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At least 721 records · Page 40Linked to original sources
PubMed2003

Is the LIM-domain protein HaWLIM1 associated with cortical microtubules in sunflower protoplasts?

Flowering plants express several LIM-domain proteins related to the animal cystein-rich proteins. The expression of sunflower LIM genes was followed by RT-PCR in cultured sunflower protoplasts. A transcript was detected only for HaWLIM1, but not for the other two genes HaPLIM1 and HaPLIM2. Polyclonal antibodies raised against either full length recombinant HaWLIM1 protein or peptides recognized a 27 kDa polypeptide on Western blots. Immunocytolocalization studies showed that HaWLIM1 is located in the cytoplasm and in the nucleus. In the cytoplasm, HaWLIM1 is localized in punctate structures, distributed along microtubule bundles. Depolymerizing microtubules with oryzalin resulted in a strong modification of the HaWLIM1 cortical pattern. In contrast, treatment of protoplasts with latrunculin B, which disrupts actin filaments, had no effect on HaWLIM1 localization. HaWLIM1 was also located within the nucleus of interphase protoplasts. During mitosis, nuclear labelling was observed in prophase, which decreased in metaphase, disappeared in anaphase, and recovered in telophase. These results suggest a dual role for HaWLIM1: in the cytoplasm, as a component of molecular complexes which may interact with microtubules, and in the nucleus, as a partner of transcription factors during interphase.

Base Sequence↗
PubMed1999

Naturally induced secretions of the potato cyst nematode co-stimulate the proliferation of both tobacco leaf protoplasts and human peripheral blood mononuclear cells.

Naturally induced secretions from infective juveniles of the potato cyst nematode Globodera rostochiensis co-stimulate the proliferation of tobacco leaf protoplasts in the presence of the synthetic phytohormones alpha-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP). With the use of a protoplast-based bioassay, a low-molecular-weight peptide(s) (< 3 kDa) was shown to be responsible for the observed effect. This mitogenic oligopeptide(s) is functionally dissimilar to auxin and cytokinin and, in addition, it does not change the sensitivity of the protoplasts toward these phytohormones. In combination with the mitogen phytohemagglutinin (PHA), cyst nematode secretions also co-stimulated mitogenesis in human peripheral blood mononuclear cells (PBMC). The stimulation of plant cells isolated from nontarget tissue--these nematodes normally invade the roots of potato plants--suggests the activation of a general signal transduction mechanism(s) by an oligopeptide(s) secreted by the nematode. Whether a similar oligopeptide-induced mechanism underlies human PBMC activation remains to be investigated. Reactivation of the cell cycle is a crucial event in feeding cell formation by cyst nematodes. The secretion of a mitogenic low-molecular-weight peptide(s) by infective juveniles of the potato cyst nematode could contribute to the redifferentiation of plant cells into such a feeding cell.

Adenine↗
PubMed2001

Ectopic expression of an Arabidopsis calmodulin-like domain protein kinase-enhanced NADPH oxidase activity and oxidative burst in tomato protoplasts.

Among plant defense responses to pathogen attack, the release of active oxygen species (AOS), termed the oxidative burst, may affect the attacking pathogen and the host plant cells at the infection site, thereby limiting the spread of the pathogen. Plasma membrane-associated NADPH oxidase represents a key enzyme in mediating the oxidative burst. The mechanisms of NADPH oxidase activation, however, remains unclear. Ectopic expression of AK1-6H, an Arabidopsis calmodulin-like domain protein kinase (CDPK) in tomato protoplasts enhanced plasma membrane-associated NADPH oxidase activity. Arabidopsis protein phosphatase 2A abolished this enhancement, whereas Arabidopsis dual-specificity protein tyrosine phosphatase 1 or maize protein phosphatase 1 had no effect tMEK2MUT, a constitutively activated, mitogen-activated protein kinase kinase from tomato, did not enhance NADPH oxidase activity when overexpressed. In a cell-free system, AK1-6H moderately stimulated the NADPH oxidase activity on plasma membrane. AK1-6H, but not tMEK2MUT, also enhanced production of AOS in intact protoplasts. Our results show that ectopic expression of a heterologous CDPK can enhance NADPH oxidase activity and stimulate an oxidative burst in tomato protoplasts.

