Flexible Coupling of Phosphate Uptake in Dunaliella acidophila at Extremely Low pH Values

The acidophilic alga Dunaliella acidophila exhibits optimalgrowth at pH 1. We have investigated the regulation of phosphateuptake by this alga using tracer techniques and by performingintracellular phosphate measurements under different growthconditions including phosphate limitation. In batch culturewith 2·2 mol m^–3 phosphate in the medium the uptakeof phosphate at micromolar phosphate concentrations followeda linear time dependence in the range of minutes and rates werein the range of 1 µmol phosphate mg^–1 chl h^–1,only. However, under discontinuous phosphate-limited growthconditions, tracer influx revealed a biphasic pattern at micromolarphosphate concentrations: An initial burst phase resulted ina 10⁴-fold internal phosphate accumulation and levelled offafter about 10 s. A double reciprocal plot of the initial influxrates obtained for phosphate-limited and unlimited algae exhibitedMichaelis-Menten kinetics. Phosphate limitation caused a significantactivation of the maximum velocity of uptake, yielding V_maxup to 1 mmol mg^–1 chl h^–1 as compared to valuesin the order of 50 µmol phosphate mg^–1 chl h^–1for the second phase (this magnitude is also representativefor non-limited batch cultures). Concomitantly the Michaelisconstant was altered from 4 mmol m^–3 to 0·7 mmolm^–3. The rapid uptake of phosphate was inhibited by arsenateand FCCP and was not stimulated by Na⁺. The pH dependence oftracer accumulation and measurements of the intracellular phosphatepool under different growth conditions indicate that at lowpH and low external phosphate concentrations the high protongradient present under these conditions is utilized for a H₃PO₄uptake or a H⁺/H₂PO^–4 cotransport. However, when the externalphosphate concentration was increased to levels sufficientlyhigh for transport to be driven by the positive membrane potential(10 mol m^–3 phosphate), the pH dependence of phosphateuptake was more complex, but could be explained by the uptakeof H₃PO₄ or a H⁺/H₂PO^–₄-cotransport at low pH and a differenttype H₂PO^–4-transport (with unknown type of ion coupling)at high pH-values. It is suggested that this flexible couplingof phosphate transport is of essential importance for the acidresistance of Dunaliella acidophila. Key words: Acid resistance, Dunaliella acidophila, phosphate cotransport, phosphate limitation, plasma membrane, sodium     

Proton Transport and pH Control in Sinapis alba Root Hairs: A Study carried out with Double-Barrelled pH Micro-electrodes

Continuous measurements of cytoplasmic pH (pH_c) in Sinapis roothairs have been carried out with double-barrelled pH-micro-electrodesin order to gain information on translocation of protons acrossthe plasmalemma and cytoplasmic pH control. (i) The cytoplasmicpH of Sinapis (7–33 ? 0–12, standard conditions)changes no more than 0.1 pH_c, per pH_o-unit, regardless of whethercyanide is present or not. (ii) Weak acids rapidly acidify pH_cand hyperpolarize, while weak bases alkalize pH_c and depolarizethe cells, (iii) 1.0 mol M,³ NaCN acidifies the cytoplasm by0.4 to 0.7 pH-units, but alkalizes the vacuole. (iv) 20 mmolm^–3 CCCP has no significant effect on pH_c, if added atpH 9.6 or 7.2, but acidifies pH_c by 1.3 units at pH 4.3. Inthe presence of CCCP, cyanide acidifies the cytoplasm, (v) Chloridetransiently acidifies pH_c, while K⁺, Na⁺, and have no significant effects, (vi) Cytoplasmic buffer capacityforms a bell-shaped curve versus pH_c with an optimum of about50 mol m^–3 H⁺pH_c-unit. The modes of proton re-entry and the effects of active and passiveproton transport on cellular pH control are critically discussed.It is suggested that the proton leak, consisting of H⁺-cotransport(e.g. H⁺/Cl^–) rather than H⁺-uniport, is no threat topH_c. The proton export pump, although itself reacting to changesin pH_c, influences pH_c only to a minor extent. It is concludedthat buffer capacity and membrane transport play moderate rolesin pH_c control in Sinapis, while the interlocked H⁺-producingand -consuming reactions of cellular metabolism are the mainregulating factors. This makes pH control in Sinapis quite differentfrom bacterial and animal cells. Key words: Cytoplasmic pH, double-barrelled pH micro-electrode, pH control, proton transport, Sinapis     

