Isolated Plant Cells Research Articles

The in vitro physiological phenotype of tomato resistance to Fusarium oxysporum f. sp. lycopersici

With the aim of dissecting host-parasite interaction processes in the system Lycopersicon aesculentum-Fusarium oxysporum f. sp. lycopersici we have isolated plant cell mutants having single-step alterations in their defense response. A previous analysis of the physiological phenotypes of mutant cell clones suggested that recognition is the crucial event for active defence, and that polysaccharide content, fungal growth inhibition, peroxidase induction in in vitro dual culture and ion leakage induced by cultural filtrates of the pathogen can be markers of resistance. In this paper we present the results of a similar analysis carried out on cell cultures from one susceptible ('Red River'), one tolerant ('UC 105') and three resistant ('Davis UC 82', 'Heinz', 'UC 90') tomato cultivars. Our data confirm that the differences in the parameters considered are correlated with resistance versus susceptibility in vivo. Therefore, these parameters can be used for early screening in selection programmes. These data, together with those obtained on isolated cell mutants, suggest that the selection in vitro for altered fungal recognition and/or polysaccharide or callose content may lead to in vivo - resistant genotypes. The data are thoroughly discussed with particular attention paid to the importance of polysaccharides in active defense initiation.

Molecular organization and clustering of cell-wall-bound enzymes as a source of kinetic apparent co-operativity.

When fixed charges and enzyme molecules are not homogeneously distributed in a matrix, the degree of organization of charges, of enzyme molecules and of charges with respect to enzyme molecules modulate the enzyme reaction rate. The overall reaction velocity of the bound enzyme system may be expressed in terms of monovariate moments of the charge density distribution and of the bivariate moments of the charge and enzyme density distributions. With respect to the situation where fixed charges and enzyme molecules are randomly distributed in the matrix, the molecular organization, as expressed by the monovariate and bivariate moments results in an increase or a decrease, of the overall reaction rate, as well as in the appearance of a kinetic cooperativity. The degree of spatial organization of objects may be expressed quantitatively through the concept of minimal spanning tree. This concept may thus be applied to the quantification of the degree of order that may exist in the bidimensional distribution of enzyme molecules in a charged matrix. Primary walls of isolated plant cells in sterile culture behave as a polyanion and contain different enzymes. The spatial distribution in sycamore cell walls of an acid phosphatase has been studied through the concept of minimal spanning tree and shown to be non-randomly distributed in the polyanionic matrix, but clustered in that matrix. This spatial organization results in a modulation of the reaction rate of the cell-wall-bound phosphatase reaction. Both the theoretical and experimental results presented in this study leave little doubt as to the validity of the idea that in situ the organization of fixed charges and enzyme molecules modulate the overall dynamics of enzyme reactions.

Signal Transduction of Auxin on Isolated Plant Cell Membranes: Indications for a Rapid Polyphosphoinositide Response Stimulated by Indoleacetic Acid

Summary Microsomal membranes prepared from Daucus carota cell suspension cultures were able to utilize [γ- 32 P]-ATP as a substrate for rapid phosphorylations of endogenous lipids resulting in the labeling of phosphatidic acid, phosphatidylinositol 4-monophosphate, and phosphatidylinositol 4,5-bisphosphate. In the presence of 1μM indoleacetic acid (IAA) the [ 32 P]-label of the phospholipids was significantly reduced within 30s, which was caused by a release of [ 32 P]-labeled inositolphosphates indicating an auxin effect on a phospholipase C-like reaction. Since the kinetics as well as the dose response curves of both IAA-mediated effects were found to correspond well, it is a further confirmation of our previous conclusions that auxin may control a rapid phosphatidylinositol turnover in plant cells, which may be similar to the wellknown phosphoinositide response in animal cells. As discussed, the experimental system presented is certainly a suitable tool for the further analysis of the detailed steps concerning the signal transduction mechanism of auxin on membranes.

Attachment ofAzospirillumto isolated plant cells

Azospirillum brasilense cells were shown to attach to isolated plant cells. A rapid semi-quantitative assay to measure plant cell attachment, as was described previously for Agrobacterium, was found to work especially well for Azospirillum. Attachment was measured in an indirect way and confirmed by direct observations through scanning electron microscopy.

