Force–displacement measurements of earlywood bordered pits using a mesomechanical tester

The elastic properties of pit membranes are reported to have important implications in understanding air‐seeding phenomena in gymnosperms, and pit aspiration plays a large role in wood technological applications such as wood drying and preservative treatment. Here we present force–displacement measurements for pit membranes of circular bordered pits, collected on a mesomechanical testing system. The system consists of a quartz microprobe attached to a microforce sensor that is positioned and advanced with a micromanipulator mounted on an inverted microscope. Membrane displacement is measured from digital image analysis. Unaspirated pits from earlywood of never‐dried wood of Larix and Pinus and aspirated pits from earlywood of dried wood of Larix were tested to generate force–displacement curves up to the point of membrane failure. Two failure modes were observed: rupture or tearing of the pit membrane by the microprobe tip, and the stretching of the pit membrane until the torus was forced out of the pit chamber through the pit aperture without rupture, a condition we refer to as torus prolapse.     

Mechanism of water stress-induced xylem embolism

We investigated the hypothesis that water stress-induced xylem embolism is caused by air aspirated into functional vessels from neighboring embolized ones (e.g. embolized by physical damage) via pores in intervessel pit membranes. The following experiments with sugar maple (Acer saccharum Marsh.) support the hypothesis. (a) Most vessels in dehydrating stem segments embolized at xylem pressures < −3 megapascals; at this point the pressure difference across intervessel pits between air-filled vessels at the segment's ends and internal water-filled vessels was >3 megapascals. This same pressure difference was found to be sufficient to force air across intervessel pits from air injection experiments of hydrated stem segments. This suggests air entry at pits is causing embolism in dehydrating stems. (b) Treatments that increased the permeability of intervessel pits to air injection also caused xylem to embolize at less negative xylem pressures. Permeability was increased either by perfusing stems with solutions of surface tension below that of water or by perfusion with a solution of oxalic acid and calcium. The mechanism of oxalic-calcium action on permeability is unknown, but may relate to the ability of oxalate to chelate calcium from the pectate fraction of the pit membrane. (c) Diameter of pores in pit membranes measured with the scanning electron microscope were within the range predicted by hypothesis (≤0.4 micrometer).     

Contrasting hydraulic architecture and function in deep and shallow roots of tree species from a semi-arid habitat

Background and Aims

Despite the importance of vessels in angiosperm roots for plant water transport, there is little research on the microanatomy of woody plant roots. Vessels in roots can be interconnected networks or nearly solitary, with few vessel–vessel connections. Species with few connections are common in arid habitats, presumably to isolate embolisms. In this study, measurements were made of root vessel pit sizes, vessel air-seeding pressures, pit membrane thicknesses and the degree of vessel interconnectedness in deep (approx. 20 m) and shallow (<10 cm) roots of two co-occurring species, Sideroxylon lanuginosum and Quercus fusiformis.

Methods

Scanning electron microscopy was used to image pit dimensions and to measure the distance between connected vessels. The number of connected vessels in larger samples was determined by using high-resolution computed tomography and three-dimensional (3-D) image analysis. Individual vessel air-seeding pressures were measured using a microcapillary method. The thickness of pit membranes was measured using transmission electron microscopy.

Key Results

Vessel pit size varied across both species and rooting depths. Deep Q. fusiformis roots had the largest pits overall (>500 µm) and more large pits than either shallow Q. fusiformis roots or S. lanuginosum roots. Vessel air-seeding pressures were approximately four times greater in Q. fusiformis than in S. lanuginosum and 1·3–1·9 times greater in shallow roots than in deep roots. Sideroxylon lanuginosum had 34–44 % of its vessels interconnected, whereas Q. fusiformis only had 1–6 % of its vessels connected. Vessel air-seeding pressures were unrelated to pit membrane thickness but showed a positive relationship with vessel interconnectedness.

Conclusions

These data support the hypothesis that species with more vessel–vessel integration are often less resistant to embolism than species with isolated vessels. This study also highlights the usefulness of tomography for vessel network analysis and the important role of 3-D xylem organization in plant hydraulic function.     

Changes in the Distribution of Plasmodesmata in Developing Fibre-tracheid Pit Membranes of Sorbus aucuparia L.