Modifying Effects of Non-toxic Levels of Aluminium on the Uptake and Transport of Phosphate in Ryegrass

Apparent uptake and transport of H₂³²PO₄^– from nutrientsolutions containing 100 mmol m^–3 phosphate were characterizedasfunctions of time, concentration and pH in ryegrass seedlings.On a log/log plot, concentration versus uptake to the root resolvedintotwo linear phases, suggesting a change in uptake mechanism orefflux at the break. These results were compared with thosefor ³²P uptake and transport in solutions containing Al rangingfrom 0–185 mmol m^–3. Al addition depressed pH, butbecauseuptake of P was unaffected by pH below 5–0, noadjustments were attempted. Uptake time-courses revealed clearlythe usualinitial adsorption shoulder in the uptake curve, increasingwith Al concentration up to 37 mmol m^–3. Beyond about2 h, P uptaketo the root became linear, at rates increasingwith external Al concentration up to 37 mmol m^–3. Concentrationsof Al muchabove 100 mmol m^–3 were toxic. Al treatmentsdid not affect P transport to the shoot and absorbed Al wasconfined to the root.The quantities of P and Al taken up intothe root indicated storage in cortex cell vacuoles, lockingup significant amounts of P.Experiments with tillering plantsshowed similar characteristics to those with seedlings. Sequesteringof P with Al within the rootcortex cells was evident, particularlyin plants which had been grown in nutrient containing Al fromsoon after germination. Aland P solution chemistry is discussedin the context of this work and the consequences of effectson P uptake for the economy ofphosphate poor upland soils wereconsidered. Key words: Phosphate, aluminium, adsorption, uptake, Lolium perenne L     

Ion Composition of the Chara Internode

Ion compositions of the cytoplasm and the vacuole of Chara australiswere analyzed according to Kishimoto and Tazawa (1964) and Kiyosawa(1979a). The ions in the cytoplasm and the vacuole analyzedwere K⁺, Na⁺, Ca²⁺, Mg²⁺, Cl^–, NO₃^– and H₂PO₄^–.Assuming that the volume of the cytoplasm V_p is 10% of thatof the whole cell V, the concentrations of K⁺, Na⁺, Ca²⁺, Mg²⁺,Cl^–, NO₃^– and H₂PO₄^– in the cytoplasm averaged70, 15, 13, 4.6, 31, 2.2 and 16 mM, respectively. If the volumeof the cytoplasm was assumed to be 5% of that of the whole cell,their averaged concentrations were 139, 31, 25, 9.2, 62, 4.4and 33 mM, respectively. The averaged ion compositions of thecell sap were K⁺, 111; Na⁺, 47; Ca²⁺, 4.4; Mg²⁺, 8.9; Cl^–,91; NO^–₃, 3.3 and H₂PO₄^–, 6.0 mM. These values,taking the concentrations and the charges of the protein (Kiyosawa1979b) and amino acids (Sakano and Tazawa 1984) into accountand assuming the presence of some uni- or oligovalent anionsand/or small nonelectrolyte molecules, could explain fairlywell both the electroneutrality and the osmotic pressure ofthe cell, except when V_p/V = 5%. (Received May 18, 1987; Accepted September 29, 1987)     

Growth and Phosphate Transport in Barley and Tomato Plants During the Development of, and Recovery from, Phosphate-stress

Barley and tomato plants were cultured in nutrient solutionsincluding 0.15 mol m^–3 H²PO^–₄. The phosphate supplywas discontinued and the subsequent effects on growth, internalphosphorus concentrations, phosphate absorption and translocationwere measured at frequent intervals. Growth rates were at firstunchanged and the internal phosphorus concentration decreased.During this phase the rate of phosphate transport by the rootssometimes increased significantly. Growth slowed more in shootsthan in roots during a second phase of stress development andvisual symptoms of deficiency appeared in tomato but not inbarley. During this phase, enhancement of phosphate uptake capacityreached a maximum in both species. The subsequent decline inuptake capacity was associated with visible symptoms of deficiencydeveloping in barley and intensifying in tomato. When stressedplants were returned to a solution containing 0.15 mol m^–3H₂PO^–₄ rapid absorption continued for several days afterthe internal phosphorus concentration had returned to the levelof the controls. Phosphate toxicity may have been the causeof leaf lesions and necrosis during the ‘recovery’phase. Stomatal conductance in tomato was decreased at an early stageof stress development. Foliar-applied phosphate was absorbedmore rapidly by P-stressed barley leaves than by their controlsand much larger amounts were translocated from the leaves tothe roots.     