Arabidopsis↗
PubMed1991

Tubular structures involved in movement of cowpea mosaic virus are also formed in infected cowpea protoplasts.

In cowpea plant cells infected with cowpea mosaic virus, tubular structures containing virus particles are formed in the plasmodesmata between adjacent cells; these structures are supposedly involved in cell-to-cell spread of the virus. Here we show that similar tubular structures are also formed in cowpea protoplasts, from which the cell wall and plasmodesmata are absent. Between 12 and 21 h post-inoculation, tubule formation starts in the periphery of the protoplast at the level of the plasma membrane. Upon assembly, the virus-containing tubule is enveloped by the plasma membrane and extends into the culture medium. This suggests that the tubule has functional polarity and makes it likely that a tubule 'grows' into a neighbouring cell in vivo. On average, 75% of infected protoplasts were shown to possess tubular structures extending from their surface. The tubule wall was 3 to 4 nm thick and they were up to 20 microns in length, as shown by fluorescent light microscopy and negative staining electron microscopy. By analogy to infected plant cells, both the viral 58K/48K movement and capsid proteins were located in these tubules, as determined by immunofluorescent staining and immunogold labelling using specific antisera against these proteins. These results demonstrate that the formation of tubules is not necessarily dependent on the presence of plasmodesmata or the cell wall, and that they are composed, at least in part, of virus-encoded components.

Capsid↗
PubMed1992

Electrotransfection of protoplasts from tomato, wild tomato, barley and chrysanthemum with tobacco mosaic virus RNA.

Protoplasts isolated from tomato, wild tomato, barley and chrysanthemum were electrotransfected with tobacco mosaic virus (TMV) RNA under almost the same optimum electric conditions: five square DC pulses of 50 microseconds duration at 500 to 800 V/cm, with the protoplasts suspended at 2 x 10(5)/ml in 0.5 M-mannitol containing 100 microM-MgCl2 and 10 to 20 micrograms/ml TMV RNA. ELISAs of these transfected protoplasts showed that the yields and the growth curves of the virus were quite similar, indicating a lack of host specificity in the initially infected cells of these plants.

Chrysanthemum cinerariifolium↗
PubMed1993

Efficient production of human gamma interferon in tobacco protoplasts by genetically engineered brome mosaic virus RNAs.

We succeeded in producing human gamma interferon (IFN-gamma) in tobacco protoplasts in quantity using genetically engineered brome mosaic virus (BMV strain ATCC66). This strain of BMV produces two types of coat protein, a full-length coat protein (20K) and a truncated coat protein (19K) which are translated from the first and second initiation codons, respectively. We replaced the truncated coat protein gene with the IFN-gamma gene and synthesized BMV-IFN-gamma chimera RNAs using an in vitro transcription system. The BMV-IFN-gamma chimera RNAs were used to inoculate tobacco protoplasts together with BMV RNA 1 and RNA 2 and produced IFN-gamma to a level of 5 to 10% of total extracted proteins per infected protoplast after 24 h of incubation. The efficient production of IFN-gamma was attributed to the high translation activity of the BMV-IFN-gamma chimera RNA. We demonstrate that 24 nucleotides coding for the N-terminal amino acids of the full-length coat protein were probably involved in the high translation activity of the BMV-IFN-gamma chimera RNA.

Base Sequence↗
PubMed1997

Replication of wild-type and mutant clones of satellite tobacco mosaic virus in Nicotiana benthamiana protoplasts.

RNA transcribed from cloned satellite tobacco mosaic virus (STMV) cDNA replicated in Nicotiana benthamiana protoplasts when co-inoculated with tobacco mild green mosaic virus (TMGMV) genomic RNA, but degraded when inoculated alone. STMV genomic RNA extracted from wild-type virions replicated in protoplasts when co-inoculated with TMGMV, tobacco mosaic virus (TMV) or tomato mosaic virus (ToMV). Transcripts from clones of two STMV coat protein (CP) mutants accumulated to the same level as wild-type transcripts in protoplasts when co-inoculated with TMGMV, whereas a third mutant accumulated to detectable levels in some, but not all, experiments. These results confirm that STMV RNA requires helper virus for replication, and that the helper specificity exhibited by cloned STMV reflects a specific requirement for the TMGMV replicase. It also demonstrates that the low accumulation of STMV CP mutants observed previously in whole plants cannot be attributed to inefficient RNA replication.

