Hidde B. A. Prins

The kinetics of NH4 + and NO3 − uptake by Douglas fir from single N-solutions and from solutions containing both NH4 + and NO3 −

The kinetics of NH4+ and NO3− uptake in young Douglas fir trees (Pseudotsuga menziesii [Mirb.] Franco) were studied in solutions, containing either one or both N species. Using solutions containing a single N species, the Vmax of NH4+ uptake was higher than that of NO3− uptake. The Km of NH4+ uptake and Km of NO3− uptake differed not significantly. When both NH4+ and NO3− were present, the Vmax for NH4+ uptake became slightly higher, and the Km for NH4+ uptake remained in the same order. Under these conditions the NO3− uptake was almost totally inhibited over the whole range of concentrations used (10–1000 μM total N). This inhibition by NH4+ occurred during the first two hours after addition. ei]{gnA C}{fnBorstlap}

Modulation of H+-ATPase Activity by Fusicoccin in Plasma Membrane Vesicles from Oat (Avena sativa L.) Roots (A Comparison of Modulation by Fusicoccin, Trypsin, and Lysophosphatidylcholine)

The fungal phytotoxin fusicoccin affects various transport processes in the plasma membrane of plant cells. The plasma membrane (PM) H+-ATPase (EC 3.6.1.35) seems to be the primary target of fusicoccin action. The kinetics of the stimulation of the PM H+-ATPase by fusicoccin was studied in PM vesicles isolated from oat (Avena sativa cv Adamo) roots by aqueous two-phase partitioning. Considerable stimulation of activity was observed only when roots were treated with fusicoccin prior to the PM isolation. Fusicoccin treatment shifted the pH optimum of the ATPase toward more alkaline values and increased Vmax. No effects on Km were observed. Treatment with trypsin resulted in stimulation of ATPase activity in control vesicles but not in the fusicoccin-treated vesicles. The characteristics of stimulation by trypsin in control vesicles were comparable with those of stimulation by fusicoccin. This result and the change of the polypeptide pattern on western blots suggest the involvement of the C-terminal inhibitory domain in the fusicoccin signal transduction chain. On the other hand, stimulation by lyso-PC demonstrated other characteristics than stimulation by fusicoccin. Lyso-PC was able to stimulate ATPase activity at both acidic and alkaline pH values. Kinetic analysis of the pH dependency curves revealed different mechanisms for activation by fusicoccin and by lyso-PC. Whereas fusicoccin shifted the pH dependency of formation of phosphorylated intermediate to more alkaline values, lyso-PC seemed to increase dephosphorylation independently of pH.

Patch clamp analysis of the dominant plasma membrane K+ channel in root cell protoplasts of Plantago media L. Its significance for the P and K state

Ion channels in the plasma membrane of root cell protoplasts of Plantago media L. were studied with the patch clamp technique in the cell-attached patch and outside-out patch configuration. An outward rectifying potassium channel was dominantly present in the plasma membrane. It appears responsible for the diffusional part, dominated by the K+ diffusion potential, of the cell membrane potential, in vivo. This channel is activated at potentials near to and more positive than the K+ diffusion potential. The dependence of this ion channel on K+ activity and voltage has been characterized. The current-voltage relationships of the open channel at various K+ concentrations are described by a four-state model. The membrane potential of intact protoplasts appears either dominated by the K+ diffusion potential, the protoplast is then said to be in the K state, or by the pump potential generated by the plasma membrane-bound proton pump/H+ ATPase, the P state. An experimental procedure is described to determine in cell-attached patch mode the state of the protoplast, either K or P state.

ELECTROPHYSIOLOGICAL MEMBRANE-CHARACTERISTICS OF THE SALT TOLERANT PLANTAGO-MARITIMA AND THE SALT SENSITIVE PLANTAGO-MEDIA

Membrane potential, electrical conductance and plasmamembrane permeability for Na+, K+ and Cl+t- of root cells of the salt tolerant Plantago maritima and the salt sensitive P. media were compared. Plants were grown with (25 mol m-3) or without NaCl. No differences were found in the membrane potentials between the species when measured in growth medium, either plus or minus NaCl. When plants were grown in 25 mol m-3 NaCl membrane potentials in both species were slightly more positive.Na and K ions both caused depolarization of the membrane potential. These were not equal for the two species, depolarization caused by K+ ions was about 3.5 times greater than that caused by Na+ ions. Membrane permeability for both ions waemed less in the salt tolerant species. Passive Na+ permeability, i.e. in the presence of the uncoupler CCCP, was about 25% of the K+ permeability in P. media, and 60% in P. maritima. From comparison of permeabilities under ‘active’ and ‘passive’ conditions and the related ion concentrations in the root cells it was concluded that in both species active, i.e. against the electrochemical gradient, Na+ efflux pumping must exist at the cortex medium boundary.

