changes in both the fluorescence and light scatter of regenerating protoplasts treated with inhibitors of fungal chitin synthase were also quantitated by flow cytometry.

Aminoglycosides↗
PubMed1984

Effects of mitotic and tubulin mutations on microtubule architecture in actively growing protoplasts of Aspergillus nidulans.

We used immunofluorescent microscopy to characterize microtubule (MT) architecture in wild-type and mutant protoplasts of Aspergillus nidulans at interphase and at mitosis. Because the visualization of MTs by immunofluorescence is technically difficult in intact hyphae of A. nidulans, we developed a method for removing the cell wall under conditions that do not perturb cell physiology, as evidenced by the fact that the resulting protoplasts undergo nuclear division at a normal rate and that cell cycle mutant phenotypes are expressed at restrictive temperature. Interphase cells exhibited an extensive network of cytoplasmic MTs. During mitosis the cytoplasmic MTs mostly disappeared and an intranuclear mitotic spindle appeared. We have previously shown that the benA 33 beta-tubulin mutation causes hyperstabilization of the mitotic spindle, and we have presented additional indirect evidence that suggested that the tubA1 and tubA4 alpha-tubulin mutations destabilize spindle MTs. In this paper, we show that the benA33 mutation increases the stability of cytoplasmic MTs as well as spindle MTs and that the tubA1 and tubA4 mutations destabilize both spindle and cytoplasmic MTs.

Aspergillus nidulans↗
PubMed1994

External pH effects on the depolarization-activated K channels in guard cell protoplasts of Vicia faba.

Previous studies reveal that the pH of the apoplastic solution in the guard cell walls may vary between 7.2 and 5.1 in closed and open stomata, respectively. During these aperture and pH changes, massive K+ fluxes cross the cellular plasma membrane driving the osmotic turgor and volume changes of guard cells. Therefore, we examined the effect of extracellular pH on the depolarization-activated K channels (KD channels), which constitute the K+ efflux pathway, in the plasma membrane of Vicia faba guard cell protoplasts. We used patch clamp, both in whole cells as well as in excised outside-out membrane patches. Approximately 500 KD channels, at least, could be activated by depolarization in one protoplast (density: approximately 0.6 micron-2). Acidification from ph 8.1 to 4.4 decreased markedly the whole-cell conductance, GK, of the KD channels, shifted its voltage dependence, GK-EM, to the right on the voltage axis, slowed the rate of activation and increased the rate of deactivation, whereas the single channel conductance was not affected significantly. Based on the GK-EM shifts, the estimated average negative surface charge spacing near the KD channel is 39 A. To quantify the effects of protons on the rates of transitions between the hypothesized conformational states of the channels, we fitted the experimental macroscopic steady state conductance-voltage relationship and the voltage dependence of time constants of activation and deactivation, simultaneously, with a sequential three-state model CCO. In terms of this model, protonation affects the voltage-dependent properties via a decrease in localized, rather than homogeneous, surface charge sensed by the gating moieties. In terms of either the CO or CCO model, the protonation of a site with a pKa of 4.8 decreases the voltage-independent number of channels, N, that are available for activation by depolarization.

Cell Membrane↗
PubMed1995

Protoplast fusion method for efficient transformation of mouse spermatogenic cells.

The efficiencies of methods for introducing DNAs into spermatogenic cells were investigated. The calcium phosphate method has been most commonly used to transfect DNA into cultured cells, but this method severely damages spermatogenic cells. Therefore, we investigated the efficiencies of transfection of spermatogenic cells by DEAE-dextran, modified liposome, and protoplast fusion methods, which seemed to cause less damage to spermatogenic cells than the calcium phosphate method. The protoplast fusion method was the most suitable for transfection of spermatogenic cells.

Animals↗
PubMed2006

Cation currents in protoplasts from the roots of a Na+ hyperaccumulating mutant of Capsicum annuum.

A wilty mutant (scabrous diminutive, sd) of Capsicum annuum L. hyperaccumulates Na+ in all tissues and has a lower K+ content in the roots. This has been shown to be due to a greater efflux of (86)Rb+ (K+) and influx of (22)Na+ in the mutant. In this study, the transporters responsible for these fluxes were investigated by applying patch clamp techniques to protoplasts derived from root cortical cells. Inwardly rectifying K+ currents were comparable in the two genotypes, but a characteristically bigger outward K+ current was observed in protoplasts from mutant roots, correlating with a bigger efflux of (86)Rb+ from mutant plants. Whole-cell currents due to the movement of Na+ have also been studied in both genotypes. The magnitude of the time-independent inward currents that conduct Na+ at hyperpolarizing voltages were comparable in both genotypes. However, microelectrode measurements of membrane potentials in cortical cells of roots in high Na+ conditions revealed that the membrane potentials of the root cells in the mutants were approximately 60 mV more negative than in wild-type root cells. Quantitatively, this hyperpolarization is calculated to be sufficient to account for the increased Na+ influx in the mutants.