Mutation↗
PubMed1979

Factors affecting recombinant frequency in protoplast fusions of Streptomyces coelicolor.

The optimum concentration of polyethylene glycol 1000 (PEG) for the production of recombinants through protoplast fusion in Streptomyces coelicolor was about 50% (w/v). The addition of 14% (v/v) dimethyl sulphoxide to the fusion mixture enhanced recombination frequencies, but only at sub-optimal PEG concentrations. After treatment of protoplasts with 50% PEG for 1 min, the frequency of recombinants in a multi-factor 'cross' sometimes exceeded 20% of the total progeny. The frequency of recombinants in the progeny could be significantly enhanced by ultraviolet irradiation of the parental protoplast suspensions immediately before fusion.

Dimethyl Sulfoxide↗
PubMed1981

Human serum complement requirements for bacterial killing and protoplast lysis of Escherichia coli ML308 225.

Normal human serum kills Escherichia coli ML308 225 and lyses protoplasts derived from this organism. Human serum which is depleted of complement component C9 or deficient in component C8 is not bactericidal, but C9-depleted serum will lyse protoplasts whereas C8-deficient serum will not. Bacterial lipopolysaccharide, which can protect bacteria from the serum bactericidal reaction, does not protect protoplasts from lysis by serum.

Bacteriolysis↗
PubMed1982

Attempts to infect plant protoplasts with Toxoplasma gondii.

The interaction of tachyzoites of Toxoplasma gondii with plant protoplasts was compared with their interaction with cultures of HeLa cells. Unlike the HeLa cells, the plant protoplasts were refractory to invasion under all conditions tested. The lack of invasion was not an artefact of the protoplast isolation technique. This is the first description of a cell type refractory to Toxoplasma invasion; hence possible mechanisms for the specificity of parasite-host cell interaction can now be studied.

Cellulase↗
PubMed1987

Formation of a new cell wall by protoplasts of Candida albicans: effect of papulacandin B, tunicamycin and Nikkomycin.

Incorporation of polysaccharides into the walls of regenerating protoplasts of Candida albicans was followed in the presence of papulacandin B, tunicamycin and nikkomycin. With the first drug, chitin was incorporated normally whereas incorporation of glucans and mannoproteins was significantly decreased. Tunicamycin decreased incorporation of all wall polymers when added at the beginning of the regeneration process but blocked only mannan and alkali-insoluble glucan incorporation when added after 5 h. Nikkomycin inhibited chitin synthesis, and the walls formed by the protoplasts were enriched in alkali-soluble glucan. Pulse-chase experiments suggested that a precursor-product relationship between the alkali-soluble and alkali-insoluble glucans existed in the wall. The results obtained with the antibiotics were confirmed and extended by cytological studies using wheat-germ agglutinin labelled with colloidal gold and concanavalin A-ferritin as specific markers of chitin and mannoproteins respectively. The results support the idea that regeneration of walls by protoplasts occurs in two steps: firstly, a chitin microfibrillar skeleton is formed, and in a later step glucan-mannoprotein complexes are added to the growing structure. The chitin skeleton probably allows the orderly spatial arrangement of the other polymers giving rise to the regenerated cell wall.

Aminoglycosides↗
PubMed1976

The interaction of amphotericin B methyl ester with protoplasts of Candida albicans.

The interaction of amphotericin B methyl ester (AME) with protoplasts of Candida albicans was measured indirectly by following the incorporation of [U-14C]phenylalanine into the acid-insoluble material. The inhibitory effects of AME at the minimum inhibitory concentration were prevented by the addition of 85 mM-KCl and 45 mM-MgCl2, as shown by Liras & Lampen (1974) for Saccharomyces cerevisiae. In C. albicans, pretreatment of the yeast before anti-biotic addition was unnecessary. KCl and MgCl2 did not prevent AME from binding to the protoplast membrane. This interaction was reversed by incubating the protoplasts in the presence of the protecting salts.