Photosynthetic bicarbonate utilization in the aquatic angiospermsPotamogeton andElodea

SummaryAs the CO2 supply often limits photosynthesis a number of aquatic species use HCO3− as carbon source as well. The use of HCO3− leads to the production of one OH− for every molecule/CO2 fixed. The OH− is excreted into the medium. We studied the mechanism of HCO3− utilization in the leaves ofElodea andPotamogeton. In the so-called polar leaves of these plants the HCO3− uptake takes place at the lower and OH−-release at the upper epidermis. This flux of negative charge is balanced by a kation flux in the same direction. The use of HCO3− and the influx of kations is accompanied by a pH drop. The release of OH− and kations at the upper epidermis causes a raise of the pH there. The pH changes and the kation concentrations (in the present experiments K+) are measured by means of miniature electrodes. From this the CO2 (including H2CO3), HCO3− and CO3= concentrations were calculated. When the light is turned on, after a dark period, the pH increases simultaneously at both sides for 5–10 minutes. During this so-called a-polar phase there is no K+ transport through the leaf. Experiments at different ambient pHs and comparison with other aquatic species shows that this initial pH raise results from CO2 fixation. After 5–10 minutes the polar phase and HCO3− utilization start. At the lower side the pH and [K+] drop, at the upper side pH and [K+] increase. During the a-polar phase [CO2] at the lower epidermis decreased, as expected. Whereas in the a-polar phase the CO2 concentration at this side very markedly increased. This sharp increase of [CO2] may be explained either by CO2 diffusion from the leaf cells previously taken up as HCO3− or by a proton (H+) extrusion at the lower epidermis causing conversion of HCO3− into CO2 in the cell wall. This latter mechanism is discussed in more detail.

Kinetic analysis of two simultaneously activated K+ currents in root cell protoplasts of Plantago media L.

Two different, simultaneously activated outward rectifying K+ currents were analyzed in the plasmalemma of root cortex protoplasts of Plantago media. Their gating is dependent on the diffusion potential for K+ (EK).The threshold potential was more negative than EKallowing small inward currents at potentials below EKthereby keeping cells with little pump activity in the K state (Vogelzang & Prins, 1994). Time and voltage dependence of the outward rectifying K+ currents have been analyzed with Hodgkin-Huxley-like (HH) models. Dynamic responses of whole cell currents to pulse potentials were analyzed with two voltage dependent functions, the Boltzmann distribution for open probability per gate and the transition rate towards the open state (α). The transition rate in the opposite direction (β), was calculated from a and the Boltzmann distribution. These functions were used for an integral analysis of activation and deactivation currents measured over a range of pulse potentials. Both whole cell and single channel data were used for the determination of the number of closed and open states. The effects of single channel flickering on time response and amplitude of tail currents were added to the model. The dominant K+ channel present in the plasmalemma of P. media has a characteristic nonlinear single channel I-V curve reducing the amplitude of whole cell currents at positive potentials. To compensate for this nonlinearity, a four state translocator model was added to the whole cell open probability model. The analysis presented here provides a general basis for the study and comparison of K+ channel kinetics in plant protoplasts.

Inhibition of inward rectifying tonoplast channels by a vacuolar factor: physiological and kinetic implications.

