Changes in the Cytoplasmic and Vacuolar pH in Intact Cells of Mung Bean Root-Tips under High-NaCl Stress at Different External Concentrations of Ca2+ Ions

The cytoplasmic pH and the vacuolar pH in root-tip cells ofintact mung bean seedlings under high-NaCl stress were measuredby in vivo ³¹P-nuclear magnetic resonance (³¹P-NMR) spectroscopy.When roots were incubated with high levels (100 mM) of NaClat the control external concentration (0.5 mM) of Ca²⁺ ions,the vacuolar pH increased rapidly from 5.6 to 6.2 within 3 h,while the cytoplasmic pH only decreased by a mere 0.1 pH uniteven after a 24-h incubation under high-NaCl conditions. Theincrease in vacuolar pH induced by the high-NaCl stress wasdiminished by an increase in the external concentration of Ca²⁺ions from 0.5 mM to 5 mM. The intracellular concentration ofNa⁺ ions in the root-tip cells increased dramatically upon perfusionof the root cells with 100 mM NaCl, and high external levelsof Ca²⁺ ions also suppressed the in flow of Na⁺ ions into thecells. The vacuolar alkalization observed in salt-stressed rootsmay be related to the inhibition of an H⁺-translocating pyrophosphatasein the tonoplast, caused by the increase in the cytoplasmicconcentration of Na⁺ ions. It is suggested that, although thevacuolar pH increased markedly under salt stress, the cytoplasmicpH was tightly regulated by some unidentified mechanisms, suchas stimulation of the H⁺-translocating ATPase of the plasmalemma,in roots of mung bean under salt stress. (Received April 18, 1992; Accepted July 6, 1992)     

Effect of Sudden Salt Stress on Ion Fluxes in Intact Wheat Suspension Cells

Although salinity is one of the major problems limiting agriculturalproduction around the world, the underlying mechanisms of highNaCl perception and tolerance are still poorly understood. Theeffects of different bathing solutions and fusicoccin (FC),a known activator of plasma membrane ATPase, on plasma membranepotential (E_m) and net fluxes of Na⁺, K⁺and H⁺were studied inwheat suspension cells (Triticum aestivum) in response to differentNaCl treatments. E_mof cells in Murashige and Skoog (MS) mediumwas less negative than in cells exposed to a medium containing10 mM KCl + 0.1 m M CaCl₂(KSM) and to a basic salt medium (BSM),containing 1 m M KCl and 0.1 m M CaCl₂. Multiphasic Na⁺accumulationin cells was observed, peaking at 13 min after addition of 120m M NaCl to MS medium. This time scale was in good agreementwith net Na⁺flux changes measured non-invasively by moving ion-selectivemicroelectrodes (the MIFE system). When 120 m M NaCl was addedto all media studied, a quick rise of Na⁺influx was reversedwithin the first 20 min. In both 120 and 20 m M NaCl treatmentsin MS medium, net Na⁺efflux was observed, indicating that activeNa⁺transporters function in the plant cell response to saltstress. Lower external K⁺concentrations (KSM and BSM) and FCpre-treatment caused shifts in Na⁺fluxes towards net influxat 120 m M NaCl stress. Copyright 2000 Annals of Botany Company Sodium, potassium, proton, membrane potential, fusicoccin, salt stress, wheat, Triticum aestivum     