Ion Fluxes in 'Isolated' Guard Cells of Commelina communis L.

Ion fluxes have been measured in ‘isolated’ guardcells of Commelina communis L. using ⁸⁶RbCl and K⁸²Br, in epidermalstrips in which all cells other than guard cells have been killedby treatment at low pH. To avoid problems of slow free spaceexchange most fluxes have been measured at pH 3.9, at whichstomata open well in K(Rb) Cl(Br) and are stable for many hours.At pH 3.9 the intracellular ⁸⁶Rb exchanged as a single compartmentwith a half-time of 2–3 h, independent of external concentration(C_o). The influx of ⁸⁶Rb rose with concentration, to a V_maxof about 23 pmol mm^–2 h^–1. The efflux curve of ⁸²Brcould be well fitted by two exponential terms, with half-timesof 38 min (independent of C_o), and 5–35 h (falling withincreasing C_o). Bromide contents of cytoplasm and vacuole (Q_cand Q_v), and fluxes at plasmalemma and tonoplast, were calculatedfrom the efflux kinetics. Over C_o 20–60 mM, as the apertureincreased from 7 µm to 17 µm, the tonoplast flux(0.5–11.5 pmol mm^–2h^–1) was always much lessthan the plasmalemma flux (7–77 pmol mm^–2 h^–1).Q_c and Q_v both increased with aperture. The increase in Q_c of10.3 pmol mm^–2 µm^–1 is adequate to accountfor the osmotic changes required to change the aperture, aspreviously estimated. However, the change in vacuolar contentof only 5.9 pmol mm^–2 µm^–1 is much too smallto account for the osmotic changes required, or to balance thecytoplasmic changes. It appears therefore that increasing KBroutside not only increases the cytoplasmic salt content, andthe Br flux at the tonoplast, but also stimulates the vacuolaraccumulation of some other solute.     

Light-Induced H+ Accumulation in the Vacuole of Nitellopsis obtusa

The effects of light on the pH in the vacuole and the electricpotential difference across the plasmalemma and the tonoplastof Nitellopsis obtusa were investigated by means of conventionaland H⁺-specific glass or antimony microelectrodes. Illuminationis found to bring about a decrease in the pH of the vacuolarsap by 0.1–0.5 units concomitant with a depolarizationof the cell. The light-induced changes of the potential differenceand the vacuolar pH depend in different ways on the pH of theexternal medium (pH_o). At pH_o 9.0 cells exhibit great light-inducedpotential changes (up to 100 mV), but only small pH changesof the vacuolar sap. At neutral or slightly acidic pH_o valuesthe amplitude of the light-induced pH changes in the vacuoleincreases up to 0.3–0.5 pH units, but the amplitudes ofthe potential changes at the plasmalemma are relatively small.At pH_o 9.0 a transient acidification of the medium is observedupon illumination whereas at lower pH values light-induced alkalinizationwas only seen. Transfer of the cells from pH_o 9.0 to pH_o 7.5results in a cell hyperpolarization by 60–80 mV and adecrease of the vacuolar pH by 0.4–0.5 units under lightconditions but has no significant effect on the potential andthe vacuolar pH in the darkness. It is proposed that mechanismsof active H⁺ extrusion from the cytoplasm are located both inthe plasmalemma and the tonoplast. The observed acidificationin the vacuole appears to be determined by a light-induced increaseof the concentration of H⁺ in the cytoplasm. The H⁺ conductionof the plasmalemma seems to increase on illumination. The patternof the light-induced H⁺ fluxes across the tonoplast and theplasmalemma depends crucially on the extent of the light-inducedchanges in the H⁺ conductance and on the electrochemical gradientfor H⁺ at the plasmalemma.     