SummaryRegulation of ion-channel activity must take place in order to regulate ion transport. In case of tonoplast ion channels, this is possible on both the cytoplasmic and the vacuolar side. Isolated vacuoles of youngVigna unguiculata seedlings show no or hardly any channel activity at tonoplast potentials >80 mV, in the vacuole-attached configuration. When the configuration is changed to an excised patch or whole vacuole, a fast (excised patch) or slow (whole vacuole) increase of inward rectifying channel activity is seen. This increase is accompanied by a shift in the voltage-dependent gating to less hyperpolarized potentials. In the whole vacuole configuration the level of inward current increases and also the activation kinetics changes. Induction of channel activity takes up to 20 min depending on the age of the plants used and the diameter of the vacuole. On the basis of the estimated diffusion velocities, it is hypothesized that a compound with a mol wt of 20,000 to 200,000 is present in vacuoles of young seedlings, which shifts the population of channels to a less voltage-sensitive state.

pH-Induced Proton Permeability Changes of Plasma Membrane Vesicles

Abstract. In vivo studies with leaf cells of aquatic plant species such as Elodea nuttallii revealed the proton permeability and conductance of the plasma membrane to be strongly pH dependent. The question was posed if similar pH dependent permeability changes also occur in isolated plasma membrane vesicles. Here we report the use of acridine orange to quantify passive proton fluxes. Right-side out vesicles were exposed to pH jumps. From the decay of the applied ΔpH the proton fluxes and proton permeability coefficients (PH+) were calculated. As in the intact Elodea plasma membrane, the proton permeability of the vesicle membrane is pH sensitive, an effect of internal pH as well as external pH on PH+ was observed. Under near symmetric conditions, i.e., zero electrical potential and zero ΔpH, PH+ increased from 65 × 10−8 at pH 8.5 to 10−1 m/sec at pH 11 and the conductance from 13 × 10−6 to 30 × 10−4 S/m2. At a constant pHi of 8 and a pHo going from 8.5 to 11, PH+ increased more than tenfold from 2 to 26 × 10−6 m/sec. The calculated values of PH+ were several orders of magnitude lower than those obtained from studies on intact leaves. Apparently, in plasma membrane purified vesicles the transport system responsible for the observed high proton permeability in vivo is either (partly) inactive or lost during the procedure of vesicle preparation. The residue proton permeability is in agreement with values found for liposome or planar lipid bilayer membranes, suggesting that it reflects an intrinsic permeability of the phospholipid bilayer to protons. Possible implications of these findings for transport studies on similar vesicle systems are discussed.

EFFECT OF HIGH PH ON THE PLASMA-MEMBRANE POTENTIAL AND CONDUCTANCE IN ELODEA-DENSA

SummaryIn leaves of Elodea densa the membrane potential measured in light equals the equilibrium potential of H+ on the morphological upper plasma membrane. The apoplastic pH on the upper side of the leaf is as high as 10.5–11.0, which indicates that alkaline pH induces an increased H+ permeability of the plasmalemma. To study this hypothesis in more detail we investigated the changes in membrane potential and conductance in response to alterations in the external pH from 7 (= control) to 9 or 11 under both light and dark conditions.Departing from the control pH 7 condition, in light and in dark the application of pH 9 resulted in a depolarization of the membrane potential to the Nernst potential of H+. In the light but not in the dark, this depolarization was followed by a repolarization to about −160 mV. The change to pH 9 induced, in light as well as in dark, an increase in membrane conductance.The application of pH 11, which caused a momentary hyperor depolarization depending on the value at the time pH 11 was applied, brought the membrane potential to around −160 mV. The membrane conductance also increased, in comparison to its value at pH 7, as a result of the application of pH 11, irrespective of the light conditions.

SIMULATION OF THE LIGHT-INDUCED OSCILLATIONS OF THE MEMBRANE-POTENTIAL IN POTAMOGETON LEAF-CELLS

SummaryAn attempt has been made to simulate the light-induced oscillations of the membrane potential of Potamogeton lucens leaf cells in relation to the apoplastic pH changes. Previously it was demonstrated that the membrane potential of these cells can be described in terms of proton movements only. It is hypothesized that the membrane potential is determined by an electrogenic H+-ATPase with a variable H+/ATP stoichiometry. The stoichiometry shifts from a value of two in the dark to a value of one in the light. Moreover, this H+ pump shows the characteristics of either a pump or a passive H+ conductance: the mode of operation of the H+ translocator is considered to be regulated by the external pH. The pump conductance is assumed to be dominant at low or neutral pH, while the passive H+ conductance becomes more significant at alkaline pH. The pH dependence of the transport characteristic is expressed by protonation reactions in the plasma membrane. The proposed model can account for most features of the light-induced oscillations but not for the absolute level of the membrane potential.