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PubMed1981

Hormonal Control of Mitotic Development in Tobacco Protoplasts: TWO-DIMENSIONAL DISTRIBUTION OF NEWLY-SYNTHESIZED PROTEINS.

Two-dimensional separation of proteins newly synthesized by tobacco mesophyll protoplasts cultivated in vitro allows us to detect, reproducibly, 257 spots. The pattern is extremely stable throughout the three days of culture, the intensity of only 24 spots varying during this time. The absence of cytokinin (N(6)-benzyladenine) in the culture medium prohibits entry into S phase but does not modify the pattern, indicating that none of the observed proteins is specifically synthesized in S, G(2), or M phases. The presence of 2,4-dichlorophenoxyacetic acid is necessary for the mitotic development of protoplasts. It induces the appearance of one protein, increases the level of another, and reduces that of eight others. All proteins sensitive to auxin belong to the group of proteins the levels of which vary during culture.

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PubMed1982

Transport of the herbicide 3-amino-1,2,4-triazole by cultured tobacco cells and leaf protoplasts.

Transport of the herbicide amitrole (3-amino-1,2,4-triazole) by suspension cultured cells and leaf protoplasts of Nicotiana tabacum cv. Wisconsin 38 has been investigated. Cells were batch-cultured and routinely assayed 3 days after subculturing. Uptake rate was pH-independent, energy independent, and culture growth phase-dependent, with growing cells exhibiting the highest rates. At a concentration of 0.2 millimolar amitrole, uptake rates yielded a Q(10) of 1.6 in the 18 to 28 degrees C temperature range. Amitrole was not concentrated over a 48-hour period and showed unsaturable kinetics over the concentration range of 0.01 to 50.0 millimolar. Uptake was not significantly influenced by a 100-fold higher concentration of several amino acids (l-Asp, gamma-amino-n-butyric acid, l-His, l-Leu, l-Met, l-Trp), sucrose, glucose, fructose, and oxaloacetic acid. Uptake rate inhibition by malic acid and stimulation by NH(4)SCN were statistically significant. Amitrole was bound to cellular material, but uptake of amitrole by tobacco leaf protoplasts demonstrated that cell walls did not qualitatively influence uptake. These results indicate that amitrole enters the cells via simple diffusion.

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PubMed1982

Effect of Fusicoccin on Dark CO(2) Fixation by Vicia faba Guard Cell Protoplasts.

When Vicia faba guard cell protoplasts were treated with fusicoccin, dark (14)CO(2) fixation rates increased by as much as 8-fold. Rate increase was saturated with less than 1 micromolar fusicoccin. Even after 6 minutes of dark (14)CO(2) fixation, more than 95% of the incorporated radioactivity was in stable products derived from carboxylation of phosphoenolpyruvate (about 50% and 30% in malate and aspartate, respectively). The relative distribution of (14)C among products and in the C-4 position of malate (initially more than 90% of [(14)C]malate) was independent of fusicoccin concentration. After incubation in the dark, malate content was higher in protoplasts treated with fusicoccin. A positive correlation was observed between the amounts of (14)CO(2) fixed and malate content.It was concluded that (a) fusicoccin causes an increase in the rate of dark (14)CO(2) fixation without alteration of the relative fluxes through pathways by which it is metabolized, (b) fusicoccin causes an increase in malate synthesis, and (c) dark (14)CO(2) fixation and malate synthesis are mediated by phosphoenolpyruvate carboxylase.

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PubMed1983

Mechanical properties of the plasma membrane of isolated plant protoplasts : mechanism of hyperosmotic and extracellular freezing injury.

The volume of isolated protoplasts of rye (Secale cereale L. cv Puma) in a suspending solution at constant concentration is shown to be negligibly changed by tensions in the plasma membrane which approach that tension necessary to lyse them. This allows a detailed investigation of the plasma membrane stress-strain relation by micropipette aspiration.Over periods less than a second, the membrane behaves as an elastic two-dimensional fluid with an area modulus of elasticity of 230 millinewtons per meter. Over longer periods, the stress-strain relation approaches a surface energy law-the resting tension is independent of area and has a value of the order 100 micronewtons per meter. Over longer periods the untensioned area, which is defined as the area that would be occupied by the molecules in the membrane at any given time if the tension were zero, increases with time under large imposed tensions and decreases under sufficiently small tension. It is proposed that these long term responses are the result of exchange of material between the plane of the membrane and a reservoir of membrane material. The irreversibility of large contractions in area is demonstrated directly, and the behavior of protoplasts during osmotically induced cycles of contraction and expansion is explained in terms of the membrane stress-strain relation.