Capsicum↗
PubMed1985

Isolation of a fraction from cauliflower mosaic virus-infected protoplasts which is active in the synthesis of (+) and (-) strand viral DNA and reverse transcription of primed RNA templates.

Sub-cellular fractions, isolated from cauliflower mosaic virus (CaMV)-infected turnip protoplasts, are capable of synthesising CaMV DNA in vitro on an endogenous template and of reverse transcribing oligo dT-primed cowpea mosaic virus RNA. The activity was not detected in mock-inoculated protoplasts. In vitro-labelled DNA hybridized to single-stranded M13 clones complementary to the putative origins of (-) and (+) strand CaMV DNA synthesis and to restriction endonuclease fragments encompassing more than 90% of the CaMV genome. The synthesis of (-) and (+) strand DNA appeared asymmetric. The template(s) for in vitro CaMV DNA synthesis are in a partially nuclease-resistant form. Fractions capable of in vitro CaMV DNA synthesis contained CaMV RNA both heterogeneous and as discrete species; they also contained a range of different sizes of CaMV DNA. Several lines of evidence indicate that this range of in vitro-labelled CaMV DNA, extending from 0.6kb to 8.0kb in length, represents elongating (-) strand DNA. These are discussed in relation to their role as possible replicative intermediates.

Cloning, Molecular↗
PubMed2001

Osmotic stress induces inactivation of photosynthesis in guard cell protoplasts of Vicia leaves.

Guard cell protoplasts isolated from Vicia leaves showed a strong suppression of the photosynthesis under hypotonic conditions, as reflected by changes in the chlorophyll fluorescence characteristics. The response was reversible as well. Mesophyll cell protoplasts did not show any lowering of photosynthetic activity under hypo-osmotic conditions. This result indicates that the response was guard cell specific.

Fabaceae↗
PubMed2001

Auxin- and abscisic acid-dependent osmoregulation in protoplasts of Phaseolus vulgaris pulvini.

Protoplasts isolated from the laminar pulvinus of Phaseolus vulgaris and bathed in a medium containing KCl as the major salt were found to swell in response to IAA and to shrink in response to ABA. The protoplasts of flexor cells and those of extensor cells responded similarly. The results indicate that the cellular content of osmotic solutes is enhanced by IAA and reduced by ABA. The IAA-induced swelling was abolished when either the K(+) or the Cl(-) of the bathing medium was replaced by an impermeant ion or when the medium was adjusted to neutral pH (instead of pH 6). The response was inhibited by vanadate. It is concluded that the swelling is caused by enhanced influxes of K(+) and Cl(-), which probably occur through K(+) channels and Cl(-)/H(+) symporters, respectively. The ABA-induced shrinking was inhibited by 5-nitro-2-(3-phenylpropylamino)-benzoic acid, an anion-channel inhibitor, suggesting that it is caused by Cl(-) efflux through anion channels and charge-balancing K(+) efflux through outward-rectifying K(+) channels. It appears that the two plant hormones act on pulvinar motor cells to regulate their turgor pressure, as they do in stomatal guard cells. The findings are discussed in relation to the pulvinar movements induced by environmental stimuli.

Abscisic Acid↗
PubMed2003

Differences in aquaporin levels among cell types of radish and measurement of osmotic water permeability of individual protoplasts.

We investigated tissue- and cell-specific accumulation of radish aquaporin isoforms by immunocytochemical analysis. In taproots, the plasma membrane aquaporins (RsPIP1 and RsPIP2) were accumulated at high levels in the cambium, while the tonoplast aquaporin (RsTIP) was distributed in all tissues. The three isoforms were highly accumulated in the central cylinder of seedling roots and hypocotyls, and rich in the vascular tissue of the petiole of mature plants. The results suggest that RsPIP1 and RsPIP2 are abundant in the cells surrounding the sieve tube of the radish plant. The swelling rate of protoplasts in a hypotonic solution was determined individually by a newly established method to compare the osmotic water permeability of different cell types. All cells of the cortex and endodermis in seedlings showed a high water permeability of more than 300 microm s(-1). There was no marked difference in the values between the root endodermis and cortex protoplasts, although the RsPIP level was lower in the cortex than in the endodermis. This inconsistency suggests two possibilities: (1) a low level of aquaporin is enough for high water permeability and (2) the water channel activity of aquaporin in the tissues is regulated individually. The uneven distribution of aquaporins in tissues is discussed along with their physiological roles.

Algorithms↗