Characterization of an Anion Transporter in the Plasma Membrane of Barley Roots

To examine the relationship between H⁺-ATPase and the transportof anions, we investigated the effects of various inhibitorson the activity of the H⁺-ATPase, the transport of protons,and the transport of Cl^- ions using plasma membrane vesiclesprepared from barley roots. Some inhibitors, namely, 4,4-diisothiocyano-2,2-stilbenedisulfonate (DIDS) and Zn²⁺ ions markedly inhibited H⁺- ATPaseactivity. Other compounds, such as phenylglyoxal (PGO) and niflumicacid (NIF), inhibited H⁺-ATPase activity by 20-30%, while anthracene-9-carboxylate(A-9-C) and tetraethylammonium chloride (TEA-Cl) had littleeffect on this activity. The ATP-dependent acidification ofthe interior of vesicles was strongly dependent on the presenceof permeant anions, such as chloride (Cl^-) and nitrate (NO₃^-),and it was completely inhibited by 0.2 mM NIF. Other compounds,namely, A-9-C of 0.1 mM and TEA-Cl of 10 mM, did not affectH⁺-transport activity. The inhibition of H⁺-transport activityby NIF was observed even when the activity was assayed in thepresence of KCl, KNO₃, or bis-tris-propane (BTP)-Cl. Using ³⁶cl,we quantified Cl^--transport activity by measuring the uptakeof Cl^- ions into the plasma membrane vesicles. The uptake dependedon the potential difference across the membrane that was generatedby H⁺-ATPase; it was enhanced by an inside-positive potentialgradient. At 0.1 mM, NIF completely blocked the voltage-dependentCl^--transport activity. From these properties of the Cl^- transporterand the inhibition of H⁺-transport activity by NIF, we suggestthat H⁺-transport activity across the plasma membrane mightbe modulated by the transport of anions via a NIF-sensitiveanion-permeable transporter that acts to collapse the inside-positivepotential generated by H⁺-ATPase. (Received September 7, 1995; Accepted July 23, 1996)     

In vivo Treatments That Modulate PPi-Dependent H+ Transport Activity of Tonoplast-Enriched Membrane Vesicles from Barley Roots

The PP_i-dependent H⁺ transport activity of tonoplast-enrichedmembrane vesicles prepared from barley roots was greatly reducedwhen the plants were grown for 4 or 5 days with an additional3 raM KC1 in growth medium that contained only 0.1 mM CaCl₂in water. To characterize the mechanism of this reduction inactivity, we attempted to treat barley roots with K⁺ ions, Cl^-ions(or acetate), and A23187 [GenBank] (with or without Ca²⁺ ions), whichmight be expected to cause alkalization, acidification and mobilizationof Ca²⁺ ions in the cytoplasm, respectively. One-day treatmentof barley roots with K⁺ ions significantly decreased PP_i--dependentH⁺ transport activity of prepared tonoplast-enriched membranevesicles, while treatment with Cl^- ions or acetate significantlyincreased the activity. A similar increase in the activity alsooccurred by treatment with Ca²⁺ ions alone or in combinationwith A23187 [GenBank] . Determination of the PP_i-hydrolyzing activity ofmembrane vesicles showed that changes in this activity by thevarious treatments were similar to those in the PP_i-dependentH⁺ transport activity. The changes in ATP-dependent H⁺ transportactivity of membrane vesicles caused by these treatments weresmall. These results indicate that the in vivo treatments hadsignificant effects on the H⁺ transport activity of H⁺-PP_i-ase,one of the two active vacuolar H⁺-pumps (H⁺-PP_iase and H⁺-ATPase).In addition, these results suggest the possibility that changesin levels of cytoplasmic H⁺ or Ca²⁺ ions may be involved inmodulation of the H⁺ transport activity of the vacuolar H⁺-PP_iaseduring plant growth. (Received September 14, 1992; Accepted March 1, 1993)     

Possible Involvement of Abscisic Acid in Increases in Activities of Two Vacuolar H+-Pumps in Barley Roots under Aluminum Stress

Levels of abscisic acid (ABA) in barley roots increased upontreatment with AlCl₃. Treatment with AlCl₃ or ABA increasedboth ATP-dependent and PP_i-dependent H⁺-pumping activities intonoplast-enriched membrane vesicles. Increase in the H⁺-pumpingactivities caused by aluminum stress could result from increasedlevels of ABA. ¹Present address: Department of Botany, Faculty of Science,Hirosaki University, Hirosaki, Aomori, 036 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).     

