Surface Of Protoplasts Research Articles

Involvement of an acid phosphatase on cell wall regeneration of tobacco protoplasts

Tobacco protoplasts begin to regenerate their own cell walls, the major components of which are β‐glucans, soon after they are transferred into an adequate medium. During the cell wall regeneration the protoplasts secrete two isoforms of acid phosphatase (APase) in time‐dependent manner. We determined that one of the isoforms, the Brefeldin A (BFA) sensitive one, is the cell wall resident APase (WP‐II) by immunoblotting of the isoform with anti‐WP‐II antibody. We hypothesized that the WP‐II may participate in the deposition of β‐glucan microfibrils on the protoplast surface during cell wall regeneration. In order to examine this hypothesis, the protoplasts were cultivated in the cell wall regeneration medium containing the same amount of the BFA‐sensitive APase (230 µg protein) as is secreted by the observed number of protoplasts (1.4 × 105 protoplasts) per plate (30‐mm‐diameter) during a 3‐h cultivation after transfer to the cell wall regeneration medium. The addition of WP‐II to the cell wall regeneration medium stimulated the deposition of β‐glucan microfibrils on the surface of the protoplasts during cell wall regeneration. To determine the stimulative effect of the 60 kDa polypeptide of WP‐II, protoplasts were cultivated in the medium containing the amount of anti‐WP‐II IgG (230 µg protein) equivalent to the BFA‐sensitive APase. These results suggested that the 60 kDa polypeptide of WP‐II is the BFA‐sensitive APase which is responsible for the enhanced deposition of β‐glucan microfibrils on the surface of the protoplasts.

Potato Cell Plasma Membrane Receptors to Ring Rot Pathogen Extracellular Polysaccharides

AbstractThe interaction of extracellular polysaccharides (EPS) of the potato ring rot bacterial pathogen Clavibacter michiganensis ssp. sepedonicus (Spieck. et Kott.) Skaptason et Burkh. (Cms) with protoplasts isolated both from leaf cells of plants grown in vitro and microsomal membrane fractions obtained from cell suspension cultures of two potato (Solanum tuberosum L.) cultivars contrasted by their resistance to this pathogen was studied. The EPS intensively bind to protoplast surfaces and microsomal membranes of the susceptible cultivar but not to those of the resistant cultivar. Treatment with protease, excess of unlabelled EPS, and with dextran, did not lead to the binding of fluorochrome‐labelled EPS to protoplasts and microsomal membranes (from both cultivars). It is proposed that (a) a great number of receptors to EPS Cms are present in the plasma membranes of potato cells of susceptible cultivars, (b) these receptors contain proteinaceous sites exposed on the external side of the plasma membrane which participate in EPS binding, and (c) the plasma membranes of cells of resistant cultivars contain a small but sufficient quantity of receptors to EPS able to induce defensive responses in plants.

High-resolution microscopy of cell wall formation in regenerating Mougeotia (Chlorophyceae) protoplasts

Field emission scanning electron microscopy (FESEM) preparation techniques have been successfully adapted for visualization of the internal and external ultrastructure of Mougeotia filaments and protoplasts. FESEM of the innermost layer of cell wall in Mougeotia filaments revealed that microfibrils are deposited parallel to each other in an interconnected mesh and are oriented perpendicular to the direction of elongation. For the first time, the surface of protoplasts at different stages of regeneration has been observed using FESEM. Nascent microfibril deposition occurs between 1 and 2 h after isolation and arrangement of these microfibrils is random for at least 8 h. Observation of the inner surface of the plasma membrane in burst protoplasts showed that microtubules are not strongly attached for at least 3 h after protoplast isolation.

The Cytoskeleton and the Secretory Pathway Are Not Involved in Targeting the Cowpea Mosaic Virus Movement Protein to the Cell Periphery

The movement protein (MP) of cowpea mosaic virus (CPMV) forms tubules on infected protoplasts and through plasmodesmata in infected plants. In protoplasts the MP fused to GFP (MP-GFP) was shown to localize in peripheral punctate structures and in long tubular structures extending from the protoplast surface. Using cytoskeletal assembly inhibitors (latrunculin B and oryzalin) and an inhibitor of the secretory pathway (brefeldin A), targeting of the MP to the peripheral punctate structures was demonstrated not to be dependent on an intact cytoskeleton or functional secretion pathway. Furthermore it was shown that a disrupted cytoskeleton had no effect on tubule formation but that the addition of brefeldin A severely inhibited tubule formation. The results presented in this paper suggest a role for a plasma membrane host factor in tubule formation of plant viral MPs.

