Roger M. Spanswick

Evidence for an electrogenic ion pump in Nitella translucens. I. The effects of pH, K+, Na+, light and temperature on the membrane potential and resistance

Abstract 1. 1. The effects of external pH and K + in the light and dark were investigated to establish conditions in which the membrane potential of Nitella translucens was more negative than the diffusion potential for the major ions. 2. 2. At high external K + concentrations there is an apparent increase in K + permeability and the membrane potential becomes equal to the calculated K + equilibrium potential. It is then possible to calculate the K + equilibrium potential for individual cells in solutions having low K + concentrations. 3. 3. In a solution containing 0.5 mM K + at pH 6, the membrane potential in the dark is close to the K + equilibrium potential. At a light intensity of 1.0 mW · cm −2 the membrane potential is hyperpolarized by 50 mV and there is a decrease in the membrane resistance from 153 kΩ · cm 2 to 17 kΩ · cm 2 . 4. 4. In the dark, a decrease in the temperature depolarizes the membrane by 0.97 mV · °C − and there is no significant effect on the membrane resistance. In the light, the temperature coefficient for the membrane potential is 2.5 mV · °C −1 and the membrane resistance is approximately doubled by a 10 °C decrease in temperature. 5. 5. In the light, the current required to reduce the potential across thee plasmalemma to the K + equilibrium potential was equivalent to a flux of about 20 pmoles · cm −2 · s −1 . This was tentatively identified with the flux through the electrogenic H + pump postulated by H. Kitasato ( J. Gen. Physiol. , 52 (1968) 60). 6. 6. It is suggested that the results cannot be accounted for by the diffusion of the major ions or by an electrogenic pump in combination with a large passive H + flux. An alternative analysis in terms of a voltage-dependent, light-stimulated electrogenic pump (S.I. Rapoport, Biophys. J. 10 (1970) 246) is presented.

Enhanced anthocyanin production in grape cell cultures

Abstract Cultured grape cells (Bailey Alicant A Vitis hybrid) that produce and accumulate anthocyanins provide a convenient model system to study secondary metabolite formation and compartmentation. A reduction in anthocyanin production in suspension cultures occurred after a series of subcultures in liquid Gamborgs B5 (GB5) maintenance medium. In contrast, newly derived suspensions from selected red calluses retained their ability to produce anthocyanin at a sustained high level. A production medium with reduced nitrate and elevated sucrose induced very high levels of anthocyanin (up to 2.9 g/l) in suspension cultures derived from selected callus. This medium also induced increased pigment production in faded suspension cultures and in callus cultures of Vignoles [Ravat 51, Vitis hybrid] which normally do not produce anthocyanin in culture. Reduction of nitrate to a critical level resulted in a ‘switchlike’ (rather than gradual) enhancement of anthocyanin production which may represent the removal of an inhibitory effect. Improvements obtained with sucrose were probably due to increased medium osmolarity. The combined effects of low nitrate and high sucrose synergistically improved anthocyanin volumetric productibility (up to 0.15 g/l · day) and surpassed previously published values for cultured grape cells. Our results indicate a possible involvement of a nitrate-sensitive tonoplast ATPase in the accumulation of anthocyanin in grape vacuoles.

Evidence for an electrogenic ion pump in Nitella Translucens. II. Control of the light-stimulated component of the membrane potential

Abstract 1. 1. It was established previously that there is a light-stimulated electrogenic ion pump in Nitella translucens (Spanswick R. M. (1972) Biochim. Biophys. Acta 288.73). The effects of CO2 and inhibitors have been used to study the relationship of the pump to photosynthesis under conditions (pH 6.0, 0.5 mM external K+) where it produces a hyperpolarization beyond the limit for a diffusion potential. 2. 2. Under CO2-free conditions the light-stimulated hyperpolarization is maximal and constant. Addition of 1 mM(CO2 + HCO3−) to the external solution produces a slow reversion of the membrane potential and resistance to the dark levels. If 1 mM(CO2 + HCO3−) is present throughout, light has only a small effect. It was concluded that the products of CO2 fixation are not involved in supplying energy to the pump. 3. 3. Further experiments with inhibitors were conducted under CO2-free conditions. 3,3-Dichlorophenyl-N,N-dimethylurea (DCMU) at concentrations of 0.5 and 1.0 μM had no significant effect on the membrane potential in the light but the higher concentration increased the membrane resistance. These experiments suggested that the pump was not directly linked to non-cyclic electron flow. 4. 4. The remaining possibility was that the pump is driven by ATP produced by cyclic photophosphorylation. This was consistent with the effect of the uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP) which, at a concentration of 1.0 μM, depolarized the cell to the dark level and increased the resistance. 1 mM azide also reversibly depolarized the cell but its site of action is not known. 5. 5. Dicyclohexylcarbodiimide (DCCD) produced a permanent depolarization to the dark level and reduced the subsequent response of the potential to external pH changes. It is suggested that it may affect the ATPase in the cell membrane. 6. 6. 710-nm light also produces a hyperpolarization, confirming the dependence on cyclic photophosphorylation.