Proton/Pyruvate Cotransport into Mesophyll Chloroplasts of C4 Plants

Light-enhanced active pyruvate uptake into mesophyll chloroplastsof C₄ plants was reported to be mimicked by either of the twotypes of cation jump: H⁺-jump in maize and phylogenically relatedspecies (H⁺-type) and Na⁺-jump in all the other C₄ species tested(Na⁺-type) [Aoki, N., Ohnishi, J. and Kanai, R. (1992) PlantCell Physiol. 33: 805]. In this study, medium and stromal pH was monitored in the suspensionof C₄ mesophyll chloroplasts. Medium alkalization lasting for5 to 10 seconds after pyruvate addition was detected by a pHelectrode and observed only in the light and only in mesophyllchloroplasts from H⁺-type species, Zea mays L. and Coix lacryma-jobiL., but not in those from Na⁺-type species Panicum miliaceumL., Setaria italica (L.) Beauv. and Panicum maximum Jacq. Theinitial rate of H⁺ consumption showed good correlation with[¹⁴C]pyruvate uptake measured by silicone oil filtering centrifugation,both being inhibited by N-ethylmaleimide and 7-chloro-4-nitrobenzo-2-oxa-l,3-diazole to the same degree. The ratio of the rate of H⁺ uptaketo that of pyruvate uptake was always about 1. Pyruvate-inducedacidification of the stroma was observed in maize mesophyllchloroplasts. These results show one to one cotransport of H⁺and pyruvate anion into mesophyll chloroplasts of H⁺-type C₄species in the light. (Received January 5, 1994; Accepted May 6, 1994)     

Increased vacuolar and plasma membrane H+-ATPase activities in Salicornia bigelovii Torr. in response to NaCl

The halophyte Salicornia bigelovii Torr. shows optimal growthand Na⁺ accumulation in 200 mM NaCl and reduced growth underlower salinity conditions. The ability to accumulate and compartmentalizeNa⁺ may result, in part, from stimulation of the H⁺ -ATPaseson the plasma membrane (PM-ATPase) and vacuolar membranes (V-ATPase).To determine if these two primary transport systems are involvedin salt tolerance, shoot fresh weight (FW) and activity of thePM- and V-ATPases from shoots in Salicornia grown in 5 and 200mM NaCI were compared. Higher PM-ATPase activity (60%) and FW(60%) were observed in plants grown in 200 mM NaCI and thesestimulations in growth and enzyme activity were specific forNa⁺ and not observed with Na⁺ added in vitro. V-ATPase activitywas significantly stimulated in vivo and in vitro (26% and 46%,respectively) after exposure to 200 mM NaCl, and stimulationwas Na⁺ -specific. Immunoblots indicated that the increasesin activity of the H⁺ -ATPases from plants grown in 200 mM NaCIwas not due to increases in protein expression. These studiessuggest that the H⁺-ATPases in Salicornia are important in salttolerance and provide a biochemical framework for understandingmechanisms of salt tolerance in plants. Key words: Salicornia, H⁺-ATPases, salt tolerance     

Reappraisal of the Role of Sodium in the Light-Dependent Active Transport of Pyruvate into Mesophyll Chloroplasts of C4 Plants

The mechanism of light-dependent active transport of pyruvatein C₄ mesophyll chloroplasts has not been clarified, particularlyin Na⁺-type C₄ species, in which the pyruvate uptake into mesophyllchloroplasts is enhanced by illumination or by making a Na⁺gradient (Na⁺-jump) across the envelope in the dark. We re-investigatedhere the effect of Na⁺ on the active transport of pyruvate inmesophyll chloroplasts of Panicum miliaceum, a Na⁺-type C₄ species,by comparing the rate of pyruvate uptake at various externalpHs under four conditions; in the light and dark together with/withoutNa⁺-jump: (1) At neutral pH, the rate of pyruvate uptake inthe dark was enhanced by Na⁺-jump but scarcely by illumination.(2) While the enhancement effect by Na⁺-jump was independentof external pH, that by illumination increased greatly at pHover 7.4, and the effects of light and Na⁺ at the alkaline pHwere synergistic. (3) The light-enhanced pyruvate uptake wasrelated to stromal alkalization induced by illumination. Infact, pyruvate uptake was induced by H⁺-jump in the medium frompH 8.0 to 6.7. (4) Stromal pH was lowered by the addition ofK⁺-pyruvate and more by Na⁺-pyruvate into the medium at pH 7.8in the light. (5) However, the pH and ATP levels in the stromawere not affected by Na⁺-jump. Thus, we discussed possibility that besides pyruvate/Na⁺ cotransportat neutral pH in the medium, pyruvate/H⁺ cotransport enhancedby the presence of Na+ operates in mesophyll chloroplasts ofNa⁺-type C4 species at alkaline medium. ¹Present address: Biological Resources Division, Japan InternationalResearch Center for Agricultural Sciences (JIRCAS), Ministryof Agriculture, Forestry and Fisheries, 2-1 Ohwashi, Tsukuba,305 Japan     