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PubMed1983

Fusion of plant protoplasts by electric fields.

The electrical fusion technique of Zimmermann and Scheurich (1981 Planta 151: 26-32) has been used to fuse mesophyll protoplasts of Avena, Zea, Vigna, Petunia, and Amaranthus. Electrical fusion proves to be a simple, effective, and general fusion technique that can be controlled to form either dikaryons or large multinucleate fusion bodies. In addition, we show that Vigna mesophyll protoplasts that are subjected to the electrical fields used in this technique are viable in culture. The construction of the fusion chambers, necessary electrical equipment, and the fusion protocol are described in sufficient detail for reproduction of the technique.

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PubMed1983

Orientation and integrity of plasma membrane vesicles obtained from carrot protoplasts.

Two fractions enriched in plasma membrane derived from suspension-cultured carrot (Daucus carota L.) cells were examined to determine if they differed from each other either in physical nature or in orientation. Parameters studied included the protein composition of purified membranes derived from trypsinized and nontrypsinized protoplasts as well as from trypsinized purified plasma membranes, the effect of inhibitors and membrane perturbants on ATPase activity, the binding of [acetyl-(14)C]concanavalin A to purified membrane fractions, and the competitive removal of [acetyl-(14)C]concanavalin A from purified membranes derived from [acetyl-(14)C]concanavalin A-labeled protoplasts. One fraction (at density of 1.102 grams per cubic centimeter on Renografin gradients) appears to be a mixed population of ;tightly' sealed vesicles with the majority being rightside-out vesicles of plasma membrane, and the other fraction (density 1.128 grams per cubic centimeter) apparently is a population of predominantly ;leaky' vesicles and/or nonvesicular fragments of plasma membrane, a large portion of which appear to be ;leaky' inside-out vesicles. In addition, it is shown that plasma membrane-enriched fractions can be distinguished from cellular endomembranes on the basis of protein and glycoprotein composition.

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PubMed1984

Auxin-Induced Regulation of Protein Synthesis in Tobacco Mesophyll Protoplasts Cultivated In Vitro: II. Time Course and Level of Auxin Control.

When protoplasts, previously cultivated in a medium lacking auxin, are transferred to complete medium, proteins whose synthesis is stimulated by the hormone become detectable after about 30 minutes and reach a constant level 2 to 4 hours after the beginning of hormonal treatment. In contrast, proteins whose level of synthesis is reduced by auxin, are only affected after 6 hours of treatment.Short radioactive labelings in deficient medium followed by chases in complete medium show that auxin does not interfere with posttranslational processes.Analysis of in vitro translation products of protoplast RNA shows that the time courses of auxin effects on protein synthesis and mRNA accumulation are perfectly superimposable. This allows us to exclude the possibility that auxin affects the translation process, but indicates that this hormone acts by regulating the concentration of the auxin-sensitive protein mRNAs.

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PubMed1985

Translocation of photosynthates into vacuoles in spinach leaf protoplasts.

A method was developed for the isolation of vacuoles from the mesophyll protoplasts of spinach leaf, employing the discontinuous Ficoll density gradient centrifugation technique. Isolated vacuole preparations were judged to be free from other organellar fractions based on the assays of marker enzyme activities of individual organelles.Using this isolation method, a time-dependent translocation of (14)C-labeled photosynthates into vacuoles was determined. In contrast to a significant transport of (14)C organic acids such as malate and citrate within 10 to 15 minutes (14)C neutral sugars and amino acids were barely transported into vacuoles during 40 minutes incubation, in spite of the fact that a relatively large amount of these compounds are found in the vacuoles. It was also found that a majority of [(14)C]sucrose remains in the cytosol, apparently not actively moving into the vacuoles. Overall results appear to suggest that vacuoles are not actively engaged in photosynthetic carbon metabolism in spinach leaf protoplasts.

