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PubMed1985

Light Activation of NADP-Malate Dehydrogenase in Guard Cell Protoplasts from Vicia faba L.

Light-induced swelling of guard cell protoplasts (GCP) from Vicia faba was accompanied by increases in content of K(+) and malate. DCMU inhibited the increase of K(+) and malate, and consequently swelling.Effect of light on the activity of selected enzymes that take part in malate formation was studied. When isolated GCP were illuminated, NADP-malate dehydrogenase (NADP-MDH) was activated, and the activity reached a maximum within 5 minutes. The enzyme activity underwent 5- to 6-fold increase in the light. Upon turning off the light, the enzyme was inactivated in 5 minutes NAD-MDH and phosphoenolpyruvate carboxylase (PEPC) were not influenced by light. The rapid light activation of NADP-MDH was inhibited by DCMU, suggesting that the enzyme was activated by reductants from the linear electron transport in chloroplasts. An enzyme localization study by differential centrifugation indicates that NADP-MDH is located in the chloroplasts, NAD-MDH in the cytosol and mitochondria, and PEPC in the cytosol. After light activation, the activity of NADP-MDH in guard cells was 10 times that in mesophyll cells on a chlorophyll basis. The physiological significance of light-dependent activation of NADP-MDH in guard cells is discussed in relation to stomatal movement.

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PubMed1986

Possible involvement of calmodulin and the cytoskeleton in electrofusion of plant protoplasts.

Calmodulin antagonists, trifluoperazine, chlorpromazine, calmidazolium, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), strongly inhibited the electrofusion of barley (Hordeum vulgare L. cv Moor) protoplasts with a marked increase of broken fusion products, after 60 minutes of incubation. W-5, a dechlorinated analog of W-7, was found less effective for the inhibition than W-7. Ethyleneglycol-bis(beta- aminoethylether)-N,N'-tetraacetic acid a Ca(2+) chelator, La(3+), a surface Ca(2+) antagonist, and verapamil, a Ca(2+) channel blocker, also inhibited electrofusion. Dielectrophoresis was inhibited by La(3+). A microtubule inhibitor, vinblastine, inhibited electrofusion strongly while colchicine, slightly. A microfilament inhibitor, cytochalasin B, promoted fused cells to become spherical while phalloidin did not affect electrofusion.

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PubMed1986

Light Activation of Pyruvate,Pi Dikinase and NADP-Malate Dehydrogenase in Mesophyll Protoplasts of Maize : Effect of DCMU, Antimycin A, CCCP, and Phlorizin.

Pyruvate,Pi dikinase (PPDK, EC 2.7.9.1) and NADP-malate dehydrogenase (MDH, EC 1.1.1.82) were activated in the light and inactivated following a dark treatment in mesophyll protoplasts of maize. DCMU (up to 33 micromolar), an inhibitor of noncyclic electron transport, inhibited activation of MDH much more strongly than it did PPDK. Antimycin A (6.6-33 micromolar), an inhibitor of cyclic photophosphorylation, inhibited the activation of PPDK (up to 61%), but had little or no effect on activation of MDH. Carbonyl cyanide m-chlorophenylhydrazone (0.2-2 micromolar) and nigericin (0.4 micromolar), uncouplers of photophosphorylation, inhibited activation of PPDK while stimulating the activation of MDH. Phlorizin (0.33-1.7 millimolar), an inhibitor of the coupling factor for ATP synthesis, strongly inhibited activation of PPDK but only slightly effected light activation of MDH. These results suggest that noncyclic electron flow is required for activation of NADP-MDH and that photophosphorylation is required for activation of PPDK.

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PubMed1987

Expression from heterologous promoters in electroporated carrot protoplasts.

Plasmids were constructed that contained promoters of ;plant' genes fused to the bacterial gene for chloramphenicol acetyl transferase. The promoters were isolated from a developmentally regulated Zea mays seed storage protein gene and from the mannopine synthase gene of the octopine type Ti plasmid of Agrobacterium tumefaciens which is constitutively expressed in crown gall tumors. These plasmids were introduced into carrot protoplasts made permeable by electroporation. Expression of chloramphenicol acetyl transferase activity directed by both promoters was positively correlated with DNA concentration. The efficiency of gene transfer was increased by inclusion of polyethylene glycol and by optimization of the voltage used in electroporation.