Ca2+ influx through alpha1S DHPR may play a role in regulating Ca2+ release from RyR1 in skeletal muscle

Skeletal muscle fiber types differ in their contents of total phosphate, which includes inorganic phosphate (P_i) and high-energy organic pools of ATP and phosphocreatine (PCr). At steady state, uptake of P_i into the cell must equal the rate of efflux, which is expected to be a function of intracellular P_i concentration. We measured ³²P-labeled P_i uptake rates in different muscle fiber types to determine whether they are proportional to cellular P_i content. P_i uptake rates in isolated, perfused rat hindlimb muscles were linear over time and highest in soleus (2.42 ± 0.17 µmol·g^–1·h^–1), lower in red gastrocnemius (1.31 ± 0.11 µmol·g^–1·h^–1), and lowest in white gastrocnemius (0.49 ± 0.06 µmol·g^–1·h^–1). Reasonably similar rates were obtained in vivo. P_i uptake rates at plasma P_i concentrations of 0.3–1.7 mM confirm that the P_i uptake process is nearly saturated at normal plasma P_i levels. P_i uptake rate correlated with cellular P_i content (r = 0.99) but varied inversely with total phosphate content. Sodium-phosphate cotransporter (PiT-1) protein expression in soleus and red gastrocnemius were similar to each other and seven- to eightfold greater than PiT-1 expression in white gastrocnemius. That the PiT-1 expression pattern did not match the pattern of P_i uptake across fiber types implies that other factors are involved in regulating P_i uptake in skeletal muscle. Furthermore, fractional turnover of the cellular P_i pool (0.67, 0.57, and 0.33 h^–1 in soleus, red gastrocnemius, and white gastrocnemius, respectively) varies among fiber types, indicating differential management of intracellular P_i, likely due to differences in resistance to P_i efflux from the fiber. inorganic phosphate; sodium-inorganic phosphate transporters; PiT-2; inorganic phosphate efflux     

Phosphate Uptake in the Cyanobacterium Synechococcus R-2 PCC 7942

Phosphate uptake rates in Synechococcus R-2 in BG-11 media (anitrate-based medium, not phosphate limited) were measured usingcells grown semi-continuously and in continuous culture. Netuptake of phosphate is proportional to external concentration.Growing cells at pH_o 10 have a net uptake rate of about 600pmol m^–2 s^–1 phosphate, but the isotopic flux for³²P phosphate was about 4 nmol m^–2 s^–1. There appearsto be a constitutive over-capacity for phosphate uptake. TheK_m and V_max, of the saturable component were not significantlydifferent at pH_o 7.5 and 10, hence the transport system probablyrecognizes both H₂PO^–₄and HPO^2–₄. The intracellularinorganic phosphate concentration is about 3 to 10 mol m^–3,but there is an intracellular polyphosphate store of about 400mol m^–3. Intracellular inorganic phosphate is 25 to 50kJ mol^–1 from electrochemical equilibrium in both thelight and dark and at pH_o 7.5 and 10. Phosphate uptake is veryslow in the dark ( 100 pmol m^–2 s^–1) and is light-activated(pH_o 7.51.3 nmol m^–2 s^–1, pH_o 10600 pmol m^–2s^–1). Uptake has an irreversible requirement for Mg²⁺in the medium. Uptake in the light is strongly Na⁺-dependent.Phosphate uptake was negatively electrogenic (net negative chargetaken up when transporting phosphate) at pH_o 7.5, but positivelyelectrogenic at pH_o 10. This seems to exclude a sodium motiveforce driven mechanism. An ATP-driven phosphate uptake mechanismneeds to have a stoichiometry of one phosphate taken up perATP (1 PO_{4 in}/ATP) to be thermodynamically possible under allthe conditions tested in the present study. (Received June 16, 1997; Accepted September 4, 1997)     

Phosphate uptake and transport in Agrostis capillaris L Effects of non-toxic levels of aluminium and the significance of P and Al speciation