Attachment of Azospirillum to isolated plant cells

Attachment of Azospirillum to isolated plant cells

Single potassium channels in membranes of isolated mesophyll barley vacuoles

Voltage-dependent K channels could be identified in on-cell and excised patch-clamp records on membranes of isolated plant cell vacuoles. The current through a membrane patch is dominated by a channel population with a conductance of about 121 pS in symmetrical 250mm KCl solution. The single channel adopts at least two conducting levels the 121-pS state being most frequently observed. The channel shows outward rectification, representing a cation flux into the vacuoles. The rectification appears to be caused by a vanishing open probability and a short channel lifetime at hyperpolarizing voltages. A selectivity ratio of potassium over sodium of about 6 was derived as an estimate. Occasionally, an additional population of K channels with a single-channel conductance of approximately 18 pS is observed. This channel type exhibits outward rectification as well.

Effect of Carbon-4 and Carbon-5 Volatile Fatty Acids on Digestion of Plant Cell Wall In Vitro

When mixed ruminal bacteria were incubated in an artificial medium with isolated plant cell walls, cell wall digestion and ammonia utilization were increased by low concentrations (< .30mM) of carbon-4 and carbon-5vol.tile fatty acids (isobutyric, isovaleric, valeric, and 2-methylbutyric). Carbon-4 and carbon-5 acid supplementation also increased cell wall digestion of intact forages. No pattern was observed between protein content of the feed and response to carbon-4 and carbon-5vol.tile fatty acids.Only the branch-chain acids (isovaleric, isobutyric, and 2-methylbutyric) stimulated bacterial growth and plant cell wall digestion. No synergisms were observed among the branched-chain acids, and they could replace each other. Valeric acid, a straight chain compound, was without effect, and phenylpropionic and phenylacetic acids likewise did not improve digestibility. The peptide source, Trypticase, enhanced cell wall digestion more than carbon-4 and carbon-5vol.tile fatty acids alone, and this observation suggested that fiber digesting bacteria may be stimulated directly by amino acids or peptides.

Attachment of Agrobacterium tumefaciens to mechanically isolated Asparagus cells

Mechanically isolated plant cells provide a convenient single-cell system for investigating bacterial attachment during the early stages of transformation by Agrobacterium tumefaciens. Cells isolated from cladodes of Asparagus officinalis were aggregated by both virulent and non-virulent strains of Agrobacterium, indicating that the bacterium can attach to the walls of cells from this Monocotyledon. The effects of temperature, media, cell concentration and viability were investigated in relation to the attachment process.

Regulation of extracellular pectate lyase in Erwinia chrysanthemi: evidence that reaction products of pectate lyase and exo-poly-α-d-galacturonosidase mediate induction on d-galacturonan

The process by which Erwinia chrysanthemi recognizes the presence of pectic polymers and thereby increases production of extracellular pectate lyase was investigated in vitro. The bacterium produced at least 20 times more extracellular pectate lyase during growth on d-galacturonan than during growth on glycerol. Several lines of evidence indicated that the induction of pectate lyase by d-galacturonan was mediated by reaction products (4,5-unsaturated digalacturonic acid and digalacturonic acid) released from the polymer by the basal, extracellular activities of pectate lyase and (or) exo-poly-α-d-galacturonosidase. The medium of cultures growing logarithmically on d-galacturonan contained factor(s) which diffused through dialysis tubing and stimulated pectate lyase induction in unadapted cultures simultaneously presented with d-galacturonan. The rate of pectate lyase induction during incubation with d-galacturonan was similarly enhanced by the addition of digalacturonic acid. At a concentration of 0·1 mg ml-1, d-galacturonan failed to be taken up by unadapted bacteria, but both saturated and unsaturated digalacturonic acid were rapidly assimilated and both induced pectate lyase. Pectate lyase production in this phytopathogenic bacterium was also induced by isolated higher plant cell walls, but an oligogalacturonide lyase-deficient mutant that was poorly induced by d-galacturonan or its disaccharide derivatives was also poorly induced by isolated plant cell walls. Pectate lyase induction on isolated pectic polymers or plant cell walls appears to be a multienzyme, autocatalytic process in which oligogalacturonides generated extracellularly are converted intracellulary to the actual inducer.