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PubMed1987

Auxin effect on the transmembrane potential difference of wild-type and mutant tobacco protoplasts exhibiting a differential sensitiity to auxin.

The effects of 1-naphthaleneacetic acid (NAA) and other auxin analogs on the transmembrane potential difference (Em) were compared on tobacco protoplasts isolated from two genotypes differing in their sensitivity to auxins. For both types, NAA modifies Em by inducing at low doses a hyperpolarization, the amplitude of which increased with auxin concentration. Above an optimal concentration this hyperpolarization was reduced and even nullified. However, for the mutant type, this electrical response was shifted toward higher NAA concentrations, as its growth response. In the presence of structural analogs of auxin which have been showed to modify the dose-response curve for growth, the Em was altered: the growth-stimulatory molecule (picloram) initiated hyperpolarization, whereas the growth-inhibitory substance (4-bromophenylacetic acid) caused depolarization. These results provide evidence for a specific action of auxin at the membrane level related to its biological activity.

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PubMed1988

Photosynthetic Carbon Fixation in Guard Cell Protoplasts of Vicia faba L. : Evidence from Radiolabel Experiments.

Photosynthetic carbon fixation in guard cells was reexamined in experiments with highly purified guard cell protoplasts from Vicia faba L. irradiated with red light. The fate of (14)CO(2) (4.8 microcuries of NaHCO(3); final concentration: 100 micromolar) supplied to these preparations was investigated with two-dimensional paper, and thin layer chromatography. Rates of CO(2) fixation were 5- to 8-fold higher in the light than in darkness. Separation of acid-stable products into water-insoluble, neutral, and anionic fractions showed that more radioactivity was incorporated into the neutral fraction in the light than in the dark. In the dark, malate and aspartate comprised 90% of the radiolabel found in the anionic fraction, whereas in the light, radioactivity was also found in 3-phosphoglyceric acid (PGA), sugar monophosphates, sugar diphosphates, and triose phosphates. Phosphorylated compounds contained up to 60% of the label in the light-treated anionic fraction. Phosphatase treatment and rechromatography of labeled sugar diphosphate showed the presence of ribulose, a specific metabolite of the photosynthetic carbon reduction pathway (PCRP). In time-course experiments, labeled PGA was detected within 5 seconds. With time, the percentage of label in PGA decreased and that in sugar monophosphate increased. We conclude that PGA is a primary carboxylation product of the PCRP in guard cells and that the activity of the PCRP, and phosphoenolpyruvate-carboxylase is metabolically regulated.

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PubMed1988

Salinity affects intracellular calcium in corn root protoplasts.

Previous work with the fluorescent Ca probe chlorotetracycline (CTC) showed that salinity displaces Ca from membranes of root cells. Using a variety of indirect approaches, we studied whether salinity displaces Ca from the cell surface or from internal membranes of corn (Zea mays L. cv Pioneer 3377) root protoplasts. Preloading the cells with supplemental Ca counteracted subsequent NaCl effects on CTC fluorescence. CTC quenching by exogenous EGTA was not competitive with CTC quenching by NaCl. The Ca channel reagent (+)-202-791 had significant interactions with the effect of NaCl on CTC fluorescence. The effect of NaCl on CTC fluorescence was attenuated by pretreatment with Li, but was restored by inositol. Salinity increased Na influx, decreased Ca influx, and increased Ca efflux from the cells. Fluorescence anisotropy indicated that NaCl decreased the fluidity of the external face of the plasmalemma but increased the fluidity of cell membranes in general. Our results suggest that salinity displaces Ca associated with intracellular membranes through activation of the phosphoinositide system and depletion of intracellular Ca pools.

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PubMed1988

Sucrose Synthase, a Cytosolic Enzyme in Protoplasts of Jerusalem Artichoke Tubers (Helianthus tuberosus L.).

