Enhanced H Transport Capacity and ATP Hydrolysis Activity of the Tonoplast H-ATPase after NaCl Adaptation

Tonoplast enriched membrane vesicle fractions were isolated from unadapted and NaCl (428 millimolar) adapted tobacco cells (Nicotiana tabacum L. var Wisconsin 38). Polypeptides from the tonoplast enriched vesicle fractions were separated by SDS-PAGE and analyzed by Western blots using polyclonal antibodies to the 70 kilodalton subunit of the red beet tonoplast H⁺-ATPase. These antibodies cross-reacted exclusively to a tobacco polypeptide of an apparent molecular weight of 69 kilodaltons. The antibodies inhibited ATP-dependent, NO₃⁻ sensitive H⁺ transport into vesicles in tonoplast enriched membrane fractions from both unadapted and NaCl adapted cells. The relative H⁺ transport capacity per unit of 69 kilodalton subunit of the tonoplast ATPase of vesicles from NaCl adapted cells was fourfold greater than that observed for vesicles from unadapted cells. The increase in specific H⁺ transport capacity after adaptation was also observed for ATP hydrolysis.     

Growth,photosynthesis and H+-ATPase activity in two Jerusalem artichoke varieties under NaCl-induced stress

In order to evaluate differential growth, photosynthesis and H⁺-ATPase activity responses to salt-induced stress, two Jerusalem artichoke (Helianthus tuberosus L.) genotypes (Nanyu No. 1 and Qingyu No. 2) were used in sand-culture experiment with different concentrations of NaCl (0, 30, 60, 90, 120 and 150 mM). After 20 days of growth, the NaCl stress resulted in a decrease of biomass accumulation, relative leaf expansion rate and photosynthetic rate, but an increase of proline content in both genotypes. Compared with Qingyu No. 2, Nanyu No. 1 had lower biomass, photosynthetic rate, gas exchange and transpiration rate, but higher proline content, activities of plasma membrane H⁺-ATPase (PM H⁺-ATPase) and vacuolar membrane H⁺-ATPase (VM H⁺-ATPase). Hence, the NaCl adaptation strategy in Nanyu No. 1 was by lowering photosynthetic rate, stomatal conductance and transpiration rate while maintaining high H⁺-ATPase activities, whereas the adjustment of Qingyu No. 2 was by keeping much higher rate of proline accumulation and concentration of chlorophyll. The differences in salt tolerance showed that different adaptation mechanisms existed between cultivars of Jerusalem artichoke. The findings offered the possibility of selecting salt-tolerant genotypes of Jerusalem artichoke.     

Utilization of metabolic energy under saline conditions: changes in properties of ATP dependent enzymes in plant cells grown under saline conditions

The effect of growth in saline medium on the activity of two ATP utilizing enzymes was studied. Hexokinase in carrot (Daucus carota L.) cells grown in suspension culture either in the absence or presence of 150 ml NaCl, and tonoplast H⁺-ATPase in tobacco (Nicotiana tabacum L. cv. Wisconsin 38) cells grown in suspension culture either in the absence of presence of 428 mM NaCl. There was no difference in the pH profiles, NaCl sensitivity and kinetic parameters towards glucose of hexokinase activities from carrot cells grown in the presence or the absence of NaCl, but the activity from cells grown in the presence of NaCl was more resistant to inhibition by N-ethylmaleimide and to inactivation by heat. Two separate apparent Km values toward ATP were delineated in the extract from cells grown in presence of NaCl while extracts from cells grown in the absence of NaCl had only one apparent Km value. The tonoplast H⁺-ATPase from NaCl grown tobacco cells showed changed kinetic compared to this activity from cells grown in the absence of NaCl. These data may indicate that growth in NaCl results in the appearance of isozymic activity that enhances the ability of plant cells to utilize metabolic energy more efficiently.     

Salinity adaptation of plasma membrane H+-ATPase in the salt marsh plant Spartina patens: ATP hydrolysis and enzyme kinetics

