Suberin production by isolated tomato fruit protoplasts

The multilamellar wall secreted by protoplasts isolated from locule tissue of tomato (Lycopersicon esculentum L.) fruit was purified, and an extract was obtained after depolymerization with BF₃-methanol. Analysis of this extract using thin layer chromatography demonstrated the presence of fatty acid methyl esters, fatty alcohols, dicarboxylic acid dimethyl esters, and ω-hydroxy acid methyl esters. These components were quantified using an Iatroscan thin layer chromatography-flame ionization detection system. The different chain lengths in each group were identified and quantified using gas chromatography. The results clearly indicated the presence of suberin.     

Sugar uptake by protoplasts isolated from tomato fruit tissues during various stages of fruit growth

The uptake of radioactive glucose and sucrose by protoplasts isolated from pericarp and placenta tissues of tomato ( Lycopersicon esculentum cv. Counter) fruit was investigated in relation to the dry matter accumulation rates of these tissues. Uptake of glucose by protoplasts isolated from pericarp tissue was highest in fruit of around 20 g fresh weight or 25 days after anthesis. Sucrose uptake by pericarp protoplasts was lower than that of glucose and did not show a peak of uptake. The maximum rate of glucose uptake by protoplasts from the pericarp was at the time when the tomato fruit was accumulating dry matter at the highest rate. Glucose uptake by placenta protoplasts was lower and at a similar level as sucrose.
Protoplast uptake of glucose, but not of sucrose, was partially inhibited by (1) p -chloromercuribenzene sulphonic acid, a sulphydryl group modifier; (2) erythrosin B, an H⁺-ATPase inhibitor; and (3) valinomycin, a K⁺-ionophore, suggesting that membrane transport of glucose by tomato fruit sink cells may be a carrier-mediated, energy-dependent process.
The main route of carbohydrate accumulation by tomato fruit during the period of rapid fruit growth may be by cleavage of sucrose by apoplastic acid invertase prior to hexose transport across the plasma membrane.     

A mechanism for the pinocytosis of latex spheres by tomato fruit protoplasts

Plant protoplasts isolated from tomato fruit locule tissue take up 0·12 polystyrene latex spheres by an endocytotic process. Freeze-etching enables the process to be clearly visualized in the electron microscope and provides important data from face view membrane fractures. When protoplasts are glutaraldehyde fixed prior to mixing with latex, the spheres adhere to the plasmalemma and cause some localized membrane distortion. This shows that adhesive forces are sufficient to initiate the invagination process. Freeze-etch membrane fracture faces carry numerous granules, the density of which is lower in the invaginating region. This reduction is in accordance with the expected value if only that part of the membrane which comes in intimate contact with the sphere expands to surround it. There is no change in the granule densities in the surrounding membrane. In fractured membrane surface views distinct phases can be delimited, and their relative occurrence indicates that the second half of the invagination process (i.e. after the diameter of the sphere has reached the membrane plane) is some thirty times quicker than the first half. It is proposed this indicates operation of surface tension forces during the fast phase, and it may be that cellular energy is required only for the initial slow phase of membrane expansion.     

Fibronectin-mediated uptake of gelatin-coated latex particles by peritoneal macrophages