Investigations on the effects of low levels of Al on P adsorption,uptake and translocation in seedlings of the indigenous grassAgrostis capillaris were undertaken. Apparent uptake and transportof H₂ ³²PO₄ from nutrient solutions containing 10 or 100mmolm^–3 phosphate were characterized as functions of timeand concentration. Experiments on ³²P uptake and transport insolutions containing no Al (control) or Al ranging from 3.7to 185 mmol m^–3 at pH ranging from 4.3 to 4.6, showedthat in 10 mmol m^–3 P, effects of Al at 3.7 and 37 mmolm^–3 on the size of the initial uptake shoulder were small,but some increase in subsequent P uptake to the roots was observed,though transport to the shoots was suppressed. With 37 mmolm^–3 Al in nutrient solution containing 100 mmol m^–3P, the uptake shoulder was much increased above the control.Subsequent root uptake was stimulated but transport was unaffected.Lack of toxicity of the Al concentrations used was indicatedby a lack of significant effect on plant fresh weight. AbsorbedAl was almost totally retained in the root in all treatments.Speciation calculations showed that the major species in Alamended nutrient solution at pH 4.4 were H₂PO₄^–, AI³⁺and AIHPO₄⁺, together with substantial amounts of AISO₄⁺ andsoluble aluminium hydroxy complexes (AIOH²⁺, AI(OH)₂⁺), dependingon the relative concentrations of P and Al. The effects of Al,with 10 mmol m^–3 P, on adsorption of complexed P werepartly accounted for in terms of preferential cell wall adsorptionof Al complexes not containing P. Conclusions were drawn aboutthe P-economy of A. capillaris plants growing on soils withlow levels of P and Al. Key words: Phosphorus, aluminium, speciation, Agrostis capillahs L     

Phosphate and Membrane Electropotentials in Trifolium repens L.

In Trifolium repens L. there were immediate transient depolarizationsof the membrane electropotential (E_vo) when KH₂PO₄ was addedto phosphate-free media, but these were of the same magnitudeas the controls (K²SO⁴ and KCI). Furthermore, the extents ofdepolarization were the same as the expected effect of the addedK⁺ calculated using the Goldman equation. There was no significantdepolarization on adding H₃PO₄ to buffered media. Consequently,there was no evidence for a depolarization caused by phosphate.This result provides evidence that the H⁺–H₂PO₄ symportin roots of T. repens operates with a stoichiometry of 1: 1. In a group of control plants ( + P plants) and a group whichwere stressed by reducing the supply of phosphate (– Pplants), the – P plants had lower values for E_vo than+P plants (– 118 mV and – 130 mV, respectively).The absence of phosphate from the measurement media also reducedE_vo (mean effect = 9 mV). A significant difference in E_vo between– P and + P plants persisted when phosphate was addedto – P plants. The electropotential difference acrossthe tonoplast (E_vo) in – P plants became more positivewith time. Key words: White clover, membrane transport, roots, tonoplast, symport     

Effects of Light/Dark on Anion Fluxes in Isolated Guard Cells of Commelina communis L.

The effects of light/dark on anion fluxes in isolated guardcells of Commelina communis L. have been studied, using ⁸²Brand ³⁶Cl. Transfer of open guard cells from light to dark hasno effect on the ⁸²Br influx, but produces a marked transientstimulation of ⁸²Br or ³⁶Cl efflux, similar to the effect ofsuch transfer on the ⁸⁶Rb fluxes, and to the effects on both⁸⁶Rb and ⁸²Br fluxes of adding ABA. On return of guard cellsto light, after the transient, there is a further reductionin Cl/Br efflux. It is argued that control of a specific processof ion extrusion is important in regulating the ability of guardcells to stay open. In three out of four batches of steady-statetissue labelled with ⁸²Br, the plasmalemma fluxes were highenough, relative to the tonoplast fluxes, for the efflux kineticsto be separable into two exponential components, allowing estimationof bromide contents in cytoplasm and vacuole (Q_c and Q_v), andfluxes at plasmalemma and tonoplast. With opening in light,Q_c increased by 3.9 ± 0.4 pmol mm^–2 µm^–1and Qy by 5.2 ± 0.6 pmol mm^–2 µm^–1(change in content per mm² of epidermis perµm change inaperture). Using rough estimates for the volumes of cytoplasmand vacuole these figures suggest that at 6.1 µm in thedark the concentrations were about 63 mol m^–3 in the cytoplasmand 35 mol m^–3 in the vacuole, rising to about 185 molm^–3 in the cytoplasm and 125 mol m^–3 in the vacuole,at 16.7 µm aperture in light. Neither increase can providean adequate increase in salt concentration to account for theosmotic change required, and some solute other than potassiumsalt must also be involved. In one experiment with 82Br andin the only experiment with 36Cl the plasmalemma flux was lower,and not high enough relative to the tonoplast flux to allowseparation of two phases in the efflux curves, and calculationof cytoplasmic and vacuolar contents and fluxes. The effectsof transfer from light to dark were, nevertheless, similar inboth types of tissue. Key words: Commelina communis L., Light/dark effects, Anion fluxes, Guard cells     