Na+/H+ Antiporter in Tonoplast Vesicles from Rice Roots

The Na⁺/H ⁺ antiporter in vacuolar membranes transports Na⁺from the cytoplasm to vacuoles using a pH gradient generatedby proton pumps; it is considered to be related to salinitytolerance. Rice (Oryza sativa L.) is a salt-sensitive crop whosevacuolar antiporter is unknown. The vacuolar pH of rice roots,determined by ³¹P-nuclear magnetic resonance (NMR), increasedfrom 5.34 to 5.58 in response to 0.1 M NaCl treatment. Transportof protons into the tonoplast vesicles from rice roots was fluorometricallymeasured. Efflux of protons was accelerated by the additionof Na⁺. Furthermore, the influx of ²²Na⁺ into the tonoplastvesicles was accelerated by a pH gradient generated by proton-translocatingadenosine 5'-triphosphatase (H⁺-ATPase) and proton-translocatinginorganic pyro-phosphatase (H⁺-PPase). We concluded that thisNa⁺/H⁺antiporter functioned as a Na⁺ transporter in the vacuolarmembranes. The antiporter had a K_m of 10 mM for Na⁺ and wascompetitively inhibited by amiloride and its analogues. TheKⁱ values for 5-(N-methyl-N-isobutyl)-amiloride (MIA), 5-(N-ethyl-N-isopropyI)-amiloride(EIPA), and 5-(N, N-hexamethylene)-amiloride (HMA) were 2.2,5.9, and 2.9 µ M, respectively. Unlike barley, a salt-tolerantcrop, NaCl treatment did not activate the antiporter in riceroots. The amount of antiporter in the vacuolar membranes maybe one of the most important factors determining salt tolerance. ¹This work was supported by a grant from Bio-Media Project ofthe Japanese Ministry of Agriculture, Forestry and Fisheries(BMP96-III-1).     

Na+ fluxes in Chara under salt stress

The influx and efflux of Na⁺ across the plasma membrane of Characorallina and Chara longifolia were examined under mild saltstress conditions. Na⁺ influx was found to be rapid in bothspecies with the freely exchangeable cytoplasmic Na⁺ cominginto isotopic equilibrium with external ²²Na⁺ within 1 h ofexposure to isotope. Cytoplasmlc Na⁺ concentration and Na⁺ influxwere greater in C. corallina than in C. longifolla under thesame conditions. Na⁺ influx across the tonoplast was much lowerthan the flux across the plasma membrane. Na⁺ efflux was stimulatedat pH 5 relative to pH 7 by 218% in C. coralllna and 320% inC. longifolia. In both species externally applied Li⁺ inhibitedNa⁺ efflux at pH 5 but not at pH 7. Na⁺ etflux was not significantlyinhibited by amiloride. Key words: Na⁺ influx, Na⁺ efflux, Na⁺/H⁺ antiport, Chara     

Overexpression of wheat Na+/H+ antiporter TNHX1 and H+-pyrophosphatase TVP1 improve salt- and drought-stress tolerance in Arabidopsis thaliana plants