Plasmolysis as a Tool to Reveal Lead Localization in the Apoplast of Root Cells

In order to analyze the distribution of lead between cell walls and plasmalemma, two-day-old maize seedlings (Zea mays L.) were incubated for 24 h on a solution of lead nitrate at a concentration causing 50% inhibition of root growth (10–5 M). Using the histochemical technique (precipitation of lead dithizonate), the distribution of lead in plasmolyzed and nonplasmolyzed cells of the root cortex was compared. This allowed us to separate the lead bound by cell walls from the lead located on the protoplast surface and in the periplasmic space. The plasmolysis was conducted prior to histochemical reaction by the incubation of seedling roots in 0.6 M sucrose solution for 30 min. The lead precipitates were located in cell walls and on the surface of protoplast. A small amount of lead was found in periplasmic space of some cells in root cortex. It is suggested that the lead is bound not only to the cell wall matrix but also to the plasmalemma.

Characterization of abscisic acid perception sites on the surface of stomatal guard cell protoplasts(Abstracts of The 37th Annual Meeting of the Japanese Society for Chemical Regulation of Plants)

Characterization of abscisic acid perception sites on the surface of stomatal guard cell protoplasts(Abstracts of The 37th Annual Meeting of the Japanese Society for Chemical Regulation of Plants)

Characterization of abscisic acid perception sites on the surface of stomatal guard cell protoplasts

Characterization of abscisic acid perception sites on the surface of stomatal guard cell protoplasts

Umbravirus-encoded movement protein induces tubule formation on the surface of protoplasts and binds RNA incompletely and non-cooperatively.

Various functions of the cell-to-cell movement protein (MP) of Groundnut rosette virus (GRV) were analysed. The GRV ORF4-encoded protein was shown by immunofluorescence microscopy to generate tubular structures that protrude from the surface of the protoplast. The protein encoded by ORF4 was assessed also for RNA-binding properties. This protein was tagged at its C terminus with six histidine residues, produced in Escherichia coli using an expression vector and purified by affinity chromatography. Gel retardation analysis demonstrated that, in contrast to many other viral MPs, including the 3a MP of Cucumber mosaic virus (CMV), the ORF4-encoded protein bound non-cooperatively to viral ssRNA and formed complexes of low protein:RNA ratios. Competition binding experiments showed that the ORF4-encoded protein bound to both ssRNA and ssDNA without sequence specificity, but did not bind to dsDNA. UV cross-linking and nitrocellulose membrane-retention assays confirmed that both the GRV and the CMV MPs formed complexes with ssRNA and that these complexes showed similar stability in NaCl. Probing the MP-RNA complexes by atomic force microscopy demonstrated that the ORF4-encoded protein bound RNA incompletely, leaving protein-free RNA segments of varying length, while the CMV 3a protein formed highly packed complexes. The significance of the two properties of limited RNA binding and tubule formation of the umbraviral MP is discussed.

Role of the alfalfa mosaic virus movement protein and coat protein in virus transport.

The movement protein (MP) and coat protein (CP) encoded by Alfalfa mosaic virus (AMV) RNA 3 are both required for virus transport. RNA 3 vectors that expressed nonfused green fluorescent protein (GFP), MP:GPF fusions, or GFP:CP fusions were used to study the functioning of mutant MP and CP in protoplasts and plants. C-terminal deletions of up to 21 amino acids did not interfere with the function of the CP in cell-to-cell movement, although some of these mutations interfered with virion assembly. Deletion of the N-terminal 11 or C-terminal 45 amino acids did not interfere with the ability of MP to assemble into tubular structures on the protoplast surface. Additionally, N- or C-terminal deletions disrupted tubule formation. A GFP:CP fusion was targeted specifically into tubules consisting of a wild-type MP. All MP deletion mutants that showed cell-to-cell and systemic movement in plants were able to form tubular structures on the surface of protoplasts. Brome mosaic virus (BMV) MP did not support AMV transport. When the C-terminal 48 amino acids were replaced by the C-terminal 44 amino acids of the AMV MP, however, the BMV/AMV chimeric protein permitted wild-type levels of AMV transport. Apparently, the C terminus of the AMV MP, although dispensable for cell-to-cell movement, confers specificity to the transport process.

Intracellular localization and movement phenotypes of alfalfa mosaic virus movement protein mutants.