Spartina patens, an intertidal C4 grass, grows in the upper salt marsh and tolerates coastal seawater salinity. The regulation of ion movement across the plasma membrane (PM) for plant salt tolerance is thought to be achieved by an electrochemical gradient generated by plasma membrane H⁺-ATPase. In this study, the change of PM H⁺-ATPase in response to NaCl was characterized for S. patens callus. Callus was cultured for 10 weeks under salinity levels of 0 mM, 170 mM, 340 mM, and 510 mM NaCl. Plasma membrane was isolated from a Dextran/PEG aqueous polymer two-phase system and the purity was demonstrated with membrane enzyme markers. There was a significant increase (up to 2-3 fold) of PM H⁺-ATPase activity when callus was grown on media containing NaCl. The incremental activation of PM H⁺-ATPase activity would enable the cell to tolerate higher cytoplasmic NaCl concentrations. PM H⁺-ATPase appeared to have a higher Vmax and a lower substrate concentration (Km to reach Vmax. When growth medium salinity increased from 0 mM to 170 and 340 mM, the Vmax of H⁺-ATPase increased from 0.64 to 1.00 and 1.73, respectively, while the Km decreased from 3.58 to 2.07 and 2.44 mM, respectively. In vitro NaCl inhibition kinetic data revealed a pattern of non-competitive inhibition by NaCl on PM H⁺-ATPase. The response of PM H⁺-ATPase in S. patens callus suggests that this species has evolved mechanisms that can regulate this important enzyme when cells are exposed to NaCl.     

Salt-induced changes on H<Superscript>+</Superscript>-ATPase activity,sterol and phospholipid content and lipid peroxidation of root plasma membrane from dwarf-cashew (<Emphasis Type="Italic">Anacardium occidentale</Emphasis> L.) seedlings

The effects of NaCl stress on the growth, water relation, gas exchange, tissue mineral content, and on H⁺-ATPase activity, lipid composition and peroxidation of root plasma membrane-enriched fractions of two genotypes (CCP06 and BRS189, sensitive and tolerant to salt stress, respectively) of dwarf-precocious cashew were studied. Growth reduction was higher in CCP06 than in BRS189. Net photosynthesis decreased in both genotypes, CCP06 being more affected. Roots of BRS189 accumulated higher amount of Na⁺ than those of CCP06 at both salt treatments, whereas Cl⁻ increase was higher only at 8 dS m⁻¹. NaCl at 8 dS m⁻¹ did not modify the plasma membrane H⁺-ATPase activity in CCP06 roots, but significantly increased it in BRS189 roots. Lipid peroxidation was lower in BRS189 than in CCP06 roots. Salinity induced higher accumulation of proline in BRS189 roots. Total phospholipids and free sterols content increased significantly in root plasma membrane of CCP06. However, in BRS189, a slight reduction of free sterols content and no changes in total phospholipids content were observed. Thus, the results suggest that the ability of cashew seedlings to adapt to salt stress is, at least in part, dependent upon the maintenance of integrity and protection against oxidative damage of plasma membrane, which could favor the activation of plasma membrane H⁺-ATPase, as a cellular mechanism to regulate ion exclusion from the shoot.     

Modulation of Na/H Antiporter Activity by Extreme pH and Salt in the Halotolerant Alga Dunaliella salina

The effect of different growth conditions on the activity of the Na⁺/H⁺ antiporter in Dunaliella salina has been investigated. Adaptation of D. salina cells to ammonia at alkaline pH or to high NaCl concentrations is associated with a pronounced increase in the plasma membrane Na⁺/H⁺ exchange activity. The enhanced activity is manifested both in vivo, by stimulation of Na⁺ influx into intact cells in response to internal acidification, and in vitro, by a larger ²²Na accumulation in plasma membrane vesicles in response to an induced pH gradient. Kinetic analysis shows that the stimulation does not result from a change of the K_m for Na⁺ but from an increase in the V_max. In contrast, adaptation of cells to a high LiCl concentration (0.8 m) depresses the activity of the Na⁺/H⁺ antiporter. Adaptation to ammonia is also associated with a large increase of three polypeptide bands in purified plasma membrane preparations, indicating that they may compose the antiporter polypeptides. These results suggest that adaptation to ammonia or to high salinity induces overproduction of the plasma membrane Na⁺/H⁺ antiporter in Dunaliella.     

NaCl increases the activity of the plasma membrane H+-ATPase in C3 halophyte Suaeda salsa callus

Suaeda salsa calli treated with different concentrations of NaCl were used to examine the response of the plasma membrane (PM) H⁺-ATPase to NaCl and its role in salt tolerance. The optimum concentration of NaCl for growth of the calli was 50 mM, while growth was significantly inhibited at 250 mM NaCl. The ion and organic solute contents of calli increased with increasing NaCl. Activity of the PM H⁺-ATPase increased when the calli were treated with NaCl over a certain concentration range (0–150 mM NaCl). However, the activity reached its maximum with 150 mM NaCl. Immunoblotting analysis of the PM H⁺-ATPase protein from calli cultures with anti-Zea mays H⁺-ATPase serum (monoclonal 46E5B11D5) identified a single polypeptide of ~90 kDa. The peptide levels increased in the calli treated with NaCl at 150 mM NaCl compared to control, but the increase at 50 mM NaCl was less pronounced. Northern blot analysis showed that the expression of the PM H⁺-ATPase also increased after the calli were treated with NaCl. These results suggest that the increase in PM H⁺-ATPase activity is due to both an increase in the amount of PM H⁺-ATPase protein and an up-regulation of the PM H⁺-ATPase gene, which is involved in the salt tolerance of S. salsa calli.     