Effects of in Vivo Treatment with Abscisic Acid and/or Cytokinin on Activities of Vacuolar H+ Pumps of Tonoplast-Enriched Membrane Vesicles Prepared from Barley Roots

We investigated the effects of in vivo treatment (1 day) ofbarley roots with abscisic acid (ABA) and/or a cytokinin (6-benzyladenine;BA) on the ATP- and PP_i-dependent H⁺ transport activities oftonoplast-enriched membrane vesicles prepared from the roots.Treatment with ABA significantly increased the two H⁺ transportactivities. By contrast, treatment with BA significantly decreasedPP_i-dependent H⁺ transport activity, while the change in ATP-dependentH⁺ transport activity was small. Increases in the two H⁺ transportactivities caused by treatment with ABA were suppressed duringtreatment with ABA and BA. Changes in the NO^–-inhibitableATPase activity and the Na⁺-inhibitable PP_iase activity of membranevesicles after treatment of roots with phytohormone(s) (ABA,BA, ABA + BA) were similar to changes in the ATP- and PP_i dependentH⁺ transport activities of the membrane vesicles, respectively.Immunoblot analysis with antibodies raised against the functionalcatalytic subunits of the vacuolar H⁺ pumps (H⁺- ATPase andH⁺-PP_iase) of mung bean revealed that only the level of thefunctional catalytic subunit of the H⁺-PP_iase of the membranevesicles was significantly increased by treatment with ABA aloneand in combination with BA. These results suggest that treatmentwith ABA has a stimulatory effect on the activities of the twoH⁺ pumps of the vacuolar membrane of barley roots, with increasein the level of the catalytic subunit of the H⁺-PP_iase, andthat treatment with BA has an inhibitory effect on the two H⁺pump activities of the vacuolar membrane without changes inthe levels of the catalytic subunits of either H⁺ pump, withthe limitation that treatment with BA has an inhibitory effectonly when the activity of the H⁺-ATPase has been increased bytreatment with ABA. ³Present address: Department of Biology, Faculty of Science,Hirosaki University, Hirosaki, 036 Japan     

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)     

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     

Repression of Proton extrusion from Intact Cucumber Roots and the Proton Transport Rate of Microsomal Membrane Vesicles of the Roots due to Ca2+ Starvation

Proton extrusion from cucumber roots decreased markedly duringCa²⁺ starvation in the presence of KC1. Vesicles with ATP-dependentproton transport activity were prepared from the microsomalmembrane fraction of control and Ca²⁺-starved roots. The protontransport rate of the vesicles from Ca²⁺-starved roots was repressedto less than half of the vesicles prepared from the controlroots. K⁺-Mg²⁺-ATPase activity associated with the vesiclesprepared from Ca²⁺-starved roots was approximately one-thirdof the activity associated with those prepared from controlroots. K_m values of the proton transport rate and K⁺-Mg²⁺-ATPasefor ATP were much higher in vesicles prepared from Ca²⁺-starvedroots. The repression of proton extrusion linked with K⁺ uptake inthe Ca²⁺-starved roots could be largely caused by the reducedproton pumping activity associated with microsomal membranesin the roots. (Received May 25, 1987; Accepted October 14, 1987)     

Characterization of Two Proton Transport Systems in the Tonoplast of Plasmalemma-Permeabilized Nitella Cells