Nitrate transport in intact wheat roots:II. Long-term effects of NO3- concentration in the nutrient solution on NO3- unidirectional fluxes and distribution within the tissues5

We have examined the long-term effects of NO₃^– concentrationson NO₃^– (¹⁵NO₃^–) fluxes and cellular pool sizesin roots of intact 30-d-old wheat (Triticum aestivum cv. Courtot)grown hydroponically. Compartmental analysis was performed understeady-state conditions at five different levels of NO₃^–concentration (from 0.1 up to 5 mol m^–3 taking into accountmetabolism and secretion into the xylem (Devienne et al., 1994).Nitrate and reduced nitrogen levels in the tissues were largelyindependent of external NO₃^– concentration although below1.5 mol m^–3 NO₃^–; concentration limited plant growth.In the chamber, marked diurnal variations in net uptake occurredand, in the light, higher NO₃^– concentrations yieldedhigher NO₃^– uptake rates. After transfer of the plantsto the laboratory, the increase in net uptake linked to elevationof NO₃^–; concentrations was even larger (from 0.1 to 8.8µmolh^–1 g^–1 FW) as a result of a marked increase (x10–11) in the unidirectional influx at the plasmalemmawhile NO₃^– efflux was less enhanced (x 4–5). Underthese conditions, influx into the vacuole was also higher (x2–4) while efflux from the vacuole was little affected(x 1–3). NO₃^– concentrations within the cell compartmentswere estimated under the clas sical assumptions. The vacuolarconcentration was a little modified by NO₃^– availabilitywhereas that in the cytosol increased from about 10 mol m^–3to about 20 mol m^–3 indicating that (1) the absolute valuefor the cytosol was high and (2) it displayed only a small increasedespite very large changes in NO₃^– fluxes. NO₃^–distribution within the cells did not seem to involve an activeaccumulation of NO₃^– in the vacuole. Key words: Wheat, ion transport, nitrate, ¹⁵N, compartmentation     

Characterization of Photosynthetic 14Carbon Assimilation by Potamogeton lucens L.

Photosynthetic assimilation of exogenous ¹⁴CO₂ and H¹⁴CO₃^–by the aquatic angiosperm Potamogeton lucens L. is reported.Equivalent maximum rates of assimilation (1.5 µmol s^–1m^–2) were obtained in the presence of saturating levelsof ¹⁴CO₂ (1.0 mol m^–3, pH 5.3) or H¹⁴CO₃^– (1.5 molm^–3, pH, 9.2). Under subsaturating ¹⁴CO₂ levels, bothgaseous diffusion and H¹⁴CO₃^– transport were shown tooperate simultaneously, such that maximal photosynthetic rateswere established. An induction lag of approximately 3 min was observed when exogenous¹⁴CO₂ was assimilated. A longer lag of approximately 12 minwas required, however, before linear assimilation rates wereestablished when H¹⁴CO₃ acted as the carbon source. The light-activatedH¹⁴CO₃^– transport system was found to be quite labile.A brief (5 min) dark treatment returned the system to the inactivestate. Bicarbonate transport was shown to be competitively inhibitedby CO₃^2–ions. The possibility is discussed that this formof inhibition may be common to many HCO₃^– assimilators. Preliminary polar cation transport studies (from lower to upperleaf surface) indicated an almost exact one to one relationshipbetween the rates of Na⁺ influx and efflux and H¹⁴CO₃^–assimilation. The possible relationship(s) between these transportprocesses and the requirement for electrical neutrality is brieflydiscussed.     