The exact subcellular location of sucrose synthase (UDP-d-glucose: d-fructose 2-alpha-d-glucosyltransferase, EC 2.4.1.13) in Helianthus tuberosus tubers was studied by comparison of its activity in protoplasts with that of vacuoles isolated from them. Assuming 100% of the beta-N-acetylglucosaminidase activity to be of vacuolar origin, less than 5% of both the sucrose synthase activity and the extravacuolar marker NAD-malate dehydrogenase was detected in the vacuole preparations. Sucrose synthase is therefore an extravacuolar enzyme. Its role in the inulin metabolism of H. tuberosus is discussed.

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PubMed1989

Photorespiratory Properties of Mesophyll Protoplasts of Nicotiana plumbaginifolia.

The photorespiratory activity of mesophyll protoplasts of Nicotiana plumbaginifolia has been clearly demonstrated by the presence of a Warburg-effect, the occurrence of an important CO(2)-sensitive O(2) uptake and the effect of some photorespiratory inhibitors on photosynthetic activity. At a nonsaturating dissolved inorganic carbon (DIC) concentration (0.1 millimolar), we observed that the rate of CO(2) fixation was 60% lower at 50% O(2) compared to that measured at 2% O(2). Using (18)O(2) and mass spectrometry, we measured O(2) exchange as a function of light intensity and of DIC concentration. Oxygen uptake measured at the CO(2) compensation point (47.4 micromoles O(2) per hour per milligram chlorophyll) was three-fold higher than that measured at a saturating CO(2) concentration. Cyanide or iodoacetamide, inhibitors of the Calvin cycle, were found to reduce the O(2) uptake to the same extent as CO(2) saturation. We conclude from these results that the major part of the CO(2)-sensitive O(2) uptake is due to photorespiration. Further, we investigated the effect on net photosynthesis of some inhibitors of the glycolate pathway. At CO(2) saturation (10 millimolar DIC), 5 millimolar aminoacetonitrile (AAN), and 1 millimolar aminooxyacetate (AOA) did not cause any significant decrease in net photosynthesis. However, when these two inhibitors were added under a period of active photorespiration (10 minutes at the CO(2) compensation point at 20% O(2)), we observed a decrease in the rate of net photosynthesis at 10 millimolar DIC measured afterward (respectively, 18 and 29%). This inhibition did not appear at 2% O(2), but was stronger at 50% O(2) (40% for AAN and 47% for AOA). With 0.05 millimolar butyl 2-hydroxy-3-butynoate (BHB) or 0.5 millimolar l-methionine-dl-sulfoximine (l-MSO), rates of net photosynthesis at 10 millimolar DIC were decreased by 10 to 15%. Additional decreases were observed after a period at the CO(2) compensation point at 20% O(2) (30% for BHB and 20% for l-MSO). From the sites of action of the four inhibitors tested, we suggest the inhibition of photosynthesis occurring after a period of active photorespiration to be due to the toxic accumulation of nonmetabolized phosphoglycolate.

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PubMed1989

Characterization of potassium-dependent currents in protoplasts of corn suspension cells.

Protoplasts obtained from corn (Zea mays) suspension cells were studied using the whole cell patch-clamp technique. One time-independent current, as well as two time-dependent currents were identified. All three currents were reduced by tetraethylammonium (9 millimolar), a K(+) channel blocker. The time-independent current had a nearly linear current-voltage relationship and its reversal potential, defined as the voltage at which there is zero current, was highly dependent on the extracellular potassium concentration. One of the two time-dependent currents was activated, with rapid kinetics, by membrane hyperpolarization to potentials more negative than -100 millivolts. The second time-dependent current was activated with a sigmoidal time course by membrane depolarization to potentials more positive than -60 millivolts. It exhibited no inactivation and was carried primarily by potassium ions. These characteristics suggest that this latter current is caused by the voltage-dependent opening of delayed-rectifier K(+) channels. These three currents, which are not generated by the plasmalemma H(+)-ATPase, are likely to assist in the regulation of the cellular K(+) fluxes and membrane potential.

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PubMed1989

Salinity stress increases cytoplasmic ca activity in maize root protoplasts.

High concentrations of NaCl immediately elevated cytoplasmic Ca activity in maize (Zea Mays L. cv Pioneer 3377) root protoplasts, as measured with the fluorescent probe Indo-1. The effect of salinity was inhibited by Li pretreatment but restored by inositol, suggesting that phosphoinositides mediate the stress response.