ATP-dependent and PP_i-dependent H⁺-transport systems of thetonoplast were characterized in plasmalemma-permeabilized Nitellacells, where direct access to the protoplasmic surface of thetonoplast was possible. Since H⁺ transport across the tonoplastcan be measured in situ, the identity of the membrane responsiblefor H⁺ pumping is unequivocal. H⁺ transport was evaluated bythe accumulation of neutral red. While both transport systemswere obligately dependent on Mg²⁺, the two transport systemsshowed completely different sensitivity to NO₃^– and K⁺,suggesting the presence of two types of H⁺-pumps in Nitellatonoplast. NO₃^– applied to the protoplasmic surface, completelyand reversibly inhibited ATP-dependent transport but had noeffect on PP_i-dependent transport. By contrast, NO₃^– appliedinto the vacuole by the vacuolar perfusion technique did notinhibit ATP-dependent or PP_i-dependent H⁺ transport. Replacementof K⁺ with the organic cation, BTP, inhibited PP_i-dependenttransport but not the ATP-dependent one, indicating that PP_i-dependenttransport is K⁺ dependent. The sensitivities of the H⁺ transportsystems found in the tonoplast of Nitella are quite similarto those of higher plant tonoplasts. ¹ Present address: Department of Botany, Faculty of Science,University of Tokyo, Hongo, Tokyo 113, Japan. (Received February 21, 1987; Accepted May 27, 1987)     

Aluminum Stress Increases K+ Efflux and Activities of ATP- and PPi- Dependent H+ Pumps of Tonoplast-Enriched Membrane Vesicles from Barley Roots

Aluminum stress significantly stimulated K⁺ effux from barleyroots that had been preloaded with K⁺ The stress also increasedPP₁- and ATP-dependent H⁺ pump activities of tonoplast enrichedmembrane vesicles from the roots. Ca²⁺ ions reduced increasesin K⁺ effux and both H⁺ pump activities under these conditions. (Received April 25, 1992; Accepted August 4, 1992)     

Salt Stress Induced Nuclear and DNA Degradation in Meristematic Cells of Barley Roots

Root growth of barley (Hordeum vulgare L., cv. Akashinriki)was inhibited by 200 raM NaCl, when 1 mM CaCl₂ was present inthe hydroponic culture solution. Increasing the CaCl₂ up to10 mM partially prevented this inhibition. However, inhibitionalso occurred with 100 mM NaCl in the presence of 0.1 mM CaCl₂.The nuclei of meristematic cells in roots in which growth hadbeen inhibited by salt stress were studied after staining withDAPI (4',6-diamino-2-phenylindol). Nuclear deformation of thecells occurred with 12 h of salt stress with 500 mM NaCl, andwas followed by degradation. The nuclear degradation was alsoobserved when the roots were exposed to more than 300 mM NaClfor 24 h. Biochemical analysis revealed that nuclear degradationwas accompanied by apoptosis-like DNA fragmentation. The intracellularmechanisms of nuclear degradation in cells after salt stressare discussed. ¹Emertius professor, Okayama University.     

External Potassium Supply is not Required for Root Growth in Saline Conditions: Experiments with Ricinus communis L. Grown in a Reciprocal Split-Root System