Thirteen mutations were introduced in the movement protein (MP) gene of Alfalfa mosaic virus (AMV) fused to the green fluorescent protein (GFP) gene and the mutant MP-GFP fusions were expressed transiently in tobacco protoplasts, tobacco suspension cells, and epidermal cells of tobacco leaves. In addition, the mutations were introduced in the MP gene of AMV RNA 3 and the mutant RNAs were used to infect tobacco plants. Ten mutants were affected in one or more of the following functions of MP: the formation of tubular structures on the surface of protoplasts, association with the endoplasmic reticulum (ER) of suspension cells and epidermal cells, targeting to punctate structures in the cell wall of epidermis cells, movement from transfected cells to adjacent cells in epidermis tissue, cell-to-cell movement, or long-distance movement in plants. The mutations point to functional domains of the MP and support the proposed order of events in AMV transport. Studies with several inhibitors indicate that actin or microtubule components of the cytoskeleton are not involved in tubule formation by AMV MP. Evidence was obtained that tubular structures on the surface of transfected protoplasts contain ER- or plasmalemma-derived material.

Transmembrane topography of plasma membrane constituents in mung bean ( Vigna radiata L.) hypocotyl cells : I. Transmembrane distribution of phospholipids

The transmembrane distribution of phospholipids (PLs) in the plasma membrane (PM) of mung bean ( Vigna radiata L.) hypocotyl cells was investigated using annexin V-fluorescein isothiocyanate, porcine pancreas phospholipase A 2, and 31P-nuclear magnetic resonance (NMR) spectroscopy. Phosphatidylserine was not located on the cell surface of mung bean protoplasts. However, phosphatidylcholine, phosphatidylethanolamine and phosphatidic acid were found to be almost symmetrically distributed across right-side-out PM vesicles obtained by aqueous two-phase partitioning by porcine pancreas phospholipase A 2 assay. 31P-NMR assay showed that the amount of PLs is about equal in the outer and the inner leaflets of the right-side-out PM vesicles. These results suggest that the topography of PM PLs might not contribute to well-known asymmetrical properties of the outer and inner surfaces of higher plant PMs. It is also indicated that inside-out PM vesicles created by Brij 58-treatment do not retain the native PL topography on dithionate reduction of 7-nitro-2,1,3-benzoxadiazol-4-yl-labeled PLs incorporated in the PM vesicles.

Isolation of cortical MTs from tobacco BY-2 cells.

We isolated the cortical microtubules (CMTs) from tobacco BY-2 cells to identify their components. By centrifugation of protoplasts homogenized in the presence of taxol, a MT-stabilizing reagent, in a density gradient of Percoll, we obtained membranous vesicles to which MTs forming a sheet-like bundle were attached. Rhodamine-conjugated Ricinus communis agglutinin I (RCA-I), a lectin that bound to the surface of protoplasts, stained these vesicles, indicating that they were plasma membrane (PM) vesicles that retained CMTs. CMTs were released by solubilization of PM vesicles with Triton X-100. A sheet-like array of CMTs was retained even after solubilization of PM vesicles. Immunoblot analysis of the isolated CMTs demonstrated the presence of tubulin, actin, the 65 kDa microtubule-associated protein (MAP) and a 130 kDa RCA-I binding protein. Purification of the isolated CMTs by the temperature dependent disassembly-reassembly cycling method revealed four polypeptides, 190, 120, 85 and 65 kDa, co-assembling with CMTs.

Short-term effects of Al3+ on the osmotic behavior of red beet (Beta vulgaris L.) protoplasts

The short-term (less than 10 min) effects of Al3+ on the biophysical properties of plasma membranes were investigated by time-series image analysis of osmotically-induced volumetric and morphologic changes of red beet (Beta vulgaris L.) protoplasts. Exposure to Al3+ under hypotonic conditions reduced the volumetric expansion of protoplasts and their resultant burst: i.e. lysis of protoplasts in a concentration-dependent manner. Under hypertonic conditions, protoplasts exposed to Al3+ underwent an enhanced volumetric contraction in cross-sectional area, while maintaining higher protoplast roundness. The residual effects of Al3+ pre-treatment on subsequent osmotic behavior were also examined, and protoplasts pre-treated with Al3+ also exhibited less lysis during subsequent exposure to hypotonic conditions and enhanced volumetric contractions and higher roundness under subsequent hypertonic conditions. Under our experimental conditions, Al3+ consistently minimized protoplast surface area by inhibiting osmotic expansion or by enhancing osmotic contraction, as well as by maintaining higher protoplast roundness. These results suggested that the electrostatic property of Al3+ might have induced the binding and possible cross-linking of negatively-charged sites on the plasma membrane surface. This may be an important factor in understanding the mechanism of Al3+ phytotoxicity.