The present study demonstrates the ability of plasma fibronectin or cold-insoluble globulin (Clg) to promote the uptake of 125I-labeled, gelatin-coated latex beads (g-Ltx*) by monolayers of peritoneal macrophages (PM). The uptake of g-Ltx* by PM was enhanced by Clg in a concentration-dependent fashion and required the presence of heparin (10 U/ml) as an obligatory cofactor for maximal particle uptake. Treatment of PM monolayers with trypsin (1 mg/ml) for 15 min at 37 degrees C after particle uptake removed less than 15% of the radioactivity incorporated by the monolayers. However, a similar trypsin treatment of the monolayers before the addition of latex particles depressed Clg-dependent uptake by greater than 75%. Pretreatment of PM monolayers with inhibitors of glycolysis effectively reduced the Clg-dependent uptake of latex. Similarly, pretreatment of monolayers with either inhibitors of protein synthesis or agents that disrupt cytoskeletal elements also significantly depressed Clg- dependent particle uptake. Phagocytosis of g-Ltx* by PM in the presence of Clg and heparin was confirmed by electron microscopy. Finally, g- Ltx* could also be effectively opsonized with Clg at 37 degrees C before their addition to the monolayers. These studies suggest that the recognition of g-Ltx* in the presence of Clg required cell surface protein(s) and that subsequent phagocytosis of these particles by PM was energy dependent and required intact intracellular cytoskeleton elements. Thus, PM monolayers provide a suitable system for further studies on the function of Clg in the recognition and phagocytosis of gelatin-coated particles by phagocytic cells.     

An electron microscopic study of the initial stages of infection of isolated tomato fruit protoplasts by tobacco mosaic virus

Summary Protoplasts were isolated from tomato fruit locule tissue and incubated with tobacco mosaic virus. Electron microscope observations on sections of suitably fixed and embedded material revealed that virus particles readily became attached to the plasmalemma, particularly in small invaginations in the surface of the protoplast. Virus particles were later observed in vesicles within the cytoplasm and it was clear that these vesicles were being formed as a result of pinocytic activity at the surface of the protoplast. Later, virus particles were observed near the nucleus. It is suggested that an initial attachment of the virus to the plasmalemma followed by a pinocytic uptake may represent the initial stages of virus infection of plant cells and that the pinocytic vesicle, containing virus, serves as the vehicle of cellular infection.     

Exposure of platelet binding sites in von Willebrand factor by adsorption onto polystyrene latex particles

Von Willebrand factor molecules are flexible linear polymers composed of repeating protomeric polypeptide subunits. In the process of primary hemostasis, von Willebrand factor promotes platelet adhesion and platelet plug formation at the site of vascular injury. This biologic activity is apparently related to the multimeric size of von Willebrand factor. We simulated von Willebrand factor binding to the subendothelial surface by adsorbing purified human von Willebrand factor onto polystyrene latex particles of two different diameters, i.e., 0.312 μm and 2.02 μm. The rate and extent of ¹²⁵I-labeled von Willebrand factor binding to polystyrene was similar with both size classes of latex particles. The von Willebrand factor-coated latex beads of 2.02 μm diameter, in contrast to the smaller size, induced rapid agglutination of formalin-fixed human platelets in the absence of any other aggregating agent. Von Willebrand factor was also adsorbed from human plasma onto latex particles coated with anti-von Willebrand factor antibodies. Again, only the large beads, carrying the von Willebrand factor-antibody complex, induced agglutination of fixed platelets. Shear stress promoted the rate of von Willebrand factor adsorption to latex particles. Our results suggest that adsorption to surface exposes binding sites in human von Willebrand factor for platelets.     

Totipotency of tomato protoplasts

Summary An efficient and reliable protocol for tomato protoplast isolation, culture, and plant regeneration has been developed. Fourteen diverse cultivars were tested. Fertile plants were regenerated from all 14 cultivars without any modification in the protocol. Plating efficiency (percentage of the protoplasts that formed minicalli) of up to 50% was achieved. Those mini-calli rapidly regenerated shoots at high frequencies. Regenerated shoots can be easily rooted on a basal medium with the appropriate auxin, and have been set to soil within two months after the isolation of the protoplasts.     