Reduced Plasma Membrane H+-ATPase Isoform OsA7 Expression and Proton Pump Activity Decrease Growth Without Affecting Nitrogen Accumulation in Rice

Plasma membrane H⁺-ATPase (PM H⁺-ATPase, EC 3.6.1.3.) is a proton pump that is necessary to promote cell growth and ion fluxes across the plasma membrane. The main goal of this study was to evaluate the role of PM H⁺-ATPase isoform OsA7 expression in rice growth and nitrogen (N) accumulation using three genetically engineered lineages with artificial micro RNA (amiRNA) targeting OsA7 (osa7.1, osa7.2, and osa7.3). PM H⁺-ATPase isoform expression in rice shoots and roots (wild-type) revealed that OsA7 is highly expressed in roots and is the most highly expressed PM H⁺-ATPase isoform. The three osa7 lineages had lower fresh weight, grain yield, height, and 1000-grain weight compared to control IRS plants. The hydroponic experiment comprised three NO₃⁻ levels over 30 days: 0.2 mM NO₃⁻–N, 2.0 mM NO₃⁻–N, and NO₃⁻ starvation for 3 days. The three osa7 lineages had lower PM H⁺-ATPase and V-H⁺-PPase activity as compared to the IRS plants. The root and shoot fresh weights were lower in osa7 lineages. The root/shoot ratio was lower in the osa7 lineages cultivated without nitrogen for 3 days and with 0.2 mM of NO₃⁻–N as compared to IRS, and did not change in plants cultivated with 2.0 mM NO₃⁻–N. The total N concentration did not change in the three osa7 lineages as compared to IRS. Overall, the results indicate that OsA7 is important for rice growth, grain production, and root growth, but does not affect N accumulation, highlighting the importance of other PM H⁺-ATPase isoforms in N uptake.

     

Nitric oxide enhances salt tolerance in maize seedlings through increasing activities of proton-pump and Na+/H+ antiport in the tonoplast

Nitric oxide (NO), an endogenous signaling molecule in animals and plants, mediates responses to abiotic and biotic stresses. Our previous work demonstrated that 100 μM sodium nitroprusside (SNP, an NO donor) treatment of maize seedlings increased K⁺ accumulation in roots, leaves and sheathes, while decreasing Na⁺ accumulation (Zhang et al. in J Plant Physiol Mol Biol 30:455–459, 2004b). Here we investigate how NO regulates Na⁺, K⁺ ion homeostasis in maize. Pre-treatment with 100 μM SNP for 2 days improved later growth of maize plants under 100 mM NaCl stress, as indicated by increased dry matter accumulation, increased chlorophyll content, and decreased membrane leakage from leaf cells. An NO scavenger, methylene blue (MB-1), blocked the effect of SNP. These results indicated that SNP-derived NO enhanced maize tolerance to salt stress. Further analysis showed that NaCl induced a transient increase in the NO level in maize leaves. Both NO and NaCl treatment stimulated vacuolar H⁺-ATPase and H⁺-PPase activities, resulting in increased H⁺-translocation and Na⁺/H⁺ exchange. NaCl-induced H⁺-ATPase and H⁺-PPase activities were diminished by MB-1. 1-Butanol, an inhibitor of phosphatidic acid (PA) production by phospholipase D (PLD), reduced NaCl- and NO-induced H⁺-ATPase activation. In contrast, applied PA stimulated H⁺-ATPase activity. These results suggest that NO acts as a signal molecule in the NaCl response by increasing the activities of vacuolar H⁺-ATPase and H⁺-PPase, which provide the driving force for Na⁺/H⁺ exchange. PLD and PA play an important role in this process.     