The distribution of plasmodesmata in cambial pit fields anddeveloping fibre-tracheid pit membranes in Sorbus aucupariaL. has been studied using conventional and high voltage transmissionelectron microscopy. In cambial pit fields, plasmodesmata arewidely separated while, during cell enlargement and the stageof secondary wall formation, they occur exclusively in a denselypacked cluster, eccentrically located on the pit membrane ina thickening of primary wall material. It is not clear at themoment whether the cluster arises by aggregation of the cambialpit field plasmodesmata, or by de novo formation in a localizedregion coupled with loss of plasmodesmata outside this region.The significance of these changes is discussed. Sorbus aucuparia L., plasmodesmata, pit membranes, xylem differentiation     

Plasmodesmata and pit development in secondary xylem elements

Developing pit membranes of secondary xylem elements in Drimys winteri, Fagus sylvatica, Quercus robur, Sorbus aucuparia, Tilia vulgaris and Trochodendron aralioides have been examined by transmission electron microscopy. Absence of plasmodesmata from the membranes of vessel elements and tracheids indicates that their pits develop independently of these structures. On the other hand, plasmodesmata are abundant in pit membranes between fibres, parenchyma cells, and combinations of these cell types in Fagus, Quercus and Tilia. In each case the plasmodesmata pass right through the developing pit membrane. In the case of Sorbus fibres, however, plasmodesmata were absent from the majority of pit membrane profiles seen in sections. Occasionally they were observed in large numbers associated with a swollen region on one side of the pit membrane between fibres and between fibres and parenchyma, radiating from a small area of the middle lamella. In the case of fibre to parenchyma pitting, this swelling was always found on the fibre side of the membrane, while on the other side a small number of plasmodesmata were present completing communication with the parenchyma cytoplasm. These observations are discussed with regard to the role of plasmodesmata in pit formation, and in the differentiation of the various cell types in secondary xylem. The significance their distribution may have for our understanding of xylem evolution is also discussed.     

Modelling the mechanical behaviour of pit membranes in bordered pits with respect to cavitation resistance in angiosperms

Background and Aims

Various correlations have been identified between anatomical features of bordered pits in angiosperm xylem and vulnerability to cavitation, suggesting that the mechanical behaviour of the pits may play a role. Theoretical modelling of the membrane behaviour has been undertaken, but it requires input of parameters at the nanoscale level. However, to date, no experimental data have indicated clearly that pit membranes experience strain at high levels during cavitation events.

Methods

Transmission electron microscopy (TEM) was used in order to quantify the pit micromorphology of four tree species that show contrasting differences in vulnerability to cavitation, namely Sorbus aria, Carpinus betulus, Fagus sylvatica and Populus tremula. This allowed anatomical characters to be included in a mechanical model that was based on the Kirchhoff–Love thin plate theory. A mechanistic model was developed that included the geometric features of the pits that could be measured, with the purpose of evaluating the pit membrane strain that results from a pressure difference being applied across the membrane. This approach allowed an assessment to be made of the impact of the geometry of a pit on its mechanical behaviour, and provided an estimate of the impact on air-seeding resistance.

Key Results

The TEM observations showed evidence of residual strains on the pit membranes, thus demonstrating that this membrane may experience a large degree of strain during cavitation. The mechanical modelling revealed the interspecific variability of the strains experienced by the pit membrane, which varied according to the pit geometry and the pressure experienced. The modelling output combined with the TEM observations suggests that cavitation occurs after the pit membrane has been deflected against the pit border. Interspecific variability of the strains experienced was correlated with vulnerability to cavitation. Assuming that air-seeding occurs at a given pit membrane strain, the pressure predicted by the model to achieve this mechanical state corresponds to experimental values of cavitation sensitivity (P₅₀).

Conclusions

The results provide a functional understanding of the importance of pit geometry and pit membrane structure in air-seeding, and thus in vulnerability to cavitation.     