Amphotericin B↗
PubMed1995

Unsaturated fatty acids are the active molecules of a glucan-synthase-inhibitory fraction isolated from entomophthoralean protoplasts.

A few entomophthoralean species are able to multiply in a protoplast form. The polysaccharide synthases which synthesize the cell wall are inactivated in this form. An inhibitor of one of the key enzymes of wall synthesis, glucan synthase, was isolated from entomophthoralean protoplasts, using silica column chromatography and HPLC. Thin-layer and gas chromatography revealed free fatty acids in the inhibitory fractions. These fatty acids, including long-chain unsaturated fatty acids, were shown to be responsible for the inhibition of glucan synthase. The fatty acids were generated during incubation of a protoplast homogenate for 36 h at 37 degrees C and were shown to be non-competitive and non-specific inhibitors of glucan synthase.

Aspergillus fumigatus↗
PubMed2002

The Role of auxin, pH, and stress in the activation of embryogenic cell division in leaf protoplast-derived cells of alfalfa.

Culturing leaf protoplast-derived cells of the embryogenic alfalfa (Medicago sativa subsp. varia A2) genotype in the presence of low (1 microM) or high (10 microM) 2, 4-dichlorophenoxyacetic acid (2,4-D) concentrations results in different cell types. Cells exposed to high 2,4-D concentration remain small with dense cytoplasm and can develop into proembryogenic cell clusters, whereas protoplasts cultured at low auxin concentration elongate and subsequently die or form undifferentiated cell colonies. Fe stress applied at nonlethal concentrations (1 mM) in the presence of 1 microM 2,4-D also resulted in the development of the embryogenic cell type. Although cytoplasmic alkalinization was detected during cell activation of both types, embryogenic cells could be characterized by earlier cell division, a more alkalic vacuolar pH, and nonfunctional chloroplasts as compared with the elongated, nonembryogenic cells. Buffering of the 10 microM 2,4-D-containing culture medium by 10 mM 2-(N-morpholino)ethanesulfonic acid delayed cell division and resulted in nonembryogenic cell-type formation. The level of endogenous indoleacetic acid (IAA) increased transiently in all protoplast cultures during the first 4 to 5 d, but an earlier peak of IAA accumulation correlated with the earlier activation of the division cycle in embryogenic-type cells. However, this IAA peak could also be delayed by buffering of the medium pH by 2-(N-morpholino)ethanesulfonic acid. Based on the above data, we propose the involvement of stress responses, endogenous auxin synthesis, and the establishment of cellular pH gradients in the formation of the embryogenic cell type.

2,4-Dichlorophenoxyacetic Acid↗
PubMed2004

Two distinct signaling pathways participate in auxin-induced swelling of pea epidermal protoplasts.

Protoplast swelling was used to investigate auxin signaling in the growth-limiting stem epidermis. The protoplasts of epidermal cells were isolated from elongating internodes of pea (Pisum sativum). These protoplasts swelled in response to auxin, providing the clearest evidence that the epidermis can directly perceive auxin. The swelling response to the natural auxin IAA showed a biphasic dose response curve but that to the synthetic auxin 1-naphthalene acetic acid (NAA) showed a simple bell-shaped dose response curve. The responses to IAA and NAA were further analyzed using antibodies raised against ABP1 (auxin-binding protein 1), and their dependency on extracellular ions was investigated. Two signaling pathways were resolved for IAA, an ABP1-dependent pathway and an ABP1-independent pathway that is much more sensitive to IAA than the former. The response by the ABP1 pathway was eliminated by anti-ABP1 antibodies, had a higher sensitivity to NAA, and did not depend on extracellular Ca(2+). In contrast, the response by the non-ABP1 pathway was not affected by anti-ABP1 antibodies, had no sensitivity to NAA, and depended on extracellular Ca(2+). The swelling by either pathway required extracellular K(+) and Cl(-). The auxin-induced growth of pea internode segments showed similar response patterns, including the occurrence of two peaks in the dose response curve for IAA and the difference in Ca(2+) requirements. It is suggested that two signaling pathways participate in auxin-induced internode growth and that the non-ABP1 pathway is more likely to be involved in the control of growth by constitutive concentrations of endogenous auxin.