Phosphorus-31 NMR Studies of Wild-Type and NaCl-Tolerant Citrus Cultured Cells

Ben–Hayyim, G. and Navon, G. 1985. Phosphorus–31NMR studies of wild–type and NaCl–tolerant Citruscultured cells.—J. exp. Bot. 36: 1877–1888. Theinternal pH of the cytoplasm and vacuole and the relative distributionof internal P_i concentrations between those two cell compartmentshave been determined by ³¹P nuclear magnetic resonance spectroscopyin wild–type and NaCl–tolerant cell lines of Shamoutiorange (Citrus sinensis L. Osbeck). Wild–type cells accumulatehigher amounts of P_i than the NaCl–tolerant cells whenexposed to equal external P_i concentrations. This additionalP_i is located mainly in the vacuole. When both types of cellsare exposed to increasing external P_i concentrations, the internalP_i concentrations increase. The cytoplasmic P_i concentrationreaches saturation at a rather low external P_i concentrationwhile the vacuolar P_i can be increased by a large factor. Transferof cells from aerobic to anaerobic conditions causes an immediateincrease of P_i in the cytoplasm and a slow acidification. Exposureof cells to NaCl during the period of their growth results inan increase in total P_i concentration with a large increasein the ratio of vacuolar to cytoplasmic P_i levels. When thesecells are exposed to NaCl for a short time, total internal P_iconcentration docs not change significantly but its proportionschange in favour of the vacuole. pH values of the cytoplasmand the vacuole under all these conditions are rather constant,the value being 5.8–6.0 for the vacuole and 7.4–7.6for the cytoplasm. Moreover, subjecting these cells to a widerange of external pH values does not change their intracellularpH. These results indicate that a strong regulation of internalpH is operating in both types of cells. The presence of a phosphorylatedmetabolite with an unusual pH titration curve, located in thevacuole, is also reported. Key words: Citrus, callus, ³¹P-NMR, NaCI tolerance, intracellular pH     

Analysis of the Diffusion Symmetry Developed by the Alkaline and Acid Bands which form at the Surface of Chara corallina Cells

The diffusion symmetry developed by the alkaline and aeid bandsof Chara corallina was studied. The alkaline system developeda diffusion pattern which could not be fitted to the equationfor a continuous point-source efflux. However, good correlationwas obtained between experimental data and the diffusion equationfor a hollow sphere. The calculated OH- efflux values, obtainedusing the equation of a continuous spherical-surface source,were checked against the influx values of H₁₄ obtained under the same experimental conditions. IndividualOH– band efflux values ranged from 0.07 to 5.95 pmol s^–1and total cell fluxes of 25 pmol cm^–2 s^–1 for OH^-and H were obtained (in the presence of 0.5 mM NaHCO₃). The acid system developed a cylindrical diffusion pattern, butthis could not be fitted to a mathematical equation. Numericalanalysis will have to be employed to obtain values of H⁺ efflux.     

Movement of Ions and Electrogenesis in Higher Plant Cells

During the past 10 years considerable information has accumulatedon the electrochemical relationships of higher plant cells duringtransport of mineral ions. Using the Nernst equation as a criterion,none of eight ions (K⁺, Na⁺, Ca⁺⁺, Mg⁺⁺, NO₃^–, Cl^–,H₂PO₄^–, and SO₄^–) is in a passive equilibrium. Na⁺,Ca⁺⁺, and Mg⁺⁺ are subject to an exclusion mechanism, and allof the anions appear to be pumped inwardly. K⁺ apparently approachesan electrochemical balance under certain conditions but probablyis actively accumulated. Compartmental analyses giving estimatesof amounts in the cytoplasm and vacuole and of unidirectionalfluxes permit application of the Ussing flux-ratio equation.The criterion in oat coleoptile cells suggests that at the plasmalemmaNa⁺ is pumped out while K⁺ and Cl^– are pumped in. K⁺ andCl^– appear to be coupled in active transport across thetonoplast into the vacuole. Good evidence has been found thatthe cell's electropotential arises from an electrogenic pump:CN^– (cyanide) and DNP (dinitrophenol) reversibly blockthe potential and ionic transport; cell potentials are higherthan can be accounted for by diffusion; the responses of respirationand potential to the concentration of CN^– are nearly parallel;and CN^– inhibited tissue approaches a fit to the Goldmanconstant field equation. Future objectives should be identificationof the ion, or ions, subject to the electrogenic pump and discoveryof the immediate energy source.     