Ricinus communis L. (castor bean) plants were grown in the absence(control) and in the presence of 100molm^–3NaCl with areciprocal split-root system, in which K⁺ was supplied to oneand NO₃^– to the other part of the root system. In theseplants shoot and, to a lesser extent, total root growth wereinhibited compared to plants with non-split roots. Without andwith NaCl, growth of roots receiving NO₃^– but noK⁺ (‘minusK/plus N-roots’) was substantially more vigorous thanunder the reverse conditions (‘plus K/minus N-roots¹).100mol m^–3 NaCl inhibited growth of minus K/plus N-roots¹to the same extent as that of non-split roots, indicating thatexternally supplied K⁺ was not required for root growth undersaline conditions. In growth media without added K⁺ the rootdepleted the external low K ⁺ levels resulting from chemicalsdown to a minimum value C_mln (1.0 to 1.4 mmol m^–3); inthe presence of 100 mol m^–3 NaCl, C_min, was higher (10–18mmol m^–3) and resulted from an initial net loss of K ⁺.C_min, was pH-dependent The distribution of K⁺, Na⁺ and Mg²⁺along the root was measured. In meristematic root tissues, K⁺ concentrations were scarcely affected by external K⁺ or byNaCl, where Na ⁺ concentrations were low, but somewhat elevatedat low external K+ and/or high NaCl. In differentiated, vacuolatedtissues K ⁺ concentrations were low and Na⁺ concentrations high,if K ⁺ was not supplied externally and/or NaCl was present.The longitudinal distribution of ions within the root was usedto estimate cytoplasmic and vacuolar ion concentrations. Thesedata showed a narrow homoeostasis of cytoplasmic K+ concentrations(100–140 mol m^–3) independent of external K ⁺ supplyeven in the presence of 100 mol m ^–3 NaCl. CytoplasmicNa ⁺ concentrations were maintained at remarkably low levels.Hence, external K⁺ concentrations above C_min, were not requiredfor maintaining K/Na selectivity, i.e. for controlling Na⁺ entry.The results are discussed with regard to mechanisms of K/Naselectivity and to the importance of phloem import of K⁺ forsalt tolerance of roots and for cytoplasmic K⁺ homoeostasis. Key words: Ricinus communis, nitrate, potassium, root (split-root), salt tolerance, phloem transport     

Characterization, Functional Reconstitution and Activation by Fusicoccin of a Ca2+-ATPase from Corydalis sempervirens Pers. Cell Suspension Cultures

Cell suspension cultures of Corydalis sempervirens have provenideal for the study of fusicoccin action [Schulz et al. (1990)Planta 183: 83] and express the fusicoccin-binding protein aswell as a plasma membrane H⁺-ATPase which is activated by thefungal toxin. Microsomal vesicles prepared from these cellsaccumulate Ca²⁺ in the presence of Mg-ATP. The protonophorecar-bonylcyanide m-chlorophenylhydrazone did not inhibit theMg-ATP dependent Ca²⁺-transport into the vesicles. This processis thus due to the activity of at least one primary active,ATP-driven, Ca²⁺-pump. The enzyme was characterized in detail.It has a pH optimum of 7.2, an apparent K_m of 0.3 mu (ATP),12pm (Ca²⁺), accepts ATP>ITP GTP>CTP UTP, and is strongly(Kⁱ, app 0.75 µmM) inhibited by erythrosine B but lessso (Kⁱ, app 95 µM) by or-thovanadate. These characteristicsare typical for the plasma membrane Ca²⁺-ATPase characterizedfrom differentiated tissues [Graf and Weiler (1990) Physiol.Plant. 75: 634]. Fusicoccin activates the erythrosine-sensitiveCa²⁺-pump by lowering its K_m for ATP, when added to living cellsprior to tissue homogenization. Thus, fusicoccin appears toactivate at least two ion-translocating ATPases in one and thesame tissue, suggesting that the toxin's mechanism of actionis complex and not restricted to activation of the H⁺-ATPase.FC has no effect when administered to microsomes. The microsomalenzyme was solubilized and reconstituted into asolec-tin liposomesin functional form. The reconstituted, erythrosine sensitiveCa²⁺-ATPase was insensitive to fusicoccin. Thus, componentsessential for toxin action are either lost or inactivated duringsubcellular fractionation. It is likely that FC action requiressoluble components. (Received April 22, 1991; Accepted July 24, 1991)     

Regulation of K+ Influx in Barley: Evidence for a Direct Control of Influx by K+ Concentration of Root Cells