Fluorescence correlation spectroscopy reveals topological segregation of the two tumor necrosis factor membrane receptors

The proinflammatory cytokine tumor necrosis factor (TNF) binds two distinct plasma membrane receptors, TNFR1 and TNFR2. We have produced different receptor mutants fused with enhanced green fluorescent protein to study their membrane dynamics by fluorescence correlation spectroscopy (FCS). TNFR1 mutants show diffusion constants of approximately 1.2 × 10^− 9 cm²/s and a broad distribution of diffusion times, which is hardly affected by ligand binding. However, cholesterol depletion enhances their diffusion, suggesting a constitutive affinity to cholesterol rich membrane microdomains. In contrast, TNFR2 and mutants thereof diffuse rather fast (D? = 3.1 × 10^− 9 cm²/s) with a marked reduction after 30 min of TNF treatment (D? = 0.9 × 10^− 9 cm²/s). This reduction cannot be explained by the formation of higher ordered receptor clusters, since the fluorescence intensity of TNF treated receptors indicate the presence of a few receptor molecules per complex only. Together, these data point to a topological segregation of the two TNF receptors in different microcompartments of the plasma membrane independent of the cytoplasmic signaling domains of the receptors.     

New Membrane Concept Applied to the Analysis of Fluid Shear- and Micropipette-Deformed Red Blood Cells

A two-dimensional elastomer material concept of the red cell membrane is applied to the analysis of fluid shear-deformed, point-attached red cells and micropipette aspiration of red cell disks. The elastic constant (corresponding to the “shear” modulus multiplied by the membrane thickness) is of the order 10^-2 dyn/cm for both cases. Additional experimental observations are in agreement with the membrane model, e.g. teardrop and “tether” formation of the sheared disks, pressure difference vs. aspirated length of the cell for micropipette experiments, etc     

Anion-Sensitive, H-Pumping ATPase of Oat Roots : Direct Effects of Cl, NO(3), and a Disulfonic Stilbene

To understand the mechanism and molecular properties of the tonoplast-type H⁺-translocating ATPase, we have studied the effect of Cl⁻, NO₃⁻, and 4,4′-diisothiocyano-2,2′-stilbene disulfonic acid (DIDS) on the activity of the electrogenic H⁺-ATPase associated with low-density microsomal vesicles from oat roots (Avena sativa cv Lang). The H⁺-pumping ATPase generates a membrane potential (Δψ) and a pH gradient (ΔpH) that make up two interconvertible components of the proton electrochemical gradient (μh⁺). A permeant anion (e.g. Cl⁻), unlike an impermeant anion (e.g. iminodiacetate), dissipated the membrane potential ([¹⁴C]thiocyanate distribution) and stimulated formation of a pH gradient ([¹⁴C]methylamine distribution). However, Cl⁻-stimulated ATPase activity was about 75% caused by a direct stimulation of the ATPase by Cl⁻ independent of the proton electrochemical gradient. Unlike the plasma membrane H⁺-ATPase, the Cl⁻-stimulated ATPase was inhibited by NO₃⁻ (a permeant anion) and by DIDS. In the absence of Cl⁻, NO₃⁻ decreased membrane potential formation and did not stimulate pH gradient formation. The inhibition by NO₃⁻ of Cl⁻-stimulated pH gradient formation and Cl⁻-stimulated ATPase activity was noncompetitive. In the absence of Cl⁻, DIDS inhibited the basal Mg,ATPase activity and membrane potential formation. DIDS also inhibited the Cl⁻-stimulated ATPase activity and pH gradient formation. Direct inhibition of the electrogenic H⁺-ATPase by NO₃⁻ or DIDS suggest that the vanadate-insensitive H⁺-pumping ATPase has anion-sensitive site(s) that regulate the catalytic and vectorial activity. Whether the anion-sensitive H⁺-ATPase has channels that conduct anions is yet to be established.     

The Kinetics of Osmotic Transport through Pores of Molecular Dimensions

This paper presents a theoretical analysis of the kinetics of osmotic transport across a semipermeable membrane. There is a thermodynamic connection between the rate of flow under a hydrostatic pressure difference and the rate of flow due to a difference in solute concentration on the two sides. One might therefore attempt to calculate the osmotic transport coefficient by applying Poiseuille's equation to the flow produced by a difference in hydrostatic pressure. Such a procedure is, however, inappropriate if the pores in the membrane are too small to allow molecules to “overtake.” It then becomes necessary to perform a statistical calculation of the transport coefficient, and such a calculation is described in this paper. The resulting expression for the number of solvent molecules passing through a pore per second is J = m D₁ δn₁/l² where m is the number of solvent molecules in the pore, l is the length of the pore, D₁ is the self-diffusion coefficient of the solute, and δn₁ the difference in solvent mole fraction on the two sides of the membrane. This equation is used for estimating the number of pores per unit area of the squid axon membrane; the result is 6 × 10⁹ pores/cm².     