Further observations on cell-wall formation around isolated protoplasts of tobacco and tomato

Freeze-etch observations of protoplasts isolated from tobacco (Nicotiana tabacum L.) mesophyll tissue and tomato (Lycopersicum esculentum Mill.) fruit locule tissue are described which clarify earlier observations (Burgess, J., Fleming, E.N., Planta 131, 173–178, 1976; Planta 133, 267–273, 1977), obtained using scanning electron microscopy. of fibres associated with projections from these cell surfaces. It is demonstrated (1) that the fibres consist of bundles of small numbers of microfibrils which have become artifactually thickened by the deposition of coating materials, and (2) that the apparent association between fibres and projections results from microfibrils being lifted preferentially from protoplast surfaces in regions rich in projections (plasmalemmasomes). With the higher resolution available using freeze-etching it can be demonstrated that microfibril deposition does not occur in discontinuous zones on these protoplast surfaces. Globules associated with microfibril termini in radish (Raphanus sativus L.) roots are illustrated and it is proposed that turgor pressure differences between isolated protoplasts and intact tissue may account for the absence of similar globules from isolated protoplast surfaces.     

DNA binding and uptake by nuclei isolated from plant protoplasts: factors affecting DNA binding and uptake

DNA binding and uptake by nuclei isolated from soybean (Glycine max L. Merr.) protoplasts were investigated using radioactive homogeneous DNA prepared from soybean cells. DNA binding to nuclei was found to decrease drastically with increased incubation time. Total uptake and acid-precipitable uptake reached a maximum after 20 minutes of incubation. Optimum DNA binding and uptake occurred at pH 6 and the process was enhanced by increasing the incubation temperature to 40 C. Salmonella typhimurium DNA and poly ([dA-dT]-[dA-dT]) competitively inhibited DNA binding whereas calf thymus DNA was less competitive; however, Micrococcus lysodeikticus DNA stimulated DNA binding and tobacco mosaic virus RNA had no effect. DNA binding and uptake was enhanced by addition of Mg ions, Ca ions, poly-l-lysine, and ATP. Increasing amounts of EDTA appeared to decrease DNA binding. Pronase strongly inhibited DNA binding and uptake.     

Enzyme activities in mitochondria isolated from ripening tomato fruit

Mitochondria were isolated from tomato (Lycopersicon esculentum L.) fruit at the mature green, orange-green and red stages and from fruit artificially suspended in their ripening stage. The specific activities of citrate synthase (EC 4.1.3.7), malate dehydrogenase (EC 1.1.1.37), NAD-linked isocitrate dehydrogenase (EC 1.1.1.41) and NAD-linked malic enzyme (EC 1.1.1.38) were determined. The specific activities of all these enzymes fell during ipening, although the mitochondria were fully functional as demonstrated by the uptake of oxygen. The fall in activity of mitochondrial malate dehydrogenase was accompanied by a similar fall in the activity of the cytosolic isoenzyme. Percoll-purified mitochondria isolated from mature green fruit remained intact for more than one week and at least one enzyme, citrate synthase, did not exhibit the fall in specific activity found in normal ripening fruit.     

Structure of aggregates of Tipula iridescent virus and polystyrene latex particles

Tipula iridescent virus aggregated with polystyrene latex particles 126 nm in diameter. There was a region of optimal proportion of the two particles. The aggregation proceeded faster and the amount of resultant aggregates was larger at the higher concentrations of the two particles, but the size of the individual aggregates was smaller. The aggregates consisted of clusters of the two particles with vacant spaces interspersed among them. A hypothetical model of the particle arrangements was presented. The aggregates were reversibly dissolved in 0.03% sodium dodecyl sulfate and 30% n-propanol and isopropanol. In the presence of lower concentrations of these solvents, the aggregation occurred at high temperatures but not at low temperatures. These results were interpreted as implicating hydrophobic interactions in the formation and stability of the aggregates.     

Contribution of damaged protoplasts to DNA uptake by purified plant protoplasts

Purified protoplast preparations contained few (5–9%) but significant numbers of non-spherical damaged protoplasts. Such damaged protoplasts rapidly saturated with relatively large amounts of exogenous DNA which was largely associated with their nuclei. The relative fraction of damaged protoplasts was found to increase with DNA uptake time and DNA concentration. Discontinuous gradient centrifugation provided a convenient method for selective removal of damaged protoplasts and reliable evaluation of exogenous DNA uptake by intact spherical protoplasts.     