Transgenic Arabidopsis plants overexpressing the wheat vacuolarNa⁺/H⁺ antiporter TNHX1 and H⁺-PPase TVP1 are much more resistantto high concentrations of NaCl and to water deprivation thanthe wild-type strains. These transgenic plants grow well inthe presence of 200 mM NaCl and also under a water-deprivationregime, while wild-type plants exhibit chlorosis and growthinhibition. Leaf area decreased much more in wild-type thanin transgenic plants subjected to salt or drought stress. Theleaf water potential was less negative for wild-type than fortransgenic plants. This could be due to an enhanced osmoticadjustment in the transgenic plants. Moreover, these transgenicplants accumulate more Na⁺ and K⁺ in their leaf tissue thanthe wild-type plants. The toxic effect of Na⁺ accumulation inthe cytosol is reduced by its sequestration into the vacuole.The rate of water loss under drought or salt stress was higherin wild-type than transgenic plants. Increased vacuolar soluteaccumulation and water retention could confer the phenotypeof salt and drought tolerance of the transgenic plants. Overexpressionof the isolated genes from wheat in Arabidopsis thaliana plantsis worthwhile to elucidate the contribution of these proteinsto the tolerance mechanism to salt and drought. Adopting a similarstrategy could be one way of developing transgenic staple cropswith improved tolerance to these important abiotic stresses. Key words: H⁺-pyrophosphatase, Na⁺/H⁺ antiporter, salt and drought tolerance, sodium sequestration, transgenic Arabidopsis plants     

Functional upregulation of H+-ATPase by lethal acid stress in cultured inner medullary collecting duct cells

The response ofH⁺-ATPase to lethal acid stress isunknown. A mutant strain (called NHE2d) was derived from cultured inner medullary collecting duct cells (mIMCD-3 cells) following three cyclesof lethal acid stress. Cells were grown to confluence on coverslips,loaded with2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein, andmonitored for intracellular pH(pH_i) recovery from an acid load. The rate of Na⁺-independentpH_i recovery from an acid load inmutant cells was approximately fourfold higher than in parent cells(P < 0.001). TheNa⁺-independentH⁺ extrusion was ATP dependent and K⁺ independent and wascompletely inhibited in the presence of diethylstilbestrol, N, N'-dicyclohexylcarbodiimide,or N-ethylmaleimide. Theseresults indicate that theNa⁺-independentH⁺ extrusion in cultured medullarycells is mediated via H⁺-ATPaseand is upregulated in lethal acidosis. Northern hybridization experiments demonstrated that mRNA levels for the 16- and 31-kDa subunits of H⁺-ATPase remainedunchanged in mutant cells compared with parent cells. We propose thatlethal acid stress results in increased H⁺-ATPase activity in innermedullary collecting duct cells. Upregulation ofH⁺-ATPase could play a protectiverole against cell death in severe intracellular acidosis.

     

Sodium-Stimulation of Phosphate Uptake in the Cyanobacterium Anabaena PCC 7119

Anabaena PCC 7119 showed higher rates of phosphate uptake whencells were under P-starvation. Phosphate uptake was energy-dependentas indicated the decrease observed when assays were performedin the dark or in the presence of inhibitors of photosyntheticelectron transport, energy transfer and adenosine triphosphataseactivity. Phosphate uptake was stimulated by Na⁺ both in P-sufficientcells and P-starved cells. Li⁺ and K⁺ acted as partial analoguesfor Na⁺. The Na⁺-stimulation of phosphate uptake followed Michaelis-Mentenkinetics, half-saturation (K) of phosphate uptake was reachedwith a Na⁺ concentration of 212 µM. The absence of Na⁺reduced the rates of phosphate uptake at all phosphate concentrationsassayed (1–20 µM). The maximum uptake rates (V_max)decreased from 658 nmol P (mg dry wt)^-1 h^-1 in the presenceof Na⁺ to 149 nmol P (mg dry wt)^-1 h^-1 in the absence of Na⁺.The absence of Na⁺ did not change significantly the concentrationof phosphate required to reach half-saturation (K) (3.01 µMin the presence of Na⁺ vs 3.21 µM in the absence of Na⁺).In the presence of Na⁺ the rate of phosphate uptake was affectedby the pH; optimal rates were observed at pH 8. In the absenceof Na⁺ phosphate uptake was not affected by the pH; low rateswere observed in all cases. Monensin, an ionophore which collapsesNa⁺-gradients, reduced the rate of phosphate uptake in Na⁺-supplementedcells. These results indicated the existence of a Na⁺-dependentphosphate uptake in Anabaena PCC 7119. (Received September 8, 1992; Accepted November 17, 1992)     