Inter-vessel pitting and cavitation in woody Rosaceae and other vesselled plants: a basis for a safety versus efficiency trade-off in xylem transport

The hypothesis that greater safety from cavitation by air-seeding through inter-vessel pits comes at the cost of less porous pit membranes with greater flow resistance was tested . Sixteen vessel-bearing species were compared: 11 from the Rosaceae, four from other angiosperm families, and one fern. Unexpectedly, there was no relationship between pit resistance (and hence the prevailing membrane porosity) and cavitation pressure. There was, however, an inverse relationship between pit area per vessel and vulnerability to cavitation (r² = 0.75). This suggests that cavitation is caused by the rare largest membrane pore per vessel, the average size of which increases with total pit area per vessel. If safety from cavitation constrains pit membrane surface area, it also limits vessel surface area and the minimum vessel resistivity. This trade-off was consistent with an approximately three-fold increase in vessel resistivity with cavitation pressure dropping from −0.8 to −6.6 MPa. The trade-off was compensated for by a reduction in the percentage of vessel wall pitted: from 10–16% in vulnerable species to 2–4% in resistant species. Across species, end-wall pitting accounted for 53 ± 3% of the total xylem resistivity. This corresponded to vessels achieving on average 94 ± 2% of their maximum possible conductivity if vessel surface area is constrained.     

Electrostatic Changes in Lycopersicon esculentum Root Plasma Membrane Resulting from Salt Stress

Salinity-induced alterations in tomato (Lypersicon esculentum Mill. cv Heinz 1350) root plasma membrane properties were studied and characterized using a membrane vesicle system. Equivalent rates of MgATP-dependent H⁺-transport activity were measured by quinacrine fluorescence (ΔpH) in plasma membrane vesicles isolated from control or salt-stressed (75 millimolar salt) tomato roots. However, when bis-[3-phenyl-5-oxoisoxazol-4-yl] pentamethine was used to measure MgATP-dependent membrane potential (ΔΨ) formation, salt-stressed vesicles displayed a 50% greater initial quench rate and a 30% greater steady state quench than control vesicles. This differential probe response suggested a difference in surface properties between control and salt-stressed membranes. Fluorescence titration of vesicles with the surface potential probe, 8-anilino-1-napthalenesulphonic acid (ANS) provided dissociation constants (K_d) of 120 and 76 micromolar for dye binding to control and salt-stressed vesicles, respectively. Membrane surface potentials (Ψ_o) of−26.0 and −13.7 millivolts were calculated for control and salt-stressed membrane vesicles from the measured K_d values and the calculated intrinsic affinity constant, K_i. The concentration of cations and anions at the surface of control and salt-stressed membranes was estimated using Ψ_o values and the Boltzmann equation. The observed difference in membrane surface electrostatic properties was consistent with the measured differences in K⁺-stimulated kinetics of ATPase activity between control and salt-stressed vesicles and by the differential ability of Cl⁻ ions to stimulate H⁺-transport activity. Salinity-induced changes in plasma membrane electrostatic properties may influence ion transport across the plasma membrane.     

Water and ion permeability of a native collagen membrane

With the help of a ribonucleoprotein it is possible to precipitate collagen in a layer of fibers with a 700 Å period. As collagen is a constituent of many membrane systems in the body, it seemed interesting to investigate the permeability of ions and water through a native collagen membrane.The experiments were carried out with the help of an acryl glass apparatus, where an osmotic pressure, a hydrostatic pressure difference or both can be maintained between the two bulk phases separated by the membrane. The diffusion coefficients for NaCl and KCl were found to be comparable with those in other biological membranes (D_s = 9 · 10⁻⁷cm² · s⁻¹) whereas there is difference of more than three orders of magnitude in the hydraulic permeability (L_p = 6 cm⁴ · J⁻¹ · s⁻¹).Volume flow measurements caused by an osmotic gradient indicated that the reflection coefficient for NaCl and KCl is very small. In hydrostatic pressure experiments, the membrane shows a preferred direction for volume flows which seems to have something to do with the mode of preparation of the membrane.     