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PubMed1990

Amino Acid Transport across the Tonoplast of Vacuoles Isolated from Barley Mesophyll Protoplasts : Uptake of Alanine, Leucine, and Glutamine.

Mesophyll protoplasts from leaves of well-fertilized barley (Hordeum vulgare L.) plants contained amino acids at concentrations as high as 120 millimoles per liter. With the exception of glutamic acid, which is predominantly localized in the cytoplasm, a major part of all other amino acids was contained inside the large central vacuole. Alanine, leucine, and glutamine are the dominant vacuolar amino acids in barley. Their transport into isolated vacuoles was studied using (14)C-labeled amino acids. Uptake was slow in the absence of ATP. A three- to sixfold stimulation of uptake was observed after addition of ATP or adenylyl imidodiphosphate an ATP analogue not being hydrolyzed by ATPases. Other nucleotides were ineffective in increasing the rate of uptake. ATP-Stimulated amino acid transport was not dependent on the transtonoplast pH or membrane potential. p-Chloromercuriphenylsulfonic acid and n-ethyl maleimide increased transport independently of ATP. Neutral amino acids such as valine or leucine effectively decreased the rate of alanine transport. Glutamine and glycine were less effective or not effective as competitive inhibitors of alanine transport. The results indicate the existence of a uniport translocator specific for neutral or basic amino acids that is under control of metabolic effectors.

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PubMed1991

Transient Gene Expression in Protoplasts of Phaseolus vulgaris Isolated from a Cell Suspension Culture.

A cell suspension culture of Phaseolus vulgaris cv Negro jamapa was established. Protoplasts isolated from this culture are suitable for transient gene expression studies after DNA transfer by either electroporation or treatment with polyethylene glycol. The optimized conditions for both methods were established, taking into consideration expression and cell viability. We have investigated two features of posttranscriptional gene regulation. We demonstrate that inclusion of the RNA leader sequence, omega (Omega), derived from tobacco mosaic virus, and a dicot intron in the leader region together enhance gene expression in this legume species about 10-fold.

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PubMed1991

Hygromycin resistance gene cassettes for vector construction and selection of transformed rice protoplasts.

Hygromycin resistance gene cassettes were designed to facilitate vector construction for plant transformation. Unique EcoRI, Pstl, and Sacll sites in the coding sequence of a hygromycin B phosphotransferase gene (hph) from Escherichia coli were eliminated. The mutated hph genes were used to form gene cassettes flanked by EcoRI-Sacll-Kpnl-Hindlll sites. Hygromycin resistance of wild-type and mutated hph genes was indistinguishable in E. coli and rice protoplast growth assay.

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PubMed1992

Redox Processes in the Blue Light Response of Guard Cell Protoplasts of Commelina communis L.

Guard cell protoplasts from Commelina communis L. illuminated with red light responded to a blue light pulse by an H(+) extrusion which lasted for about 10 minutes. This proton extrusion was accompanied by an O(2) uptake with a 4H(+) to O(2) ratio. The response to blue light was nil in darkness without a preillumination period of red light and increased with the duration of the red light illumination until about 40 minutes. However, acidification in response to a pulse of blue light was obtained in darkness when external NADH (1 millimolar) was added to the incubation medium, suggesting that redox equivalents necessary for the expression of the response to blue light in darkness may be supplied via red light. In accordance with this hypothesis, the photosystem II inhibitor 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (10 micromolar) decreased the acidification in response to blue light more efficiently when it was added before red light illumination than before the blue light pulse. In the presence of hexacyanoferrate, the acidification in response to a blue light pulse was partly inhibited (53% of control), suggesting a competition for reducing power between ferricyanide reduction and the response to blue light.

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PubMed1992

Mechanism of aluminum inhibition of net ca uptake by amaranthus protoplasts.