Respiration-Dependent Proton and Sodium Pumps in a Psychrophilic Bacterium, Vibrio sp. Strain ABE-1

Respiration-dependent proton and sodium flows in a psychrophilicbacterium, Vibrio sp. strain ABE-1, were examined. At alkalinepH, this bacterium grew without being affected by a proton conductor,carbonylcyanide m-chlorophenylhydrazone (CCCP). O₂-pulse intoanaerobic cell suspensions prepared with Na^$-free buffers inducedtransient alkalization in the presence of CCCP and acidificationat pH 8.5 and 6.5, respectively. However, using cells preparedwith Na^$-containing buffer, the transient pH changes of thecell suspension could be simultanously detected at both pHs.Several inhibitory experiments suggested that the acidificationand alkalization should be attributed to a respiration-dependentprimary H^$ pump and Na^$ pump, respectively, and that the latterwas similar to that first reported in a marine bacterium, Vibrioalginolyticus. This Na^$ pump may have supported the CCCP-resistantgrowth at alkaline pH. The H^$ and Na^$ pumps operated very actively at low temperatures,such as 5?C, and should markedly help sustain bacterial growthat low temperatures. (Received May 30, 1987; Accepted November 13, 1987)     

Stimulation of the Extrusion of Protons and H+-ATPase Activities with the Decline in Pyrophosphatase Activity of the Tonoplast in Intact Mung Bean Roots under High-NaCl Stress and Its Relation to External Levels of Ca2+ Ions

Extrusion of protons as a response to high-NaCl stress in intactmung bean roots was investigated at different external concentrationsof Ca²⁺ ions ([Ca²⁺]_ex). The extrusion of protons was graduallyenhanced in the roots exposed to 100 mM NaCl, and high [Ca²⁺]_exdiminished this enhancement of the extrusion. Vesicles of plasmalemmaand tonoplast were prepared from the roots and the H⁺-translocatingATPase (H⁺-ATPase) activities associated with the two typesof membrane and the H⁺-pyrophosphatase (H⁺-PPase) activity ofthe tonoplast were assayed. The plasmalemma ATPase was stimulatedin parallel with dramatic increases in the intracellular concentrationof Na⁺([Na⁺]_in). High [Ca²⁺]ex prevented the increase in [Na⁺]inand diminished the stimulation of ATPase activity. The tonoplastATPase showed a rapid response to salt stress and was similarlystimulated even at high [Ca²⁺]M. The activities of both ATPaseswere, however, insensitive to concentrations of Na⁺ ions upto 100 HIM. By contrast, H⁺-PPase activity of the tonoplastwas severely inhibited with increasing [Na⁺]in under salt stressand recovered with high [Ca²⁺]ex. These findings suggest thathigh-NaCl stress increases the intracellular concentration ofNa⁺ ions in mung bean roots, which inhibits the tonoplast H⁺-PPase,and the activity of the plasmalemma H⁺-ATPase is thereby stimulatedand regulates the cytoplasmic pH. (Received March 26, 1991; Accepted December 13, 1991)     

Sodium-dependent inactivation of sodium/calcium exchange in transfected Chinese hamster ovary cells