Liposome-mediated uptake of chloroplasts by plant protoplasts

Summary Liposomes, artificial lipid vesicles, have been used to induce the uptake of isolated spinach chloroplasts into protoplasts of bothNeurospora crassa andDatura innoxia. Chloroplasts showed high oxygen evolution rates preceding uptake and for extended periods of time after uptake. Survival, as measured by pigment retention and oxygen evolution, was much improved over polyethylene glycol induce uptake. Possible future application of liposome-mediated uptake in plant protoplast studies are discussed.     

Light-dependent rubidium uptake into isolated mesophyll protoplasts from leaves of Pisum sativum

A method is described for the isolation of large amounts of physiologically active protoplasts from leaves of Pisum sativum L. Rubidium uptake was determined after separation of the intact protoplasts from the loading medium by rapid centrifugation through a phthalate step gradient. In freshly isolated mesophyll protoplasts of Pisum sativum , rubidium uptake was carbonylcyanide- p -trifluoromethoxyphenylhydrazone reduced by metabolic inhibitors such as 5 μ M , 0.1 mW cyanide, 2 μ M DCMU and 5 m M arsenate and by dark incubation. Reduction of rubidium uptake by inhibition of aerobic respiration or the photosynthetic electron transport system demonstrates that both processes play a role in the energy supply for membrane transport in these protoplasts.     

Cell-wall regeneration by isolated tomato-fruit protoplasts

Summary Using both light microscopy and electron microscopy of thin sections, and surface replicas, it has been shown that isolated tomato fruit locule tissue protoplasts regenerate a new cell wall when maintained in suitable culture media.This work was supported by a special grant from the S.R.C. Dr. E. Pojnar was in receipt of an EMBO Fellowship.     

Fourier transform IR study of enzymatically isolated tomato fruit cuticular membrane

Fourier transform ir study on enzymatically isolated tomato fruit cuticle has been performed. Assignments of the observed frequencies to functional groups present in the cuticular membrane yield information on the polyester cross-links and the interactions among the different components that form this plant protective barrier. © 1992 John Wiley & Sons, Inc.     

Effects of fusicoccin and abscisic acid on glucose uptake into isolated beetroot protoplasts

Uptake of glucose, 3-O-methylglucose and sucrose into beetroot protoplasts is considerably stimulated by 10^–6M fusicoccin. This effect is decreased in the presence of 10mM Na⁺ or K⁺, 2 mM Mg²⁺ or Ca²⁺. Whereas fusicoccin causes no change in the pH-optimum of the sugar uptake (pH 5.0), the apparent K_m of this uptake which obeys a biphasic kinetics is decreased by the action of fusicoccin. In the protoplast suspension, fusicoccin induces an acidification which is suppressed by uncoupling agents. Correspondingly, uncouplers as well as vanadate and diethylstilbestrol markedly inhibit the effect of fusicoccin on sugar uptake. The present data support the view that glucose uptake into beetroot protoplasts depend on the proton-pumping activity of the plasmalemma-ATPase. cis–Abscisic acid diminishes significantly the fusicoccin-enhanced glucose uptake. By using a radioimmunoassay, the internal abscisic acid content of the protoplast was estimated to be in the range of 10^–6 M. Protoplasts isolated from bundle tissue contain twice as much abscisic acid as those derived from storage parenchyma. Because protoplasts from the bundle tissue were shown to take up sugars much faster than those from the storage cells, the observed effect of abscisic acid might reflect an involvement of this hormone in the regulation of carbohydrate partitioning in the beet.Abbreviations ABA cis–abscisic acid - bundle protoplast protoplasts isolated from the conducting tissue of beetroots - DES diethylstilbestrol - FC fusicoccin - 3-OMG 3-O-methylglucopyranose - PCMBS p–chloromercuribenzenesulfonic acid - storage protoplasts protoplasts isolated from storage parenchyma