Calcium ions serve as a second messenger in signal transduction and metabolic regulation. Effects of Al on calcium homeostasis remain to be elucidated. Short-term net (45)Ca(2+) uptake by Amaranthus tricolor protoplasts was monitored from uptake media prepared to test the influence of pH, Al, and various inhibitors. Accumulation of (45)Ca(2+) increased during the first 3 to 6 minutes and then leveled off or declined. Al and Ca(2+) channel blockers (verapamil and bepridil) decreased net (45)Ca(2+) uptake. This decrease was more pronounced when Al and bepridil were both present in uptake media, but Al did not aggravate verapamil-induced reduction of net (45)Ca(2+) uptake. Erythrosin B and calmidazolium each increased net (45)Ca(2+) uptake, probably by interfering with Ca(2+) efflux. This effect was undetectable in the presence of Al. Mycophenolic acid decreased net (45)Ca(2+) uptake; guanosine alleviated this effect. Al-induced reduction of net (45)Ca(2+) uptake was not aggravated by mycophenolic acid. Net (45)Ca(2+) uptake was generally less at pH 4.5 than at 5.5 for all treatments. It is concluded that Al ions affect net (45)Ca(2+) uptake by binding to the verapamil-specific channel site that is different from the bepridil-specific one, as well as by interfering with the action of guanosine 5'-triphosphate-binding proteins.

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PubMed1992

K Channels Are Responsible for an Inwardly Rectifying Current in the Plasma Membrane of Mesophyll Protoplasts of Avena sativa.

In whole-cell recording, the conductance of the plasma membrane of protoplasts isolated from mesophyll cells of leaves of oat (Avena sativa) was greater for inward than outward current. The inward current in both the whole-cell mode and with isolated patches was dependent on [K(+)](o). When the membrane voltage was more positive than -50 millivolts, the membrane conductance in the whole-cell mode was low, and K(+) channels in cell-attached or outside-out patches had a low probability of being open. At a membrane voltage more negative than -50 millivolts, the membrane conductance increased by sevenfold in the whole-cell mode, and the probability of the channels being open increased. The inward current was highly selective for K(+) compared with Cs(+), Na(+), choline or Cl(-). Low concentrations of [Cs(+)](o) or [Na(+)](o) blocked the inward current in a strongly voltage-dependent fashion. Comparison of single-channel with the macroscopic current yields an estimate of about 200 inwardly rectifying K(+) channels per cell at a density of 0.035 per square micrometer. At physiological membrane voltages and [K(+)](o) about 10 millimolar, the influx through these channels is sufficient to increase the internal [K(+)] by 2 millimolar per minute. These K(+) channels are activated by membrane voltages in the normal physiological range and could contribute to K(+) uptake whenever the membrane is more negative than the K(+) equilibrium potential.

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PubMed1992

Involvement of Calmodulin and Calmodulin-Dependent Myosin Light Chain Kinase in Blue Light-Dependent H Pumping by Guard Cell Protoplasts from Vicia faba L.

Signal transduction processes involved in blue light-dependent proton pumping were investigated using guard cell protoplasts from Vicia faba.N-[2-(Methylamino)ethyl]-5-isoquinolinesulfonamide, an inhibitor of cyclic AMP- and cyclic GMP-dependent protein kinases, had no effect. 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine (H-7) and calphostin C, inhibitors of protein kinase C, produced slight inhibition of the blue light-dependent proton pumping. 1-[N, O-Bis(5-isoquinolinesulfonyl)-N-methyl-l-tyrosyl] -4-phenylpiperazine, a specific inhibitor of Ca(2+)/calmodulin (CaM)-dependent protein kinase II, did not inhibit the proton pumping, but 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine and 1-(5-chloro-naphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-9), inhibitors of Ca(2+)/CaM-dependent myosin light chain kinase, strongly suppressed the proton pumping. A CaM antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), inhibited blue light-dependent proton pumping, whereas its less active structural analog, N-(6-aminohexyl)-1-naphthalenesulfonamide (W-5), had little effect on the response. Other CaM antagonists, trifluoperazine, compound 48/80, prenylamine, and 3-(2-benzothiazolyl)-4,5-dimethoxy-N-[3-(4-phenyl-piperidinyl)- propylbenzenesulfonamide inhibited the proton pumping. In accord with these results, light-induced stomatal opening in the epidermis of Commelina benghalensis ssp. was inhibited by ML-9 and W-7, but not by H-7 and W-5. Thus, it is concluded that CaM and Ca(2+)/CaM-dependent myosin light chain kinase are the components of the signal transduction process in blue light-dependent proton pumping in guard cells.

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