A precise localization of cardiolipin in plant cells

Cardiolipin and cytochrome aa 3 contents of isolated plant cells (sycamore cells) and their purified mitochondria were measured. Since the cardiolipin/cytochrome aa 3 ratio was the same in the intact cells and in the isolated mitochondria it was strongly suggested that cardiolipin is present only in the mitochondria. Furthermore, outer and inner mitochondria membranes of purified sycamore cells and mung bean hypocotyl mitochondria were separated and it was shown that cardiolipin is localized in the inner mitochondrial membrane.

Binding of exogenous DNA by chromatin from isolated plant cell nuclei

DNA-binding and uptake by nuclei isolated from protoplasts of Petunia hybrida were investigated using radioactive DNA prepared from E. coli. Isolated nuclei were incubated with bacterial DNA. After washing the nuclei the nuclear sap was extracted with 0.14 M NaCl/buffer. Chromatin was released from the nuclei by sonication and banded by isopycnic centrifugation in metrizamide gradients. Detection of chromatin was achieved by fluorescence measurement with ethidium bromide. In the nuclear sap bacterial DNA was found and an association between chromatin and bacterial DNA was demonstrated. A covalent integration of the bacterial DNA into the plant DNA could not be detected.

Influence of monuron on photosystem II and light-dependent 14CO 2 fixation in isolated cells of C 3 and C 4 plants

Cells were isolated from the developing leaves of Ipomoea aquatica (water spinach), a C 3 plant, and three kinds of C 4 plants, namely, Digitaria sanguinalis (NADP +-specific malate dehydrogenase type), Panicum miliaceum (NAD +-specific malic enzyme type), and Panicum texanum (phosphoenopyruvate carboxy kinase type), to study the effect of monuron on light-dependent 14CO 2 fixation and oxygen evolution. Bundle sheath cells, except for those of D. sanguinalis, and mesophyll cells of all plants fixed approximately the same amount of 14CO 2. Monuron, at the range used (2 to 10 × 10 −7 M), showed strong inhibition in the mesophyll cells of water spinach and in bundle sheath cells of P. miliaceum and P. texanum and moderate inhibition in the mesophyll cells of all C 4 plants. In the bundle sheath cells of D. sanguinalis the low rate of 14CO 2 fixation was stimulated to some extent by the addition of malate and ribose 5-phosphate. The I 50 value was 6 × 10 −7 M for the sensitive cells. Monuron inhibited the oxygen evolution of all seven cell types and their I 50 values varied between 3 × 10 −7 to 6 × 10 −7 M. The differential response of isolated plant cells from different species to light-dependent CO 2 fixation in the presence of monuron may also be involved in urea herbicide selectivity and undoubtedly is due to the differential photosynthetic pathways present nn them.

Effect of α-Tomatine on the Integrity and Biochemical Activities of Isolated Plant Cell Organelles

Effect of α-Tomatine on the Integrity and Biochemical Activities of Isolated Plant Cell Organelles

Purification of an Endo-β-1,4 Galactanase Produced by Sclerotinia sclerotiorum: Effects on Isolated Plant Cell Walls and Potato Tissue

Purification of an Endo-<i>β</i>-1,4 Galactanase Produced by Sclerotinia sclerotiorum: Effects on Isolated Plant Cell Walls and Potato Tissue

Xylanase from Trichoderma pseudokoningii: Purification, Characterization, and Effects on Isolated Plant Cell Walls

Xylanase from Trichoderma pseudokoningii: Purification, Characterization, and Effects on Isolated Plant Cell Walls

Membrane-surfactant Interactions in Lipid Micelles Labeled with l-Anilino-8-naphthalenesulfonate

Sonicated lipid micelles, formed from phospholipids isolated from yolks of fresh hen eggs, were used as a model membrane system for studying the effects of several surfactants on membrane properties. The interactions of the surfactants with the model system were followed through the fluorescence of the hydrophobic probe l-anilino-8-naphthalenesulfonate. The surfactants investigated were polyoxyethylene sorbitan monolaurate (Tween 20), polyoxyethylene thioether (Sterox SK), mono-calcium salt of polymerized aryl alkyl sulfonic acids (Daxad 21), and alkylbenzyl quaternary ammonium halide (AHCO DD 50). All surfactants enhanced fluorescence of the membrane-bound probe, and the degree of this enhancement correlated with the previously established phytotoxicity of these substances. The results indicate that surfactants can produce distinct changes in artificial phospholipid membranes and suggest that this lipid interaction may account for altered membrane permeability characteristics in surfactant-treated plants. The effects are observable for surfactant concentrations as low as 0.0001% (w/v), representing an approximate 10-fold increase in sensitivity for detecting surfactant effects compared with previous results on permeability changes in isolated plant cells.