Electrical properties of chloride transport across theNecturus proximal tubule

Summary The chloride conductance of the basolateral cell membrane of theNecturus proximal tubule was studied using conventional and chloride-sensitive liquid ion exchange microelectrodes. Individual apical and basolateral cell membrane and shunt resistances, transepithelial and basolateral, cell membrane potential differences, and electromotive forces were determined in control and after reductions in extracellular Cl^–. When extracellular Cl^– activity is reduced in both apical and basolateral solutions the resistance of the shunt increases about 2.8 times over control without any significant change in cell membrane resistances. This suggests a high Cl^– conductance of the paracellular shunt but a low Cl^– conductance of the cell membranes. Reduction of Cl^– in both bathing solutions or only on the basolateral side hyperpolarizes both the basolateral cell membrane potential difference and electromotive force. Hyperpolarization of the basolateral cell membrane potential difference after low Cl^– perfusion was abolished by exposure to HCO ₃ ^– -free solutions and SITS treatment. In control conditions, intracellular Cl^– activity was significantly higher than predicted from the equilibrium distribution across both the apical and basolateral cell membranes. Reducing Cl^– in only the basolateral solution caused a decrease in intracellular Cl^–. From an estimate of the net Cl^– flux across the basolateral cell membrane and the electrochemical driving force, a Cl^– conductance of the basolateral cell membrane was predicted and compared to measured values. It was concluded that the Cl^– conductance of the basolateral cell membrane was not large enough to account for the measured flux of Cl^– by electrodiffusion alone. Therefore these results suggest the presence of an electroneutral mechanism for Cl^– transport across the basolateral cell membrane of theNecturus proximal tubule cell.     

Phenylalanine uptake in isolated renal brush border vesicles

The uptake of l-phenylalanine into brush border microvilli vesicles and basolateral plasma membrane vesicles isolated from rat kidney cortex by differential centrifugation and free flow electrophoresis was investigated using filtration techniques.Brush border microvilli but not basolateral plasma membrane vesicles take up l-phenylalanine by an Na⁺-dependent, saturable transport system. The apparent affinity of the transport system for l-phenylalanine is 6.1 mM at 100 mM Na⁺ and for Na⁺ 13 mM at 1 mM l-phenylalanine. Reduction of the Na⁺ concentration reduces the apparent affinity of the transport system for l-phenylalanine but does not alter the maximum velocity.In the presence of an electrochemical potential difference for Na⁺ across the membrane (η_Na0 >η_Na1) the brush border microvilli accumulate transiently l-phenylalanine over the concentration in the incubation medium (overshoot phenomenon). This overshoot and the initial rate of uptake are markedly increased when the intravesicular space is rendered electrically more negative by membrane diffusion potentials induced by the use of highly permeant anions, of valinomycin in the presence of an outwardly directed K⁺ gradient and of carbonyl cyanide p-trifluoromethoxyphenylhydrazone in the presence of an outward-directed proton gradient.These results indicate that the entry of l-phenylalanine across the brush border membrane into the proximal tubular epithelial cells involves cotransport with Na⁺ and is dependent on the concentration difference of the amino acid, on the concentration difference of Na⁺ and on the electrical potential difference. The exit of l-phenylalanine across the basolateral plasma membranes is Na⁺-independent and probably involves facilitated diffusion.     