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PubMed1985

Linear sucrose transport in protoplasts from developing soybean cotyledons.

Previous studies with isolated soybean cotyledon protoplasts revealed the presence of a saturable, simple diffusion, and nonsaturating carrier-mediated uptake of sucrose into soybean cotyledon cells. A proton/sucrose cotransport may be involved in the saturable sucrose uptake (Lin et al. 1984 Plant Physiol 75: 936-940 and Schmitt et al. 1984 Plant Physiol 75: 941-946). In this study, we investigated the linear sucrose uptake mechanism by treating isolated protoplasts with 15 micromolar p-trifluoromethoxy-carbonylcyanide phenylhydrazone (FCCP) or 100 micromolar p-chloromecuribenzenesulfonic acid to eliminate the saturable uptake. We found: (a) increasing external pH decreases the linear sucrose uptake; (b) fusicoccin at 20 micromolar stimulates and FCCP at 15 micromolar inhibits this linear sucrose uptake; and (c) the ratio of the initial influx of proton to sucrose is close to one in both saturable and nondiffusive linear (difference between the total linear and diffusive components) uptakes. The results suggest that a proton/sucrose cotransport is also involved in the nondiffusive linear sucrose uptake into soybean cotyledon cells.

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PubMed1986

Amino Acid transport in protoplasts isolated from soybean leaves.

We isolated large quantities of mesophyll protoplasts from source and sink leaves of soybean plants and examined them for amino acid uptake. Accumulation of amino acids in isolated protoplasts was linear for at least 40 minutes. Uptake kinetics revealed the presence of both saturable and linear components. Increasing external pH decreases the uptake. The uncoupler, carbonyl cyanide p-trifluoromethoxyphenylhydrazone at 15 micromolar inhibited and fusicoccin at 10 micromolar stimulated amino acid uptake. Our data are consistent with a proton-cotransport mechanism for the uptake of l-glutamine and alpha-amino isobutyric acid into soybean mesophyll cells.

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PubMed1986

Exoamylase activity in vacuoles isolated from pea and wheat leaf protoplasts.

Vacuoles isolated from pea (Pisum sativum), and wheat (Triticum aestivum) leaf protoplasts contained considerable activities of electrophoretically highly mobile exoamylases. Vacuoles from spinach (Spinacia oleracea) leaf and photoautotrophic Chenopodium rubrum suspension culture cell protoplasts were devoid of amylolytic activity. Endoamylase activity was in all cases associated primarily with the chloroplast.

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PubMed1987

Auxin reduces the synthesis of major vacuolar proteins in tobacco mesophyl protoplast.

We have established that polypeptides whose synthesis is reduced by 2,4-dichlorophenoxyacetic acid during in vitro culture of tobacco mesophyll protoplasts are secreted into the vacuole where they constitute the bulk of labeled proteins. In addition, these proteins continue to be synthesized in protoplast-derived cultured cells and their synthesis is strictly correlated with the size of the cell, i.e. with vacuolar size.

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PubMed1987

Iron Specifically Protects Corn Protoplasts from T-Toxin of Cochliobolus heterostrophus.

Ferric ion reduced the damaging effects of T-toxin, a series of linear beta-polyketols produced by the pathogenic fungus Cochliobolus heterostrophus, on leaf mesophyll protoplasts from susceptible T-cytoplasm corn. Of nine metals tested, only ferric and ferrous ions had this effect. Despite the presence of 12 available oxygen atoms in each T-toxin molecule, there was no evidence for the formation of an aqueous Fe(2+)- or Fe(3+)-T-toxin complex. The protective effect of iron was eliminated by a molar excess of EDTA. Iron had no effect on the sensitivity of T-cytoplasm mitochondria to T-toxin, even at a 1000-fold molar excess, nor did it protect roots of T-cytoplasm corn seedlings from inhibition by T-toxin. The mechanism by which iron specifically protects protoplasts from T-toxin is not understood, but time lapse experiments suggest that iron acts on some intracellular site to modify T-toxin sensitivity and not on a transport system at the cell surface.