Chloride Transport in Chara corallina and the Electrochemical Potential Difference for Hydrogen Ions

The pH of the cytoplasm of Chara corallina cells has been measuredwith the weak acid 5,5-dimethyloxazolidine-2,4-dione (DM0).Over an external pH range 4·5–9·5 the resultsfit the regression equation pH_cytoplasm=6·28+0·22pH_out. Using measured values of the electric potential difference acrossthe plasmalemma we have calculated the electrochemical potentialdifference across this membrane for H⁺ and Cl^–. Thesedata are used to test the hypothesis that the inward transportof Cl^– is coupled to the inthix of H⁺ or, which comesto the same thing, efflux of OH^–. One-for-one couplingwill not give net Cl^– uptake from solutions with pH greaterthan about 7·2, unless the cytoplasmic Cl^– concentrationis lower than 10 mM, or the pH just outside the membrane islower than that in the bulk solution. It is shown that net Cl^–uptake proceeds from solutions with pH up to 9. The alternative possibility is that Cl^– transport is broughtabout by co-transport of two H⁺ for each Cl^–; this isnot ruled out by the results reported. Such a mechanism mightbe detectable by its electrogenic effect: although such effectshave not been detected, it is shown that they would be smallunder most conditions. Other possible mechanisms are discussed.     

Pharmacological and biophysical isolation of K+ currents encoded by ether-à-go-go-related genes in murine hepatic portal vein smooth muscle cells

The functional properties of the Saccharomyces cerevisiae bicarbonate transporter homolog Bor1p (YNL275wp) were characterized by measuring boron (H₃BO₃), Na⁺, and Cl^– fluxes. Neither Na⁺ nor Cl^– appears to be a transported substrate for Bor1p. Uphill efflux of boron mediated by Bor1p was demonstrated directly by loading cells with boron and resuspending in a low-boron medium. Cells with intact BOR1, but not the deletant strain, transport boron outward until the intracellular concentration is sevenfold lower than that in the medium. Boron efflux through Bor1p is a saturable function of intracellular boron (apparent K_m 1–2 mM). The extracellular pH dependences of boron distribution and efflux indicate that uphill efflux is driven by the inward H⁺ gradient. Addition of 30 mM HCO₃^– does not affect boron extrusion by Bor1p, indicating that HCO₃^– does not participate in Bor1p function. Functional Bor1p is present in cells grown in medium with no added boron, and overnight growth in 10 mM H₃BO₃ causes only a small increase in the levels of functional Bor1p and in BOR1 mRNA. The fact that Bor1p is expressed when there is no need for boron extrusion and is not strongly induced in the presence of growth-inhibitory boron concentrations is surprising if the main physiological function of yeast Bor1p is boron efflux. A possible role in vacuolar dynamics for Bor1p was recently reported by Decker and Wickner (10). Under the conditions used presently, there appears to be mildly abnormal vacuolar morphology in the deletant strain. boron; SLC4; YNL275w     

Salinity-induced Malate Accumulation in Chara

Ion absorption by Chara corallina from solutions containingpredominantly KC1 or RbCl at up to 100 mol m^–3 resultedin accumulation of salts and turgor regulation. Turgor regulationdid not occur in solutions containing Na⁺ or Li⁺salts. Duringion absorption from various salts of K⁺ and Rb⁺ vacuolar cationconcentration exceeded Cl^– concentration. This differencewas shown to be balanced by the synthesis and accumulation ofmalate. Vacuolar malate concentration reached 48 mol m₃^–,with accumulation occurring at rates of up to 0.45 mol m^–3h^–1. Malate accumulation was inhibited by low externalpH and was dependent upon external HCO₃^– concentration.The synthesis of malic acid and its subsequent dissociationimposed a severe acid load on the cell. Biophysical regulationof cellular pH was achieved by a H⁺efflux at a rate of about40 nmol m^–2 s^–1from the cell. The results presentedargue against cytoplasmic Cl^–, HCO₃^– or pH regulatingmalate accumulation in Chara and it is suggested that malatetransport across the tonoplast may regulate malate accumulation. Key words: Malate, Chara corallina, pH regulation, salinity