The transport kinetics of lanthanide species in a single erythrocyte probed by confocal laser scanning microscopy

 A novel method has been developed to visualize and follow the temporal course of lanthanide transport across the membrane into a single living erythrocyte. By means of confocal scanning microscopy and the optical section technique, the entry of lanthanide ions was followed by the fluorescence quenching of fluorescein isothiocyanate (FITC)-labeled membrane and cytosol. From the difference of the quenching kinetics of the whole section and the central area, the time for diffusion through the membrane and the diffusion in the extracellular and intracellular media can be deduced. To clarify the mechanism of lanthanide-induced fluorescence quenching of FITC-labeled erythrocytes and to ensure that this reaction can be used in this method, the reaction was investigated by steady-state fluorescence techniques. The results showed that the lanthanides strongly quenched the florescence emitted by FITC covalently bound to membrane proteins and cytosolic proteins. The static quenching mechanism is responsible for the fluorescence quenching of FITC-labeled proteins by Ln species. The quenching mechanism is discussed on the basis of complex formation. The dependence of fluorescence quenching on both ion size and the total orbital angular momentum L supports the complexation mechanism. The transport time across the membrane is strikingly correlated with Ln species and extracellular concentration. For a given concentration, the transport time of [Ln(cit)₂]^3– is much shorter than that of Ln³⁺, since they enter the cells via the anion channel. This is supported by the inhibition effect of 4,4′-diisothiocyanato-2,2′-stilbenendisulfonate on the transport of [Ln(cit)₂]^3–. On the other hand, the transport of free Ln³⁺ might be attributed to the enhanced permeability of erythrocytes owing to Ln³⁺ binding. These findings strongly demonstrate the existence of the non-internalization mechanism of Ln species uptake by erythrocytes. Received: 7 January 1999 / Accepted: 7 May 1999     

Probing the role of active site histidine residues in the catalytic activity of lacrimal gland peroxidase

The role of active site histidine residues in SCN^– oxidation by lacrimal gland peroxidase (LGP) has been probed after modification with diethylpyrocarbonate (DEPC). The enzyme is irreversibly inactivated following pseudo-first order kinetics with a second order rate constant of 0.26 M^–1 sec^–1 at 25°C. The pH dependent rate of inactivation shows an inflection point at 6.6 indicating histidine derivatization. The UV difference spectrum of the modified versus native enzyme shows a peak at 242 nm indicating formation of N-carbethoxyhistidine. Carbethoxyhistidine formation and associated inactivation are reversed by hydroxylamine indicating histidine modification. The stoichiometry of histidine modification and the extent of inactivation show that out of five histidine residues modified, modification of two residues inactivates the enzyme. Substrate protection with SCN^– during modification indicates that although one histidine is protected, it does not prevent inactivation. The spectroscopically detectable compound II formation is lost due to modification and is not evident after SCN^– protection. The data indicate that out of two histidines, one regulates compound I formation while the other one controls SCN^– binding. SCN^– protected enzyme is inactive due to loss of compound I formation. SCN^– binding studies by optical difference spectroscopy indicate that while the native enzyme binds SCN^– with the K_d of 15 mM, the modified enzyme shows very weak binding with the K_d of 660 mM. From the pH dependent binding of SCN^–, a plot of log K_d vs. pH shows a sigmoidal curve from which the involvement of an enzyme ionizable group of pKa 6.6 is ascertained and attributed to the histidine residue controlling SCN^– binding. LGP has thus two distinctly different essential histidine residues – one regulates compound I formation while the other one controls SCN^– binding.     

Properties and new methods of non-equilibrium membrane bound proton fraction research under conditions of proton pump activation

Under the conditions of low-amplitude mitochondrial swelling, the oxidative phosphorylation system functions in a local coupling mode postulated by Williams in 1961. The proton pumps activation leads to the formation of non-equilibrium membrane bounded proton fraction (nef-H⁺), which is sorbed on the outer side of the inner mitochondrial membrane under these conditions. This proton fraction is crucial for the ATP synthesis. The present work is devoted to the development of the methods allowing investigations of the properties of nef-H⁺. For this purpose, a new membranotropic highly hydrophobic uncoupler 2,4,6-trichloro-3-pentadecylphenol was synthesized. In accordance with our results, it can be referred to as a new type of transmemebrane proton carriers, which interact specifically only with nef-H⁺ outer side of the inner membrane. A new method of the nef-H⁺ removal from the outer side of the inner mitochondrial membrane under the conditions when the proton pumps are active has been developed. The method is based on neutralization of nef-H⁺ by hydroxyl anions transferred by H₂PO₄⁻/OH⁻-antiporter to interfacial border. It was demonstrated that the membrane proton fraction is heterogeneous. Two components of the fraction were identified. And at the same time, it was shown that nucleotide translocator participates in the formation of one component. Also, in this series of experiments it was found that a well-known effect of respiration inhibition by high concentrations of uncoupler under certain conditions can be completely explained by the nef-H⁺ formation. We presume that one of the most important results of the studies carried out is a new independent evidence of the existence of nef-H⁺, which is formed under the conditions of proton pump activation.     