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PubMed1987

Properties of Single K and Cl Channels in Asclepias tuberosa Protoplasts.

Potassium and chloride channels were characterized in Asclepias tuberosa suspension cell derived protoplasts by patch voltage-clamp. Whole-cell currents and single channels in excised patches had linear instantaneous current-voltage relations, reversing at the Nernst potentials for K(+) and Cl(-), respectively. Whole cell K(+) currents activated exponentially during step depolarizations, while voltage-dependent Cl(-) channels were activated by hyperpolarizations. Single K(+) channel conductance was 40 +/- 5 pS with a mean open time of 4.5 milliseconds at 100 millivolts. Potassium channels were blocked by Cs(+) and tetraethylammonium, but were insensitive to 4-aminopyridine. Chloride channels had a single-channel conductance of 100 +/- 17 picosiemens, mean open time of 8.8 milliseconds, and were blocked by Zn(2+) and ethacrynic acid. Whole-cell Cl(-) currents were inhibited by abscisic acid, and were unaffected by indole-3-acetic acid and 2,4-dichlorophenoxyacetic acid. Since internal and external composition can be controlled, patch-clamped protoplasts are ideal systems for studying the role of ion channels in plant physiology and development.

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PubMed1988

Characterization of Alkaloid Uptake by Catharanthus roseus (L.) G. Don Protoplasts.

The accumulation of alkaloids by protoplasts of Catharanthus roseus (L.) G. Don var. Little Bright Eye was studied to determine the specificity of uptake and the role of ion trapping in the storage of alkaloids. Accumulation of the indole alkaloids vindoline, ajmalicine, tabersonine, and vinblastine was found to be biphasic, with an initial burst of uptake followed by a slow, prolonged phase of accumulation. The concentration and pH dependence of the initial burst of uptake for vindoline suggested that uptake occurred by simple diffusion. Uptake of nicotine was monophasic, with a half life of 5.2 minutes. The accumulation ratio (Ci/Ce) for nicotine at steady state and for the initial burst of uptake for vindoline and ajmalicine suggested that accumulation was driven by the pH gradient between the vacuole and the external assay medium. The second, sustained phase of uptake of vindoline was sensitive to inhibition by either 20 millimolar NaN(3) or 0.5 millimolar Cu(2+). In azide-treated protoplasts, the uptake for vindoline conformed to the kinetics of simple diffusion, with a half life of 4 minutes. The second phase of uptake for ajmalicine, although sensitive to inhibition by Cu(2+), was insensitive to inhibition by NaN(3). The biphasic uptake of the indole alkaloids was not due to any significant metabolism. It is concluded that accumulation and storage of the indole alkaloids is due only partly to ion trapping of the alkaloids by the low pH of the vacuole lumen. In the case of vindoline, there appears to be a specific energy-requiring uptake that is not seen with nicotine (which is not endogenous to Catharanthus). Accumulation of ajmalicine appears to involve both ion trapping and an azide-insensitive component, which may be due to complexation with organic counterions and phenolics.

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PubMed1988

Influence of Photorespiration on ATP/ADP Ratios in the Chloroplasts, Mitochondria, and Cytosol, Studied by Rapid Fractionation of Barley (Hordeum vulgare) Protoplasts.

Using the principle described by R McC Lilley, M Stitt, G Mader, HW Heldt (1982 Plant Physiol 70: 965-970), an apparatus for rapid fractionation of barley leaf (Hordeum vulgare) protoplasts by membrane filtration was built. From studies of ATP/ADP ratios, it is concluded that the quenching of metabolic reactions is very fast, making it possible to use the method for studies on metabolic interactions between different compartments in plant cells. The fractionation method was used to study the influence of photorespiration on ATP/ADP ratios in the chloroplasts, mitochondria, and cytosol of barley leaf protoplasts. The cytosolic ATP/ADP ratio was higher under photorespiratory conditions than under nonphotorespiratory conditions. Aminoacetonitrile, an inhibitor of the photorespiratory conversion of glycine to serine, had a very small effect on the ATP/ADP ratios in the different subcellular compartments during photosynthesis in nonphotorespiratory conditions (saturating CO(2)). In photorespiratory conditions (limiting CO(2)), on the other hand, aminoacetonitrile increased the ATP/ADP ratio in the chloroplasts and decreased the ATP/ADP ratios in the mitochondria and the cytosol. These results are consistent with the hypothesis, that during photorespiration glycine oxidation is coupled to oxidative phosphorylation to provide ATP to the cytosol.