High concentrations of cytosolic Na⁺ ions induce the time-dependent formation of an inactive state of the Na⁺/Ca²⁺ exchanger (NCX), a process known as Na⁺-dependent inactivation. NCX activity was measured as Ca²⁺ uptake in fura 2-loaded Chinese hamster ovary (CHO) cells expressing the wild-type (WT) NCX or mutants that are hypersensitive (F223E) or resistant (K229Q) to Na⁺-dependent inactivation. As expected, 1) Na⁺-dependent inactivation was promoted by high cytosolic Na⁺ concentration, 2) the F223E mutant was more susceptible than the WT exchanger to inactivation, whereas the K229Q mutant was resistant, and 3) inactivation was enhanced by cytosolic acidification. However, in contrast to expectations from excised patch studies, 1) the WT exchanger was resistant to Na⁺-dependent inactivation unless cytosolic pH was reduced, 2) reducing cellular phosphatidylinositol-4,5-bisphosphate levels did not induce Na⁺-dependent inactivation in the WT exchanger, 3) Na⁺-dependent inactivation did not increase the half-maximal cytosolic Ca²⁺ concentration for allosteric Ca²⁺ activation, 4) Na⁺-dependent inactivation was not reversed by high cytosolic Ca²⁺ concentrations, and 5) Na⁺-dependent inactivation was partially, but transiently, reversed by an increase in extracellular Ca²⁺ concentration. Thus Na⁺-dependent inactivation of NCX expressed in CHO cells differs in several respects from the inactivation process measured in excised patches. The refractoriness of the WT exchanger to Na⁺-dependent inactivation suggests that this type of inactivation is unlikely to be a strong regulator of exchange activity under physiological conditions but would probably act to inhibit NCX-mediated Ca²⁺ influx during ischemia. ischemia; cytosolic calcium concentration; cytosolic sodium concentration; cellular phosphatidylinositol-4,5-bisphosphate     

Deciphering PiT transport kinetics and substrate specificity using electrophysiology and flux measurements

Members of the SLC20 family or type III Na⁺-coupled P_i cotransporters (PiT-1, PiT-2) are ubiquitously expressed in mammalian tissue and are thought to perform a housekeeping function for intracellular P_i homeostasis. Previous studies have shown that PiT-1 and PiT-2 mediate electrogenic P_i cotransport when expressed in Xenopus oocytes, but only limited kinetic characterizations were made. To address this shortcoming, we performed a detailed analysis of SLC20 transport function. Three SLC20 clones (Xenopus PiT-1, human PiT-1, and human PiT-2) were expressed in Xenopus oocytes. Each clone gave robust Na⁺-dependent ³²P_i uptake, but only Xenopus PiT-1 showed sufficient activity for complete kinetic characterization by using two-electrode voltage clamp and radionuclide uptake. Transport activity was also documented with Li⁺ substituted for Na⁺. The dependence of the P_i-induced current on P_i concentration was Michaelian, and the dependence on Na⁺ concentration indicated weak cooperativity. The dependence on external pH was unique: the apparent P_i affinity constant showed a minimum in the pH range 6.2–6.8 of 0.05 mM and increased to 0.2 mM at pH 5.0 and pH 8.0. Xenopus PiT-1 stoichiometry was determined by dual ²²Na-³²P_i uptake and suggested a 2:1 Na⁺:P_i stoichiometry. A correlation of ³²P_i uptake and net charge movement indicated one charge translocation per P_i. Changes in oocyte surface pH were consistent with transport of monovalent P_i. On the basis of the kinetics of substrate interdependence, we propose an ordered binding scheme of Na⁺:H₂PO₄^–:Na⁺. Significantly, in contrast to type II Na⁺-P_i cotransporters, the transport inhibitor phosphonoformic acid did not inhibit PiT-1 or PiT-2 activity. Na⁺-P_i cotransport; two-electrode voltage clamp; surface pH electrode; SLC20; retroviral receptor     

Alloxan-induced diabetes reduces sarcolemmal Na+-K+ pump function in rabbit ventricular myocytes

The effect of diabetes on sarcolemmal Na⁺-K⁺ pump function is important for our understanding of heart disease associated with diabetes and design of its treatment. We induced diabetes characterized by hyperglycemia but no other major metabolic disturbances in rabbits. Ventricular myocytes isolated from diabetic rabbits and controls were voltage clamped and internally perfused with the whole cell patch-clamp technique. Electrogenic Na⁺-K⁺ pump current (I_p, arising from the 3:2 Na⁺-to-K⁺ exchange ratio) was identified as the shift in holding current induced by Na⁺-K⁺ pump blockade with 100 µmol/l ouabain in most experiments. There was no effect of diabetes on I_p recorded when myocytes were perfused with pipette solutions containing 80 mmol/l Na⁺ to nearly saturate intracellular Na⁺-K⁺ pump sites. However, diabetes was associated with a significant decrease in I_p measured when pipette solutions contained 10 mmol/l Na⁺. The decrease was independent of membrane voltage but dependent on the intracellular concentration of K⁺. There was no effect of diabetes on the sensitivity of I_p to extracellular K⁺. Pump inhibition was abolished by restoration of euglycemia or by in vivo angiotensin II receptor blockade with losartan. We conclude that diabetes induces sarcolemmal Na⁺-K⁺ pump inhibition that can be reversed with pharmacological intervention. sodium transport; insulin; angiotensin II; cardiomyopathy; hyperglycemia     