Intracellular distribution, cell-to-cell trafficking and tubule-inducing activity of the 50 kDa movement protein of Apple chlorotic leaf spot virus fused to green fluorescent protein.

The 50 kDa protein (50KP) encoded by ORF2 of Apple chlorotic leaf spot virus (ACLSV) fused to green fluorescent protein (GFP) was expressed transiently in cells of Nicotiana occidentalis and Chenopodium quinoa leaves. Its intracellular distribution, cell-to-cell trafficking in leaf epidermis and tubule formation on the surface of protoplasts were analysed. The 50KP-GFP fluorescence was distributed as small irregular spots or a fibrous network structure on the periphery of epidermal cells and protoplasts of both plant species. In leaf epidermis of N. occidentalis, the protein spread from the cells that produced it into neighbouring cells in both young and mature leaves and targetted plasmodesmata in these cells. In contrast, GFP was restricted to single cells in most cases in mature leaves. When 50KP and GFP were co-expressed in leaf epidermis of N. occidentalis, GFP spread more widely from the initial cells that produced it than when GFP was expressed alone, suggesting that 50KP facilitated the cell-to-cell trafficking of GFP. 50KP-GFP was able to complement local spread of 50KP-deficient virus when expressed transiently in leaf epidermis of C. quinoa. Expression of 50KP-GFP in protoplasts resulted in the production of tubular structures protruding from the surface. Mutational analyses showed that the C-terminal region (aa 287-457) was not essential for localization to plasmodesmata, cell-to-cell trafficking, complementation of movement of 50KP-deficient virus or tubule formation on protoplasts. In contrast, deletions in the N-terminal region resulted in the complete disruption of all these activities.

Formation of Surface Tubules and Fluorescent Foci in Arabidopsis thaliana Protoplasts Expressing a Fusion between the Green Fluorescent Protein and the Cauliflower Mosaic Virus Movement Protein

The movement protein (MP) of cauliflower mosaic virus (CaMV) is a multifunctional protein that potentiates the cell-to-cell and long distance movement of the virus. Functional domains in the CaMV MP were determined by analyzing deletions in green fluorescent protein (GFP)–MP fusions transfected into Arabidopsis thaliana leaf protoplasts. GFP–MP accumulated at fluorescent foci at the cell periphery and in tubular structures extending from the protoplast surface. A region located near the center of MP was required for targeting GFP–MP to foci, whereas a larger region extending nearly to the N-terminus was needed for tubule formation. Cytoskeletal assembly inhibitors did not disrupt tubule formation or the accumulation of GFP–MP at foci, but brefeldin A, which disrupts the plant cell endomembrane system, did interfere with the formation of tubules but not foci.

Functional, c-myc-tagged Cf-9 resistance gene products are plasma-membrane localized and glycosylated.

The Cf-9 resistance gene from tomato confers resistance to races of the fungal pathogen Cladosporium fulvum that express the corresponding avirulence gene, Avr9. Avr9 encodes a secreted peptide. To investigate Cf-9 function, we tagged the Cf-9 protein with a triple myc epitope at either the amino- or carboxy-terminus of the mature protein. Tobacco plants carrying these constructs activate a defence response to Avr9 peptide. The Cf-9 sequence predicts a protein of 94 kDa, with 22 glycosylation sites. Using c-myc antibodies, c-myc : Cf-9 protein was detected as a unique band with a molecular size of 160 kDa. The band shifted to approximately 105 kDa after glucosidase treatment, indicating that Cf-9 protein is highly glycosylated. Plasma membranes were isolated using two-phase partitioning, and c-myc : Cf-9 was enriched in these fractions, indicating that Cf-9 is a plasma membrane protein. This was confirmed by silver-enhanced immunogold labelling of tobacco protoplasts carrying the amino-terminal c-myc tag; a higher labelling density was observed on the surface of protoplasts derived from c-myc : Cf-9 tobacco compared to untransformed control. The presence of Cf-9 in the plasma membrane is consistent with its role in conferring recognition of the extracellular Avr9 peptide.