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PubMed1988

Calcium Transport in Protoplasts Isolated from ml-o Barley Isolines Resistant and Susceptible to Powdery Mildew.

Free cytoplasmic calcium has been postulated to play a role in preventing powdery mildew in a series of homozygous ml-o mutants of barley, Hordeum vulgare L. Protoplasts isolated from 7-day-old plants of the ml-o resistant-susceptible (R-S) barley isolines, Riso 5678/3(*) x Carlsberg II R and S, were used to test for differences in fluxes of Ca(2+) across the plasmalemma. Greater influx or lesser efflux might account for a higher free cytosolic Ca(2+) postulated to exist in ml-o R mutants. Uniform patterns of uptake were maintained for 3 hours from solutions of 0.2 and 2 millimolar Ca(2+). Washout curves of (45)Ca(2+) from R and S protoplasts revealed three compartments-presumed to represent release from the vacuole, organelles, and the cytoplasm (which included bound as well as free Ca(2+)). Uptake and washout did not differ between isolines. On the basis of recent determinations of submicromolar levels of free cytoplasmic Ca(2+) and our initial rates of (45)Ca-labeled Ca(2+) uptake, we show that measurement of the unidirectional influx of Ca(2+) across the plasmalemma is not feasible because the specific activity of the pool of free cytoplasmic calcium increases almost instantaneously to a level that would result in a significant, but unknown, efflux of label. Similarly, measurement of the efflux of Ca(2+) across the plasmalemma is not possible since the activity of the pool of free cytoplasmic calcium is a factor of 350 smaller than the most rapid component of the washout experiment. This pool of cytoplasmic free Ca(2+) will wash out too rapidly and be too small to detect under the conditions of these experiments.

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PubMed1988

Subcellular Location of NADPH-Dependent Hydroxypyruvate Reductase Activity in Leaf Protoplasts of Pisum sativum L. and Its Role in Photorespiratory Metabolism.

Protoplasts purified from pea (Pisum sativum L.) leaves were lysed and fractionated to assess the subcellular distribution of NADPH-dependent hydroxypyruvate reductase (NADPH-HPR) activity. Rate-zonal centrifugation and sucrose-gradient experiments demonstrated that most (about 70%) of the NADPH-HPR activity was located in the supernatant or cytosol fraction. Detectable, but relatively minor activities were associated with the chloroplast fraction (up to 10% on a chlorophyll basis when compared to the lysate) and with peroxisomes. The minor NADPH-HPR activity in the peroxisomes could be fully accounted for by the secondary NADPH-dependent activity of NADH-dependent HPR. The subcellular distribution of NADPH-HPR followed closely that previously determined for NADPH-dependent glyoxylate reductase (NADPH-GR), an enzyme localized predominantly in the cytosol of pea leaf protoplasts (CV Givan et al. 1988 J Plant Physiol 132: 593-599). Low activities of both NADPH-HPR and NADPH-GR were also found in purified chloroplasts prepared by mechanical homogenization of Pisum and Spinacia leaves. In pea and spinach chloroplasts, rates of both NADPH-HPR and NADPH-GR were lower than the activity of the NADH-dependent GR. The results are discussed in relation to a possible role for NADPH-HPR in the oxidative carbon pathway of photorespiration. Both NADPH-HPR and the GRs could function as auxiliary reactions to photorespiration, utilizing hydroxypyruvate and/or glyoxylate ;leaked' or otherwise exported from peroxisomes. NADPH-HPR function might be especially significant under conditions of limiting NADH supply to peroxisomes, with extraperoxisomal reduced pyridine nucleotide acting as the reductant.

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