Effect of NaCI Salinity on Flows and Partitioning of C, N, and Mineral Ions in Whole Plants of White Lupin, Lupinus albusL.

Plants of Lupinus albus L., cv. Ultra, were grown hydroponicallywith NO₃^–-nutrition for 51 d under control (0.05 mol m^–3Na⁺ and 10 mol m^–3 Cl^–) and saline (40 mol m^–3NaCI) conditions. Plants were harvested 41 and 51 d after germinationand analysed for content and net increment of C, N and the mineralcations K⁺, Na⁺, Mg²⁺, and Ca²⁺ and the anions Cl^–, NOJ,malate, phosphate, and SO₄^2–. Roots, stem interaodes,petioles and leaflets were analysed separately. During the studyperiod net photosynthesis, respiratory losses of CO₂ from shootand root and the composition of the spontaneously bleeding phloemsap and the root pressure xylem exudate were also determined.Using molar ratios of C over N in the transport fluids, incrementsof C and N, and photosynthetic gains as well as respiratorylosses of C, the net flows of C and N in the xylem and phloemwere then calculated as in earlier studies (Pate, Layzell andMcNeill, 1979a). Knowing the carbon flows, the ratios of ionto carbon in the phloem sap, and ion increments in individualorgans, net flows of K⁺, Na⁺, and Cl^– over the study periodwere also calculated. Salt stress led to a general decrease of all partial componentsof C and N partitioning indicating that inhibitions were notdue to specific effects of NaCI salinity on photosynthesis oron NO₃ uptake. However, there were differences between variouslyaged organs, and net phloem export of nitrogenous compoundsfrom ageing leaves was substantially enhanced under saline conditions.In addition, NO₃^–reduction in the roots was specificallyinhibited. Uptake and xylem transport of K⁺ was more severelyinhibited than photosynthetic carbon gain or NO₃^– uptakeby the root. K⁺ transport in the phloem was even more severelyrestricted under saline conditions. Na⁺ and Cl^– flowsand uptake, on the other hand, were substantially increasedin the presence of salt and, in particular, there were thenmassive flows of Na^– in the phloem. The results are discussedin relation to the causes of salt sensitivity of Lupinus albus.The data suggest that both a restriction of K⁺ supply and astrongly increased phloem translocation of Na⁺ contribute tothe adverse effects of salt in this species. Restriction ofK⁺ supply occurs by diminished K⁺ uptake and even more by reducedK⁺ cycling within the plant. Key words: Lupinus albus, salt stress, phloem transport, xylem transport, partitioning, carbon, nitrogen, K⁺, Na⁺, CI^–     

SEA-0400, a potent inhibitor of the Na+/Ca2+ exchanger, as a tool to study exchanger ionic and metabolic regulation

The effects of a new, potent, and selective inhibitor of the Na⁺/Ca²⁺ exchange, SEA-0400 (SEA), on steady-state outward (forward exchange), inward (reverse exchange), and Ca²⁺/Ca²⁺ transport exchange modes were studied in internally dialyzed squid giant axons from both the extra- and intracellular sides. Inhibition by SEA takes place preferentially from the intracellular side of the membrane. Its inhibition has the following characteristics: it increases synergic intracellular Na⁺ (Na_i⁺) + intracellular H⁺ (H_i⁺) inactivation, is antagonized by ATP and intracellular alkalinization, and is enhanced by intracellular acidification even in the absence of Na⁺. Inhibition by SEA is still present even after 1 h of its removal from the experimental solutions, whereas removal of the cointeracting agents of inhibition, Na_i⁺ and H_i⁺, even in the continuous presence of SEA, releases inhibition, indicating that SEA facilitates the reversible attachment of the natural H_i⁺ and Na_i⁺ synergic inhibitors. On the basis of a recent model of squid Na⁺/Ca²⁺ exchange regulation (DiPolo R and Beaugé L. J Physiol 539: 791–803, 2002), we suggest that SEA acts on the H_i⁺ + Na_i⁺ inactivation process and can interact with the Na⁺-free and Na⁺-bound protonized carrier. Protection by ATP concurs with the antagonism of the nucleotide by H_i⁺ + Na_i⁺ synergic inhibition. ionic-metabolic interactions     

