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R M Spanswick

Publications and source records attributed to R M Spanswick.

At least 19 recordsLinked to original sources
PubMed1996

Plasma membrane isolation from freshwater and salt-tolerant species of Chara: antibody cross-reactions and phosphohydrolase activities.

Plasma membranes were isolated using the aqueous polymer two-phase partition method from the algae Chara corallina and Chara longifolia, algae which differ in their ability to grow in saline environments. Enrichment of plasma membrane and depletion of tonoplast relative to the microsomal fraction was monitored using phosphohydrolase assays and cross-reactions to antibodies raised against higher plant transporters. Antibodies to the vacuolar ATPase and pyrophosphatase cross-reacted with epitopes in the microsomal fraction, but showed little affinity for the plasma membrane fraction. Pyrophosphatase activity also declined in the plasma membrane fraction relative to the microsomal fraction. The V-type H(+)-ATPase activity, sensitive to nitrate or bafilomycin, was low in both fractions, though the cross-reaction to the antibody was reduced in the plasma membrane fraction. By contrast, the antibody recognition of a P-type H(+)-ATPase amino acid sequence from Arabidopsis did not occur strongly in the anticipated 90-100 kDa range. While there was enhanced recognition of a polypeptide at around 140 kDa in the plasma membrane fraction, salt treatment of Chara longifolia resulted in plasma membrane fractions with reduced amounts of this epitope, but no change in vanadate-sensitive ATPase activity, suggesting that it does not represent the only P-type ATPase. Microsomal membranes from salt-adapted C. longifolia have higher reactivity with the antibody to the tonoplast ATPase.

Adenosine Triphosphatases↗
PubMed1993

Evidence for a membrane skeleton in higher plants. A spectrin-like polypeptide co-isolates with rice root plasma membranes.

A fraction enriched in plasma membranes was isolated from rice roots by differential centrifugation and aqueous polymer two-phase partitioning. Analysis of the fraction by SDS-PAGE showed the presence of several low mobility polypeptides (M(r) > 100 kDa). One of these polypeptides (M(r) approximately 230 kDa) was specifically recognized by polyclonal antibodies to human erythrocyte spectrin. This finding suggests that a higher plant spectrin-based membrane skeleton may be preserved and studied using high-purity plasma membrane fractions obtained by aqueous polymer two-phase partitioning.

Adenosine Triphosphatases↗
PubMed1992

Quantitative Analysis of Photosynthate Unloading in Developing Seeds of Phaseolus vulgaris L. : I. The Use of Steady-State Labeling.

The pathway and kinetics of photosynthate unloading in developing seeds of bean (Phaseolus vulgaris L.) were investigated using steady-state labeling with (14)CO(2). The continuous assimilation of (14)CO(2) at constant specific activity produced stable tracer fluxes that facilitated straightforward analyses of photosynthate import and unloading in developing seeds. The kinetics of tracer equilibration within intact seeds were compatible with a symplastic route of photosynthate unloading in the seed coat. The import and partitioning of tracer within seeds were partially disrupted by the surgical excision of the distal halves of seeds as practiced during the preparation of "empty" seed coats for perfusion.

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PubMed1992

Quantitative Analysis of Photosynthate Unloading in Developing Seeds of Phaseolus vulgaris L. : II. Pathway and Turgor Sensitivity.

Phloem import and unloading in perfused bean (Phaseolus vulgaris L.) seed coats were investigated using steady-state labeling. Though photosynthate import and unloading were significantly reduced by perfusion, measurements of photosynthate fluxes in perfused seed coats proved useful for the study of unloading mechanisms in vivo. Phloem import was stimulated by lowered seed coat cell turgor, as demonstrated by an increase in tracer and sucrose import to seed coats perfused with high concentrations of an osmoticum. The partitioning of photosynthates between retention in the seed coat and release to the perfusion solution also was turgor sensitive; increases in seed coat cell turgor stimulated photosynthate release to the apoplast at the expense of photosynthate retention within the seed coat. There was no evidence of a turgor-sensitive sucrose uptake mechanism in perfused seed coats. Thus, the turgor sensitivity of photosynthate partitioning within perfused seed coats was consistent with a turgor-sensitive efflux control mechanism. Measurements of tracer equilibration and sugar partitioning in perfused seed coats provided strong evidence for symplastic phloem unloading in seed coats.