Surfactant Effects on Isolated Plant Cells

Isolated plant cells from leaf tissue of soybean [Glycine max(L.) Merr. ‘Harosoy’] and wild onion (Allium canadenseL.) were used to study the effects of four surfactants on cell permeability as measured by loss of intracellular material and on photosynthetic14CO2fixation. Surfactant-induced alteration in cell permeability could not be related to surfactant-ionogenic class but could be related to reported phytotoxicity. Surfactants that altered cell permeability also inhibited photosynthetic14CO2fixation, indicating that surfactant effects were not restricted to the outer-cell membrane. Isolated cells from the monocotyledonous wild onion were more susceptible to surfactant-induced alterations in cell permeability than were cells from the dicotyledonous soybean. Efflux of intracellular material was modified by Ca++and Mg++to a greater extent for soybean than for wild onion. Integrity of the isolated cells, before and after surfactant treatment, was verified by microscopic observation.

Sorption of Some14C-Herbicides by Isolated Plant Cells and Protoplasts

Isolated plant cells (Zinnia elegansJacq. leaf tissue) and protoplasts (immature tomato fruit,Lycopersicon esculentumMill.) were obtained by hydrolysis of pectic substances in the middle lamella and the primary cell wall, respectively. Both types of cells were stabilized in an inorganic salt solution and used to study absorption and distribution of four14C-herbicides. Intracellular distribution within the protoplast was determined by ultracentrifugation in a discontinuous Ficoll gradient. Marked absorption ofp-nitrophenyl-α,α,α-trifluoro-2-nitro-p-tolyl ether (fluorodifen) and α,α,α-trifluoro-2,6-dinitro-N,N-dipropyl-p-toluidine (trifluralin) by the isolated cells was observed; absorption increased with time. Trifluralin was retained within the cell more strongly than fluorodifen. Similar absorption patterns of fluorodifen and trifluralin by protoplasts were observed. Trifluralin was incorporated to a greater extent than fluorodifen by the cells and protoplasts. Trifluralin was distributed evenly between the mitochondrial and the chloroplasts-nuclei fractions; whereas fluorodifen was concentrated in the chloroplast-nuclei fraction. No significant absorption of 1,1-dimethyl-3-(α,α,α-trifluoro-m-tolyl)urea fluometuron) or 3-(4-bromo-3-chlorophenyl)-1-methoxy-1-methylurea (chlorbromuron) was detected by either the isolated cells or protoplasts.

Callus formation from single protoplasts of petunia.

ISOLATED protoplasts of higher plants are becoming of greater interest as objects of plant physiology and pure and applied genetics. They have two great advantages over isolated plant cells; first, after cell wall removal protoplasts can easily take up macromolecules, small particles and viruses1,2, which cannot readily overcome the barrier of a cell wall and thus protoplasts allow direct modification of cell metabolism, transfer of genetic material and possibly transplanting of cell organelles. Second, protoplasts can be fused3,4, thus allowing the combination of genotypes of different species and of distinctly differentiated cells. To obtain the maximum information from such studies, it is necessary to grow protoplasts to flowering plants, as has been reported for protoplasts of tobacco5–7. Up till now for other plants, only nuclear8,9 and cellular10–12 division have been reported. We report here on successful culture of single mesophyll protoplasts from Petunia hybrida, growing to cell clusters and to proliferating callus masses.

Electron-Microprobe Analysis of Isolated Plant Cells

A technique is described for the study of the intracellular localization of minerals in a single isolated plant cell by Electron Microprobe X-ray Analyser. Tissue-culture cells were micro analysed for zinc, calcium, potassium, and pnosphorus. The X-ray oscillograms showed accumulation of zinc, potassimn, and phosphorus in the nuclei. Microprobe analysis is a non destructive method and allows for multiple element analysis in the same tissue, thereby having a wide application in the study of biological materials at cellular and subcellular levels.