Iron deficiency decreases the Fe(III)-chelate reducing activity of leaf protoplasts.

The ferric-chelate reductase (FC-R) activity of mesophyll protoplasts isolated from Fe-sufficient (control) and Fe-deficient sugar beet (Beta vulgaris L.) leaves has been characterized. Measurements were made in an ionic environment similar to that in the apoplastic space of the sugar beet mesophyll cells. The FC-R activity of Fe-sufficient and Fe-deficient protoplasts was dependent on light. Fe deficiency decreased markedly the FC-R activity per protoplast surface unit. The optimal pH for the activity of the FC-R in mesophyll protoplasts was in the range 5.5 to 6.0, typical of the apoplastic space. Beyond pH 6.0, the activity of the FC-R in mesophyll protoplasts decreased markedly in both Fe-sufficient and Fe-deficient protoplasts. These data suggest that both the intrinsic decrease in FC-R activity per protoplast surface and a possible shift in the pH of the apoplastic space could lead to the accumulation of physiologically inactive Fe pools in chlorotic leaves.

Mutational Analysis of the Cowpea Mosaic Virus Movement Protein

Cowpea mosaic virus moves from cell-to-cell in a virion form through tubular structures that are assembled in modified plasmodesmata. Similar tubular structures are formed on the surface of protoplasts inoculated with cowpea mosaic virus. The RNA 2-encoded movement protein (MP) is responsible for the induction and formation of these structures. To define functional domains of the MP, an alanine-substitution mutagenesis was performed on eight positions in the MP, including two conserved sequence motifs, the LPL and D motifs. Results show that these two conserved motifs as well as the central region of the MP are essential for cell-to-cell movement. Several viruses carrying mutations in the N- or C-terminal parts of their MP retained infectivity on cowpea plants. Coexpression studies revealed that mutant MPs did not interfere with the activity of wild-type MP and could not mutually complement their defects.

Are Tubules Generated by the 3a Protein Necessary for Cucumber Mosaic Virus Movement?

The 3a movement protein of cucumber mosaic virus (CMV), fused to the jellyfish green fluorescent protein (3a-GFP) generated surface punctate aggregates as well as tubules protruding from infected tobacco and Nicotiana benthamiana protoplasts. Fluorescent tubules also appeared on the surface of protoplasts prepared from transgenic tobacco plants expressing 3a-GFP, indicating that the 3a protein is the only viral component required for the formation of the tubules. CMV with a mutation in the gene encoding the 3a protein, M8 CMV, could infect tobacco systemically, but tubules were not detected protruding from infected protoplasts when the mutated 3a protein was fused to the GFP [(M8)3a-GFP]. This indicates that the ability of the 3a protein to generate tubules in the surface of protoplasts is not a function required for the spread of CMV in tobacco. On the other hand, the (M8)3a-GFP did not traffic through plasmodesmata interconnecting tobacco epidermal cells, in contrast to the wild-type 3a-GFP. This suggests that there may be a correlation between the ability of the 3a protein to assemble tubules in protoplasts and its ability to promote movement within certain tissues.

Subcellular localization and in vivo identification of the putative movement protein of olive latent virus 2.

The gene encoding the 36.5 kDa ('36K') nonstructural protein located on RNA3 of olive latent virus 2 (OLV-2) was cloned, expressed with the Escherichia coli pGEX-2T system and the purified protein used to raise a polyclonal antiserum. Immunoblot analysis of OLV-2-infected Nicotiana benthamiana plants showed that the 36K protein accumulated in the early stages of infection and was associated with a subcellular fraction enriched in cytoplasmic membranes. In infected cells there were tubular structures, some containing virus-like particles, scattered in the cytoplasm or protruding from or penetrating the cell wall at the plasmodesmata. Immunogold labelling localized the 36K protein in the plasmodesmata of OLV-2-infected cells and showed it to be associated with virus-containing tubules. Leaf trichome cells of N. tabacum plants, transformed with a 36K-green fluorescent protein (GFP) fusion construct, revealed localized fluorescence in the cell walls, possibly due to association of the fusion protein with plasmodesmata. When the same 36K-GFP fusion protein was expressed in N. tabacum protoplasts, long tubular fluorescent structures protruded from the protoplast surface, suggesting that the 36K protein is responsible for tubule induction. The conclusion is drawn that this protein is likely to be the OLV-2 movement protein, mediating cell-to-cell virus movement, and that movement is by a tubule-guided mechanism.