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PubMed1992

Measurement of Net Fluxes of Ammonium and Nitrate at the Surface of Barley Roots Using Ion-Selective Microelectrodes : II. Patterns of Uptake Along the Root Axis and Evaluation of the Microelectrode Flux Estimation Technique.

Net fluxes of NH(4) (+) and NO(3) (-) into roots of 7-day-old barley (Hordeum vulgare L. cv Prato) seedlings varied both with position along the root axis and with time. These variations were not consistent between replicate plants; different roots showed unique temporal and spatial patterns of uptake. Axial scans of NH(4) (+) and NO(3) (-) net fluxes were conducted along the apical 7 centimeters of seminal roots of intact barley seedlings in solution culture using ion-selective microelectrodes in the unstirred layer immediately external to the root surface. Theoretically derived relationships between uptake and concentration gradients, combined with experimental observations of the conditions existing in our experimental system, permitted evaluation of the contribution of bulk water flow to ion movement in the unstirred layer, as well as a measure of the spatial resolution of the microelectrode flux estimation technique. Finally, a method was adopted to assess the accuracy of this technique.

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PubMed1991

Auxin transport in suspension-cultured soybean root cells : I. Characterization.

The kinetic parameters of auxin transport in suspension-cultured soybean (Glycine max [L.] Merr.) root cells were investigated. The same processes that are responsible for polar indoleacetic acid (IAA) transport in other plant tissues were found to occur in soybean root cells. These include (a) passive diffusion of the undissociated auxin molecule across the plasma membrane, (b) uptake via a specific, saturable carrier, and (c) phytotropin-sensitive efflux. Metabolism of exogenously added IAA was rapid; at the end of a 15-minute uptake period >80% of the IAA taken up had been converted to other compounds. The time course of [(14)C]IAA uptake in the first 90 seconds revealed two phases, the first corresponding to a rate of uptake approximately twice as large as the second phase. The transition to the second phase was delayed in the presence of the phytotropins triiodobenzoic acid or naphthylphthalamic acid, suggesting that an increase in the efflux of label as IAA accumulates in the cytoplasm is responsible for the transition. Carrier-mediated uptake contributes between 50 and 60% to the total rate of auxin uptake from a 0.28 micromolar IAA solution, with passive diffusion accounting for the remainder. Kinetic analysis of carrier-mediated uptake revealed a pH optimum of 5.0 and a Michaelis-Menten constant of 0.4 micromolar at pH 5.5. Because phytotropins had no effect on the initial rate of uptake, the efflux carrier does not appear to be involved in the uptake process.

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PubMed1991

Auxin Transport in Suspension-Cultured Soybean Root Cells : II. Anion Effects on Carrier-Mediated Uptake.

To test the hypothesis that the carrier-mediated component of the indoleacetic acid (IAA) influx involves an electrogenic proton/IAA anion symport, the effects on the IAA influx of salts expected to depolarize the membrane potential were examined in suspension-cultured soybean (Glycine max [L.] Merr.) root cells. Although KCl does inhibit carrier-mediated uptake, the effect is specific to the anion at low concentrations and not due to more general processes such as changes in ionic or osmotic strength. Other anions such as bromide, iodide, and fluoride inhibit the carrier more strongly. Because potassium iminodiacetate, which is also expected to depolarize the membrane potential, has no inhibitory effect on the IAA influx, there is no evidence for the involvement of the membrane potential in carrier-mediated uptake. It is therefore most likely that in soybean cells, if carrier-mediated uptake occurs via a proton symport, the H(+):IAA- stoichiometry is 1:1. At concentrations greater than 70 millimolar, sorbitol, a nonionic osmoticum, inhibits carrier-mediated IAA uptake. The effects of specific anions and osmotic potential on the uptake carrier necessitates the reevaluation of other auxin transport studies in which KCl was routinely used as an agent with which to depolarize the membrane potential.