Siddiqi, M. Y. and Glass, A. D. M. 1987. Regulation of K⁺ influxin barley: Evidence for a direct control of influx by K⁺ concentrationof root cells.—J. exp. Bot. 38: 935–947. The kinetics of K⁺ (⁸⁶Rb⁺) influx into intact roots of barley(Hordeum vulgare L. cv. Fergus) seedlings having different combinationsof root and shoot [K⁺], different growth rates and differentroot:shoot weight ratios were studied. K⁺ influx was stronglycorrelated with root [K⁺]; shoot [K⁺], growth rates, and root:shoot ratios appeared to have little effect on K⁺ influx. Adetailed study showed that both V_max and K_m for K⁺ influx wereaffected by root [K⁺] but not by shoot [K⁺]. We have suggestedthat factors such as growth rates and root: shoot ratio mayaffect K⁺ influx indirectly primarily via their influence onroot factors such as root [K⁺]. We have reiterated that othertypes of kinetic control, e.g. increased or decreased synthesisof ‘carrier systems’, may operate in addition todirect (allosteric?) control of K⁺ influx by root [K⁺]. Thenegative feedback signal from root [K⁺] appeared to be the primeeffector in the regulation of K⁺ influx. Key words: Barley, K⁺ influx     

Ion Effects on the H+-Translocating Adenosine Triphosphatase and Pyrophosphatase Associated with the Tonoplast of Chora corallina

H⁺-translocating ATPase and pyrophosphatase (PPase) associatedwith the tonoplast of Chara corallina were isolated with theaid of a perfusion technique, and the effects of ions on theiractivities were studied. All the alkali metal cations testedstimulated the ATPase and ATPdependent H⁺ pumping activitiesonly by 10 to 40%. Anions, on the other hand, strongly affectedthe activities. Potassium salts of Cl^- and Br^- stimulated them,while F^- and NO₃^- inhibited them. By contrast, the H⁺-translocatingPPase was insensitive to anions but sensitive to cations. Theorder of cation stimulation was Rb⁺=K⁺>Cs⁺>Na⁺=Li⁺>choline⁺.NO₃^- (50 mil), thought to be a specific inhibitor of the tonoplast-typeH⁺-ATPase, inhibited the ATPdependent H⁺ pumping almost completelybut the ATPase activity by only about 50%. Na⁺ inhibited thePP₁-dependent H⁺ pumping (I_5O=5OmM) in the presence of 50 mMKCl but not the ATP-dependent one. The PPase was more sensitiveto F^- (I₅₀=400µM) than the ATPase. Both the H⁺-ATPaseand the H⁺-PPase required Mg²⁺ for their activities, althoughan excess was inhibitory to both. The different sensitivitiesof the PP₁-dependent and the ATP-dependent H⁺- pumping enzymesto ions correspond to the tonoplast enzymes of higher plantsand may be used as "markers" to distinguish between these enzymesin characean cells (Received October 2, 1987; Accepted May 18, 1988)     

Ionic Relations of Garden Orache, A triplex hortensis L.: Growth and Ion Distribution at Moderate Salinity and the Function of Bladder Hairs

The growth of garden orache, A triplex hortensis was studiedunder conditions of mild NaCl or Na₂SO₄ salinity. Growth, drymatter production and leaf size were substantially stimulatedat 10 mM and 50 mM Na⁺ salts. Increased growth, however, appearedto be due to a K⁺-sparing effect of Na⁺ rather than to salinityper se. The distribution of K⁺ and Na⁺ in the plant revealeda remarkable preference for K+ in the roots and the hypocotyl.In the shoot the K/Na ratio decreased strongly with leaf age.However, the inverse changes in K⁺ and Na⁺ content with leafage were dependent on the presence of bladder hairs, which removedalmost all of the Na⁺ from the young leaf lamina. Measurementsof net fluxes of K⁺ and Na⁺ into roots and shoots of growingAtriplex plants showed a higher K/Na selectivity of the netion flux to the root compared to the shoot. With increasingsalinity the selectivity ratio S_{K, Na}^* of net ion fluxes tothe roots and to the shoots was increased. The data suggestthat recirculation of K⁺ from leaves to roots is an importantlink in establishing the K/Na selectivity in A. hortensis plants.The importance of K⁺ recirculation and phloem transport forsalt tolerance is discussed. Key words: Atriplex hortensis, Salinity, Potassium, Sodium, K⁺ retranslocation, Bladder hairs, Growth stimulation     