Electrogenic and diffusive components of the membrane ofHydrodictyon africanum

Summary In cells of the freshwater algaHydrodictyon africanum, in solutions where [K⁺]₀=0.1mm and pH₀>7.0, the membrane in the light is hyperpolarized. The membrane potential difference {ie179-1} has values from –180 to –275 mV, more negative than any ion diffusion potential difference, and is predominantly a function of pH₀, and independent of [K⁺]₀. The hyperpolarization of the membrane appears to arise from an electrogenic efflux of H⁺, estimated from voltage-clamp data to be about 8 nmol m^–2 sec^–1 when pH₀=8.5. In the light the membrane conductanceg _m is about 0.084 S m^–2. At light-off, {ie179-2} becomes less negative, with a halftime for change of 15 to 30 sec andg _m decreases by about 0.052 S m^–2. After dark periods of up to 300 sec, {ie179-3} is largely independent of pH₀ for values greater than 6.0 and usually behaves as a combined K⁺ and Na⁺ diffusion potential with permeability ratioP _Na/P _K=0.05 to 0.2. The membrane potassium conductanceg _K has either a low value of 2–6×10^–2 Sm^–2, or a high value of up to 18×10^–2 S m^–2 depending on [K⁺]₀, the transition from low to high values occurring when {ie179-4} moves over a threshold value that is more negative than {ie179-5}, the electrochemical equilibrium potential for K⁺. The time for half-change of the transition is about 30 sec. The results are consistent with a model of the membrane in which the pump electromotive force and conductance are in parallel with diffusive electromotive forces and conductances. When the pump is operating its properties determine membrane properties, and when it is inoperative, or running at a diminished rate, the membrane properties are determined more by the diffusive pathways. Changes in both pump rate andg _K can account for a variety of characteristic changes in membrane PD and conductance occurring in response to ligh-dark changes, changes in light intensity, pasage of externally applied electric current across the membrane and changes in ionic constituents of the external medium.     

The Ca-Transport ATPase of Plant Plasma Membrane Catalyzes a nH/Ca Exchange

Microsomal vesicles from 24-hour-old radish (Raphanus sativus L.) seedlings accumulate Ca²⁺ upon addition of MgATP. MgATP-dependent Ca²⁺ uptake co-migrates with the plasma membrane H⁺-ATPase on a sucrose gradient. Ca²⁺ uptake is insensitive to oligomycin, inhibited by vanadate (IC₅₀ 40 micromolar) and erythrosin B (IC₅₀ 0.2 micromolar) and displays a pH optimum between pH 6.6 and 6.9. MgATP-dependent Ca²⁺ uptake is insensitive to protonophores. These results indicate that Ca²⁺ transport in these microsomal vesicles is catalyzed by a Mg²⁺-dependent ATPase localized on the plasma membrane. Ca²⁺ strongly reduces ΔpH generation by the plasma membrane H⁺-ATPase and increases MgATP-dependent membrane potential difference (Δψ) generation. These effects of Ca²⁺ on ΔpH and Δψ generation are drastically reduced by micromolar erythrosin B, indicating that they are primarily a consequence of Ca²⁺ uptake into plasma membrane vesicles. The Ca²⁺-induced increase of Δψ is collapsed by permeant anions, which do not affect Ca²⁺-induced decrease of ΔpH generation by the plasma membrane H⁺-ATPase. The rate of decay of MgATP-dependent ΔpH, upon inhibition of the plasma membrane H⁺-ATPase, is accelerated by MgATP-dependent Ca²⁺ uptake, indicating that the decrease of ΔpH generation induced by Ca²⁺ reflects the efflux of H⁺ coupled to Ca²⁺ uptake into plasma membrane vesicles. It is therefore proposed that Ca²⁺ transport at the plasma membrane is mediated by a Mg²⁺-dependent ATPase which catalyzes a nH⁺/Ca²⁺ exchange.