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PubMed1990

Measurement of net fluxes of ammonium and nitrate at the surface of barley roots using ion-selective microelectrodes.

Neutral carrier-based liquid membrane ion-selective microelectrodes for NH(4) (+) and NO(3) (-) were developed and used to investigate inorganic nitrogen acquisition in two varieties of barley, Hordeum vulgare L. cv Olli and H. vulgare L. cv Prato, originating in cold and warm climates, respectively. In the present paper, the methods used in the fabrication of ammonium- and nitrate-selective microelectrodes are described, and their application in the study of inorganic nitrogen uptake is demonstrated. Net ionic fluxes of NH(4) (+) and NO(3) (-) were measured in the unstirred layer of solution immediately external to the root surface. The preference for the uptake of a particular ionic form was examined by measuring the net flux of the predominant form of inorganic nitrogen, with and without the alternative ion in solution. Net flux of NH(4) (+) into the cold-adapted variety remained unchanged when equimolar concentrations (200 micromolar) of NH(4) (+) and NO(3) (-) were present. Similarly, net flux of NO(3) (-) into the warm-adapted variety was not affected when NH(4) (+) was also present in solution. The high temporal and spatial resolution afforded by ammonium- and nitrate-selective microelectrodes permits a detailed examination of inorganic nitrogen acquisition and its component ionic interactions.

Journal Article↗
PubMed1990

Evidence for cotransport of nitrate and protons in maize roots : I. Effects of nitrate on the membrane potential.

The electrical response of nitrate-grown maize (Zea mays L.) roots to 0.1 millimolar nitrate was comprised of two sequential parts: a rapid and transient depolarization of the membrane potential, followed by a slower, net hyperpolarization to a value more negative than the original resting potential. The magnitude of the response was smaller in roots of seedlings grown in the absence of nitrate, but, within 3 hours of initial exposure to 0.1 millimolar nitrate, increased to that of nitrate-grown roots. Chloride elicited a separate electrical response with a pattern similar to that of the nitrate response. However, the results presented in this study strongly indicate that the electrical response to nitrate reflects the activity of a nitrate-inducible membrane transport system for nitrate which is distinct from that for chloride. Inhibitors of the plasmalemma H(+)-ATPase (vanadate, diethylstilbestrol) completely inhibited both parts of the electrical response to nitrate, as did alkaline external pH. The magnitude of the initial nitrate-dependent, membrane potential depolarization was independent of nitrate concentration, but the subsequent nitrate-dependent hyperpolarization showed saturable dependence with an apparent K(m) of 0.05 millimolar. These results support a model for nitrate uptake in maize roots which includes a depolarizing NO(3) (-)/H(+) symport. The model proposes that the nitrate-dependent membrane potential hyperpolarization is due to the plasma membrane proton pump, which is secondarily stimulated by the operation of the NO(3) (-)/H(+) symport.

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PubMed1990

Evidence for Cotransport of Nitrate and Protons in Maize Roots : II. Measurement of NO(3) and H Fluxes with Ion-Selective Microelectrodes.