Effects of nitrogen nutrition on salt-stressed Nicotiana tabacum var. Samsun in vitro plantlets

Tobacco shoots were grown in vitro for 35 d, in MS culture mediummodified to include various sources (nitrate-N, ammonium-N ora mixture) and levels (0–120 mM) of N, and in the presenceof 0–180 mM NaCI or iso-osmotic concentrations of mannitol.Growth of control plantlets was significantly inhibited whenNH₄⁺-N was the sole N source, and at high (120 mM) NO^–₃-N supply. Under conditions of salt stress (90 and 180 mM NaCI)growth was repressed, with roots being more severely affectedthan shoots. Salinity also inhibited root emergence in vitro.The only alleviation of the salt stress by nitrate nutritionobserved in this study was on shoot growth parameters of plantletsgrown on 60 mM NO^–₃-N and 90 mM NaCI. Although both weresignificantly inhibited by NaCI, nitrate reduc-tase activitywas more severely affected than nitrate uptake. When mannitolreplaced NaCI in the culture medium, similar Inhibition of growth,nutrient uptake and enzyme activity were recorded. These observations,together with the relatively low recorded values for Na⁺ andCI^– uptake, indicate that under in vitro salt stress conditionsthe negative effects of NaCI are primarily osmotic. Key words: Growth, nitrogen metabolism, osmotic stress, salinity     

Biophysical and Biochemical Regulation of Cytoplasmic pH in Chara corallina during Acid Loads

The contribution of membrane transport to regulation of cytoplasmicpH in Chara corallina has been measured during proton-loadingby uptake of butyric acid. In the short-term (i.e. up to 20min) uptake of butyric acid is not affected by removal of externalK⁺, Na⁺ or Cl^– but over longer periods uptake is decreased(by 20–50% in different experiments) in the absence ofexternal Na⁺ or, sometimes, K⁺. Influxes of both Na⁺ and K⁺increase temporarily after addition of butyrate, Na⁺ immediatelyand K⁺ after a lag. Effects on Cl^– influx are small butCl^– efflux increases enormously after a short lag. Anapproximate comparison of internal butyrate with changes inthe concentration of K⁺, Na⁺, and Cl^– suggests that initially(i.e. for a few min) cytoplasmic pH is determined by bufferingand possibly by some decarboxylation of organic acids (biochemicalpH regulation), and that biophysical pH regulation involvingefflux of H⁺ balanced by influxes of K⁺, Na⁺ and especiallyefflux of Cl^– progressively becomes dominant. When butyric acid is washed out of the cells, cytoplasmic pHis restored completely or partially (depending on the butyrateconcentration used) and this is independent of the presenceor absence of external Cl^–. Where Cl^– is present,its influx is relatively small. It is suggested that cytoplasmicpH is then controlled biochemically, involving the synthesisof an (unidentified) organic acid and the accumulation of acidicanions in place of butyurate lost from the cell. During thesecond application of butyrate, net Cl^– efflux is small:it is suggested that control of cytoplasmic pH then involvesdecarboxylation of the organic acid anions. The questions of the source of Cl^– lost from the cell(cytoplasm or vacuole) and of possible cytoplasmic swellingassociated with the accumulation of butyrate are discussed. Key words: Chara corallina, butyric acid, cytoplasmic pH, membrane transport