Solubilization and reconstitution of a vanadate-sensitive H+-ATPase from the plasma membrane ofBeta vulgaris

SummaryA vanadate-sensitive H+-translocating ATPase isolated from red beet plasma membrane has been solubilized in active form and successfully reconstituted into artificial proteoliposomes. The H+-ATPase was solubilized in active form with deoxycholate, CHAPSO or octylglucoside in the presence of glycerol. Following detergent removal by gel filtration and reconstitution into proteoliposomes, ATP:Mg-dependent H+ transport could be measured as ionophore-reversible quenching of acridine orange fluorescence. Solubilization resulted in a three-to fourfold purification of the plasma membrane ATPase, with some additional enrichment of specific activity following reconstitution. H+ transport activity was inhibited half-maximally between 1 and 5 μM vanadate (Na3VO4) and nearly abolished by 100 μM vanadate. ATPase activity of native plasma membrane showed aKi for vanadate inhibition of 9.5 μM, and was inhibited up to 80% by 15 to 20 μM vanadate (Na3VO4). ATPase activity of the reconstituted vesicles showed aKi of 2.6 μM for vanadate inhibition. The strong inhibition by low concentrations of vanadate indicates a plasma membrane rather than a mitochondrial or tonoplast origin for the reconstituted enzyme.

Characterization of native and reconstituted plasma membrane H+-ATPase from the plasma membrane ofBeta vulgaris

SummaryCharacteristics of the native and reconstituted H+-ATPase from the plasma membrane of red beet (Beta vulgaris L.) were examined. The partially purified, reconstituted H+-ATPase retained characteristics similar to those of the native plasma membrane H+-ATPase following reconstitution into proteoliposomes. ATPase activity and H+ transport of both enzymes were inhibited by vanadate, DCCD, DES and mersalyl. Slight inhibition of ATPase activity associated with native plasma membranes by oligomycin, azide, molybdate or NO3− was eliminated during solubilization and reconstitution, indicating the loss of contaminating ATPase activities. Both native and reconstituted ATPase activities and H+ transport showed a pH optimum of 6.5, required a divalent cation (Co2+>Mg2+>Mn2+>Zn2+>Ca2+), and preferred ATP as substrate. The Mg:ATP kinetics of the two ATPase activities were similar, showing simple Michaelis-Menten kinetics. Saturation occurred between 3 and 5mM Mg: ATP, with aKm of 0.33 and 0.46mM Mg: ATP for the native and reconstituted enzymes, respectively. The temperature optimum for the ATPase was shifted from 45 to 35°C following reconstitution. Both native and reconstituted H+-ATPases were stimulated by monovalent ions. Native plasma membrane H+-ATPase showed an order of cation preference of K+>NH4+ >Rb+>Na+>Cs+>Li+>choline+. This basic order was unchanged following reconstitution, with K+, NH4+, Rb+ and Cs+ being the preferred cations. Both enzymes were also stimulated by anions although to a lesser degree. The order of anion preference differed between the two enzymes. Salt stimulation of ATPase activity was enhanced greatly following reconstitution. Stimulation by KCl was 26% for native ATPase activity, increasing to 228% for reconstituted ATPase activity. In terms of H+ transport, both enzymes required a cation such as K+ for maximal transport activity, but were stimulated preferentially by Cl− even in the presence of valinomycin. This suggests that the stimulatory effect of anions on enzyme activity is not simply as a permeant anion, dissipating a positive interior membrane potential, but may involve a direct anion activation of the plasma membrane H+-ATPase.

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 ~ 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.