We report here on an investigation of net nitrate and proton fluxes in root cells of maize (Zea mays L.) seedlings grown without (noninduced) and with (induced) 0.1 millimolar nitrate. A microelectrode system described previously (IA Newman, LV Kochian, MA Grusak, WJ Lucas [1987] Plant Physiol 84: 1177-1184) was utilized to quantify net ionic fluxes from the measurement of electrochemical potential gradients for NO(3) (-) and H(+) within the unstirred layer at the root surface. The nitrate-inducibility, pH dependence, and concentration dependence of net NO(3) (-) uptake correlated quite closely with the electrical response of maize roots to nitrate under the same experimental conditions (as described in PR McClure, LV Kochian, RM Spanswick, JE Shaff [1990] Plant Physiol 93: 281-289). Additionally, it was found that potential inhibitors of the plasmalemma H(+)-ATPase (vandate, diethylstilbestrol), which were shown to abolish the electrical response to NO(3) (-) (in PR McClure, LV Kochian, RM Spanswick, JE Shaff [1990] Plant Physiol 93: 281-289), dramatically inhibited NO(3) (-) absorption. These results strongly indicate that the NO(3) (-) electrical response is due to the operation of a NO(3) (-) transport system in the plasmalemma of maize root cells. Furthermore, the results from the H(+)-ATPase inhibitor studies indicate that the NO(3) (-) transport system is linked to the H(+)-ATPase, presumably as a NO(3) (-)/H(+) symport. This is further supported by the pH response of the NO(3) (-) transport system (inhibition at alkaline pH values) and the change in net H(+) flux from a moderate efflux in the absence of NO(3) (-), to zero net H(+) flux after exposing the maize root to exogenous nitrate. Although these results can be explained by other interpretations, the simplest model that fits both the electrical responses and the NO(3) (-)/H(+) flux data is a NO(3) (-)/H(+) symport with a NO(3) (-):H(+) flux stoichiometry >1, whose operation results in the stimulation of the H(+)-ATPase due to the influx of protons through the cotransport system.

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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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PubMed1987

The effect of vanadate on proton-sucrose cotransport in ricinus cotyledons.

The effects of orthovanadate on the uptake of sucrose by Ricinus cotyledons and on sucrose-coupled proton influx were measured in order to gain insight into the relationship to the plasma membrane proton pump. Vanadate had no effect on short-term sucrose uptake. In longterm experiments (>30 min) sucrose uptake was progressively inhibited, but only at high external sucrose concentrations. Vanadate did not affect proton efflux pumping in the absence of sucrose and neither did it change the initial rate of sucrose-coupled proton influx. However, it enhanced the maximal level of sucrose-induced alkalinization of the medium at all sucrose concentrations tested. This is interpreted as an inhibiting effect of vanadate on the proton pump that recycles protons during sucrose-proton cotransport. The sensitivity towards vanadate indicates that this proton pump is an ATPase. A second proton-translocating system, that is insensitive to vanadate, is postulated to function in the absence of sucrose.

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PubMed1986

H-pumping driven by the vanadate-sensitive ATPase in membrane vesicles from corn roots.

The initial rate of quenching of quinacrine fluorescence was used to monitor Mg:ATP-dependent H(+)-pumping in membrane vesicles from corn (Zea mays L. cv WF9 x MO17) roots and obtain a preparation in which vanadate-sensitive H(+)-pumping could be observed. Separation of membranes on a linear sucrose density gradient resulted in two distinct peaks of H(+)-pumping activity: a major one, at density 1.11 grams per cubic centimeter, was sensitive to NO(3) (-) and resistant to vanadate, while a minor one, at density 1.17 grams per cubic centimeter, was substantially resistant to NO(3) (-) and sensitive to vanadate. A membrane fraction enriched in the vanadate-sensitive H(+)-pump could be obtained by washing microsomes prepared in the presence of 10% glycerol with 0.25 molar KI. The kinetics of inhibition of H(+)-pumping by vanadate in this membrane preparation indicated that most of the H(+)-pumping activity in this fraction is sensitive to inhibition by vanadate, 50% inhibition being reached at about 60 micromolar vanadate. This value is fairly close to that observed for inhibition by vanadate of the ATPase activity in similar experimental conditions (40 micromolar). The inhibitor sensitivity, divalent cation dependence, pH optimum (6.5), and K(m) for ATP (0.7 millimolar) of the H(+)-pumping activity match quite closely those reported for the plasma membrane ATPase of corn roots and other plant materials.

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PubMed1986

Purification and properties of the h-translocating ATPase from the plasma membrane of tomato roots.