Efficiency of Potassium Utilization by Barley Varieties: The Role of Subcellular Compartmentation

Memon, A. R., Saccomani, M. and Glass, A. D. M. 1985. Efficiencyof potassium utilization by barley varieties: The role of subcellularcompartmentation.?J. exp. Bot. 36: 1860–1876. The subcellulardistributions of K⁺ in roots of three barley (Hordeum vulgareL.) varieties, grown at 10 and 100 mmol m^–3 external K⁺([K⁺]_o) were estimated by compartmental analyses. In general,increased [K⁺]_o caused a 2–3 fold increase in vacuolar[K⁺], but cytoplasmic [K⁺] increased only slightly. Nevertheless,the three varieties, which had been selected for study on thebasis of their different rates of K⁺ utilization, showed distinctdifferences in the allocation of K⁺ between cytoplasm and vacuole.At 10 mmol m^–3 [K⁺]_o var. Betzes exhibited typical K⁺deficiency symptoms while var. Fergus and var. Compana did not,even though Betzes had higher [K⁺] in shoots and roots. Theinefficient utilization of K⁺ in this variety appears to beassociated with a failure to mobilize vacuolar K⁺ into the cytoplasmiccompartment (the ratio of vacuolar: cytoplasmic K⁺ contentsfor Betzes was 4.1 compared to 2.7 and 2.5, respectively, forFergus and Compana). Fergus and Betzes, which demonstrate pronouncedgrowth responses to increased [K⁺]₀ between 10 and 100 mmolm^–3, showed significant increases of cytoplasmic [K⁺]in this range of [K⁺]_o. By contrast, cytoplasmic [K⁺] in Compana,a variety whose growth is not stimulated by increased [K⁺]₀(from 10 to 100 mmol m^–3) showed virtually no increase.It is suggested that the efficiency of K⁺ utilization and thegrowth response to [K⁺]₀ in these varieties are functions ofthe subcellular distribution of this ion between cytoplasm andvacuole. Key words: Barley varieties, K⁺ subcellular compartmentation, utilization efficiency     

Repression of Asolectin-Dependent Activation of Partially Lipid Depleted ATPase Prepared from the Plasma Membrane-Enriched Fraction of Cucumber Roots due to Ca2+ Starvation

The ATPase activity of the plasma membrane-enriched fractionwas severely inhibited by withdrawal of Ca²⁺ from the mediumfor 5 days, although the root system appeared to be unaffectedto visual inspection. Partially lipid-depleted ATPases withsimilar ratios of phospholipid to protein were prepared fromthe plasma membrane-enriched fraction of cucumber roots culturedwith control medium and one lacking Ca²⁺, and their propertieswere compared. SDS disc polyacrylamide gel electrophoresis showedthat the polypeptide components were essentially similar betweencontrol and Ca²⁺-starved roots. Partially lipid-depleted ATPasereassociated with asolectin, the lecithin from soybean, showedtypical characteristics of plasma membrane type ATPase; pH optimumat 6.5, high specificity for ATP as substrate and strong inhibitionby vanadate but not nitrate. The activity of reassociated ATPaseobtained from the control roots was apparently higher than theactivity obtained from Ca²⁺-starved roots. The amount of asolectinrequired for maximum activation of the partially lipid-depletedATPase prepared from control roots was much lower than thatprepared from Ca²⁺-starved roots. Reassociation of partiallylipid-depleted ATPase with asolectin produced higher ATPaseactivity than that with individual phospholipids. The activationof partially lipid-depleted ATPase prepared from control rootswith asolectin was not inhibited by addition of a sample preparedfrom Ca²⁺-starved roots. Thus, a decrease in the functionalassociation of ATPase with phospholipids might be one of thephysiological injuries in root cell membranes of cucumber causedby Ca²⁺ starvation. ¹Permanent address: Department of Horticulture, College of Agriculture,Chonnam National University, Chonnam 500, Korea. (Received February 23, 1988; Accepted August 18, 1988)     

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.