Solubilization and reconstitution of an anion-sensitive H+-ATPase from corn roots

SummaryThe distribution of corn root microsomal ATPase activity on continuous sucrose gradients was nearly identical to that of membrane vesicles capable of ATP-dependent H+ transport when assayed in the presence of 20 μm vanadate, 3mm azide, 5mm MgSO4 and 50mm LiCl. These assay conditions were used to monitor solubilization of an anion-sensitive H+-ATPase from a purified membrane fraction (20–30% (wt/wt) sucrose interface) believed to be derived from tonoplast membranes.The detergents N-tetradecyl-N,N-dimethyl-3-ammonio-1-propane-sulfonate (Zwittergent® 3–14) and deoxycholate were used to solubilize the H+-ATPase. In the presence of 40% glycerol, the H+-ATPase was solubilized in active form by both detergents. Glycerol was essential for the preservation of activity during solubilization and added phospholipid was required for maximal activity of the deoxycholate-solubilized, but not the Zwittergent® 3-14-solubilized, H+-ATPase. Phospholipid reactivation of the deoxycholate-solubilized H+-ATPase was maximal with mixed phospholipids, although several purified phospholipids gave substantial reactivation, indicating a relatively nonspecific interaction of the H+-ATPase with the hydrophobic lipid environment.Reconstitution of the deoxycholate-solubilized, anion-sensitive H+-ATPase was accomplished by removal of deoxycholate by gel filtration through Sephadex G-200 in the presence of added phospholipid. ATP-dependent H+ transport in reconstituted vesicles was similar to that in native vesicles with respect to substrate specificity, salt stimulation, and inhibitor sensitivity.

The effects of bicarbonate ions and external pH on the membrane potential and resistance ofNitella translucens

SummaryThe effects of bicarbonate ions on the membrane potential and resistance ofNitella translucens are shown to be primarily due to the change in pH produced by the bicarbonate acting as a buffer, and not due to the presence of an electrogenic anion pump. The mechanism by which pH affects the membrane potential is discussed in the context of recent work by other authors on this effect.

The kinetics of nitrate uptake from flowing solutions by rice : influence of pretreatment and light

Abstract The kinetics of nitrate (NO − 3 ) uptake by intact 23 day old rice plants was studied by measuring the depletion of NO − 3 in solutions flowing over the plant roots. A Michaelis-Menten kinetic model was applied, allowing the uptake kinetics to be characterized by two parameters: the apparent half-velocity constant, K m , and the apparent maximum uptake rate, V max . A propagation of uncertainty calculation revealed that the kinetic parameters could be determined with a high degree of accuracy; the standard deviation in K m was typically 15% of the K m value; the standard deviation in V max was typically 7% of the V max value. The plants were exposed to full nutrient solutions containing NO − 3 at 50, 200, 500 and 800 μ m for 24 h prior to kinetic testing, and both K m and V max were found to vary with pretreatment NO − 3 concentration, [NO − 3 ]; plants pretreated at high [NO − 3 ] had lower V max and higher K m values than plants pretreated at lower [NO − 3 ]. However, the variations in V max were more consistent than those in K m . These changes in the kinetic parameters reflect an uptake system which is capable of compensating for changes in the external [NO − 3 ] to maintain a virtually constant NO − 3 uptake rate in the range studied. Changes in K m and V max begin within 4 h of a change in [NO − 3 ]. Light deprivation during pretreatment in 200 μ m NO − 3 resulted in a complete cessation of NO − 3 uptake; 4–8 h of illumination were required before the uptake resumed, and uptake rates had not yet reached normal levels 8 h after the resumption of illumination.

Propagation of the neuroid action potential of the carnivorous plantDrosera

SummaryThe neuroid action potentials ofDrosera rotundifolia recorded from single cells resemble those recorded from the surface of tentacle stalks. They have similar amplitudes and durations and they show the same variation of duration with interval that characterizes the extracellularly recorded action potentials. All living cells of the stalk appear to be excitable since cells from both layers were observed to produce action potentials when intracellular recording techniques were used.Propagation of electrically induced action potentials down the stalk occurred at a rate of 4.3 mm/s±0.6 S.E.M. while propagation up the stalk occurred at a rate of 9.9 mm/s±2.0 S.E.M. The fact that attenuated signals from electrical processes and stimulus artifacts in distant parts of the stalk were detected in recordings indicates that the cells of the stalk were closely coupled and that propagation from cell to cell is probably by an electrotonic mechanism. This hypothesis gains additional support from the observation of numerous cytoplasmic connections (plasmodesmata) through the cell walls separating the cells which are most likely to conduct the action potential.