The proton-translocating, plasma membrane ATPase was purified from tomato roots. At the final stage of purification approximately 80% of the protein was found in a single band with an apparent molecular weight of 90 kilodaltons. Cross-linking studies indicated that the ATPase normally exists as a trimer of catalytic subunits. No evidence was found for any additional subunits. The pH optimum for ATP hydrolysis by the purified protein was 6.5. Activity was stimulated by K(+), especially at low pH, and inhibited by vanadate, N,N'-dicyclohexylcarbodiimide, and diethylstilbestrol; nitrate was weakly inhibitory. Activity was stimulated by lysolecithin but inhibited by sonicated phospholipids. The inhibition by lipids could be prevented if octylglucoside was added with the lipids; the combination of octylglucoside and lipids actually stimulated activity. The purified protein could be reconstituted into liposomes and catalyzed ATP-dependent, vanadate-sensitive proton translocation.

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PubMed1985

Characterization of Anion Effects on the Nitrate-Sensitive ATP-Dependent Proton Pumping Activity of Soybean (Glycine max L.) Seedling Root Microsomes.

The ATP-dependent proton-pumping activity of soybean (Glycine max L.) root microsomes is predominantly nitrate sensitive and presumably derived from the tonoplast. We used microsomes to characterize anion effects on proton pumping of the tonoplast vesicles using two distinctly different techniques.Preincubation of the vesicles with nitrate caused inhibition of proton pumping and ATPase activity, with similar concentration dependence. Fluoride, which preferentially inhibits the plasma membrane ATPase, inhibited ATPase activity strongly at concentrations which did not affect proton pumping activity.Addition of potassium salts, after a steady-state pH gradient is established in the absence of such salts, caused an increased pH gradient which was due to alleviation of Delta Psi and subsequent increased influx of H(+) into these vesicles. This anion-induced increase in the pH gradient could be used as a measure of the relative anion permeabilities, which were of the order Br(-) = NO(3) (-) > Cl(-) >> SO(4) (2-). Phosphate and fluoride caused no increase in the pH gradient. Since the concentration dependence of KCl- and KNO(3)-induced quenching exhibited a saturable component, and since H(+) uptake was increased by only certain anions, the data suggest that there may be a relatively specific anion channel associated with tonoplast-derived vesicles.

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PubMed1985

H-ATPase Activity from Storage Tissue of Beta vulgaris: III. Modulation of ATPase Activity by Reaction Substrates and Products.

Two distinct membrane fractions containing H(+)-ATPase activity were prepared from red beet. One fraction contained a H(+)-ATPase activity that was inhibited by NO(3) (-) while the other contained a H(+)-ATPase inhibited by vanadate. We have previously proposed that these H(+)-ATPases are associated with tonoplast (NO(3) (-)-sensitive) and plasma membrane (vanadate-sensitive), respectively. Both ATPase were examined to determine to what extent their activity was influenced by variations in the concentration of ATPase substrates and products. The substrate for both ATPase was MgATP(2-), and Mg(2+) concentrations in excess of ATP had only a slight inhibitory effect on either ATPase. Both ATPases were inhibited by free ATP (i.e. ATP concentrations in excess of Mg(2+)) and ADP but not by AMP. The plasma membrane ATPase was more sensitive than the tonoplast ATPase to free ATP and the tonoplast ATPase was more sensitive than the plasma membrane ATPase to ADP.Inhibition of both ATPases by free ATP was complex. Inhibition of the plasma membrane ATPase by ADP was competitive whereas the tonoplast ATPase demonstrated a sigmoidal dependence on MgATP(2-) in the presence of ADP. Inorganic phosphate moderately inhibited both ATPases in a noncompetitive manner.Calcium inhibited the plasma membrane but not the tonoplast ATPase, apparently by a direct interaction with the ATPase rather than by disrupting the MgATP(2-) complex.The sensitivity of both ATPases to ADP suggests that under conditions of restricted energy supply H(+)-ATPase activity may be reduced by increases in ADP levels rather than by decreases in ATP levels per se. The sensitivity of both ATPases to ADP and free ATP suggests that modulation of cytoplasmic Mg(2+) could modulate ATPase activity at both the tonoplast and plasma membrane.

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