Calcium↗
PubMed1998

Interaction of cryptochrome 1, phytochrome, and ion fluxes in blue-light-induced shrinking of Arabidopsis hypocotyl protoplasts.

Protoplasts isolated from red-light-adapted Arabidopsis hypocotyls and incubated under red light exhibited rapid and transient shrinking within a period of 20 min in response to a blue-light pulse and following the onset of continuous blue light. Long-persisting shrinkage was also observed during continuous stimulation. Protoplasts from a hy4 mutant and the phytochrome-deficient phyA/phyB double mutant of Arabidopsis showed little response, whereas those from phyA and phyB mutants showed a partial response. It is concluded that the shrinking response itself is mediated by the HY4 gene product, cryptochrome 1, whereas the blue-light responsiveness is strictly controlled by phytochromes A and B, with a greater contribution by phytochrome B. It is shown further that the far-red-absorbing form of phytochrome (Pfr) was not required during or after, but was required before blue-light perception. Furthermore, a component that directly determines the blue-light responsiveness was generated by Pfr after a lag of 15 min over a 15-min period and decayed with similar kinetics after removal of Pfr by far-red light. The anion-channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoic acid prevented the shrinking response. This result, together with those in the literature and the kinetic features of shrinking, suggests that anion channels are activated first, and outward-rectifying cation channels are subsequently activated, resulting in continued net effluxes of Cl- and K+. The postshrinking volume recovery is achieved by K+ and Cl- influxes, with contribution by the proton motive force. External Ca2+ has no role in shrinking and the recovery. The gradual swelling of protoplasts that prevails under background red light is shown to be a phytochrome-mediated response in which phytochrome A contributes more than phytochrome B.

Arabidopsis↗
PubMed1986

Polyamine metabolism and osmotic stress. II. Improvement of oat protoplasts by an inhibitor of arginine decarboxylase.

We have attempted to improve the viability of cereal mesophyll protoplasts by pretreatment of leaves with DL-alpha-difluoromethylarginine (DFMA), a specific 'suicide' inhibitor of the enzyme (arginine decarboxylase) responsible for their osmotically induced putrescine accumulation. Leaf pretreatment with DFMA before a 6 hour osmotic shock caused a 45% decrease of putrescine and a 2-fold increase of spermine titer. After 136 hours of osmotic stress, putrescine titer in DFMA-pretreated leaves increased by only 50%, but spermidine and spermine titers increased dramatically by 3.2- and 6-fold, respectively. These increases in higher polyamines could account for the reduced chlorophyll loss and enhanced ability of pretreated leaves to incorporate tritiated thymidine, uridine, and leucine into macromolecules. Pretreatment with DFMA significantly improved the overall viability of the protoplasts isolated from these leaves. The results support the view that the osmotically induced rise in putrescine and blockage of its conversion to higher polyamines may contribute to the lack of sustained cell division in cereal mesophyll protoplasts, although other undefined factors must also play a major role.

Arginine↗
PubMed1989

Effects of microgravitation on electrofusion of plant cell protoplasts.

Electrofusion of evacuolated with vacuolated mesophyll protoplasts of Nicotiana tabacum was performed as part of the German Sounding Rocket Program (TEXUS 17, 1988). The results indicate a significant increase not only in the yield of 1:1 hybrids, but also in homo- and multifusion products. Hybrids obtained under microgravity have been shown to be viable to a higher degree with respect to their ability for light-dependent O2-evolution (independent of other substrates than bicarbonate). This finding is of interest for fusion experiments where only limited numbers of fusion partners are available (e.g. protoplasts from embryogenic tissues) or where fusion yields are extremely low under 1 x gravity (e.g. protoplasts of different specific density).

Cell Fusion↗