The survival mechanism of dune reed (Phragmites communis) cultures under high sodium chloride concentration

Adaptations to salt stress were studied in embryogenic cultures from two ecotypes of reed (Phragmites communisT.). In the 600 mM NaCl treatment, relative cell viability of dune reed embryogenic cultures from a desert region was 56% greater than the control, 198% greater than swamp reed embryogenic cultures. After treatment with different NaCl concentrations, their relative growth rates (RGRs), pyridine nucleotides, activities of antioxidant enzymes and plasma membrane H⁺-ATPase (EC 3.6.1.35) were determined. The results showed that NADPH content, NADPH/NADP⁺ ratio and the activity of plasma membrane H⁺-ATPase in dune reed embryogenic cultures were higher than those of the control in the present of 600 mM NaCl. The activities of peroxidase (POD, EC 1.11.1.7) and catalase (CAT, EC 1.11.1.6) increased more in dune reed embryogenic cultures than in swamp reed embryogenic cultures. Dune reed embryogenic cultures tolerated higher concentration of NaCl than swamp reed embryogenic cultures. Under high concentration of NaCl, the survival of dune reed embryogenic cultures might be due to reductive status maintenance and ions absorption regulation in the plant cells. This phenomenon would be a result of cross-adaptation in nature.     

Sodium transport measured in plasma membrane vesicles isolated from wheat genotypes with differing K+/Na+ discrimination traits

Right-side-out plasma membrane vesicles were isolated from wheat roots using an aqueous polymer two-phase system. The purity and orientation of the vesicles were confirmed by marker enzyme analysis. Membrane potential (Ψ)-dependent ²²Na⁺ influx and sodium/proton (Na⁺/ H⁺) antiport-mediated efflux across the plasma membrane were studied using these vesicles. Membrane potentials were imposed on the vesicles using either K⁺ gradients in the presence of valinomycin or H⁺ gradients. The ΔΨ was quantified by the uptake of the lipophilic cation tetraphenylphosphonium. Uptake of Na⁺ into the vesicles was stimulated by a negative ΔΨ and had a K_m for extrav-esicular Na⁺ of 34.8 ± 5.9 mol m³. The ΔΨ-dependent uptake of Na⁺ was similar in vesicles from roots of hexaploid (cv. Troy) and tetraploid (cv. Langdon) wheat differing in a K⁺/Na⁺ discrimination trait, and was also unaffected by growth in 50 mol m^?3 NaCl. Inhibition of ΔΨ-dependent Na⁺ uptake by Ca²⁺ was greater in the hexaploid than in the tetraploid. Sodium/proton antiport was measured as Na⁺-dependent, amiloride-inhibited pH gradient formation in the vesicles. Acidification of the vesicle interior was measured by the uptake of ¹⁴C-methylamine. The Na⁺/H⁺ antiport had a K_m, for intravesicular Na⁺ of between 13 and 19 mol m^?3. In the hexaploid, Na⁺/H⁺ antiport activity was greater when roots were grown in the presence of 50 mol m^?3NaCl, and was also greater than the activity in salt-grown tetraploid wheat roots. Antiport activity was not increased in a Langdon 4D chromosome substitution line which carries a trait for K⁺/Na⁺ discrimination. It is concluded that neither of the transport processes measured is responsible for the Na⁺/K⁺ discrimination trait located on the 4D chromosome of wheat.     

Adaptation of the tonoplast V-type H+-ATPase of Mesembryanthemum crystallinum to salt stress, C3–CAM transition and plant age

Plants of the facultative halophyte and CAM species Mesembryanthemum crystallinum L. (Aizoaceae) were stressed for 8 d with 400 mol m⁻³ NaCl in the root medium. NaCl was then removed from the substratum, and the plants were watered again with NaCl-free solution. A second set of plants was maintained as controls. A small degree of CAM, as indicated by day-night changes in malate levels, was expressed during ageing of the plants. Salinity-stress-dependent CAM induction was reversible by the removal of salt, as indicated by similar Δ malate levels in previously salt-stressed plants and in non-stressed plants on day 19 of the experiment. Tonoplast vesicles were isolated from leaves during the time-course of stress application, stress removal and ageing. Parameters of the tonoplast H⁺-ATPase were correlated to the application of salinity, the expression of CAM and ageing. It was concluded, first, that a pronounced increase in the amount of tonoplast H⁺-ATPase is related to salinity per se and a smaller increase to ageing; secondly, that there is an increase in the specific activity of the enzyme related to ageing; thirdly, that the induction of two new polypeptides with molecular masses of 32 and 28 kDa is correlated in time with the expression of CAM, and, fourthly, that the two new polypeptides are part of the tonoplast H⁺-ATPase holoenzyme.     

Physiological Aspects of Adaptation of the Marine Microalga Tetraselmis (Platymonas) viridis to Various Medium Salinity

We studied the capability of the marine microalga Tetraselmis (Platymonas) viridis to adapt to low and high medium salinity. The normal NaCl concentration for growth of this alga is 0.5 M. It was shown that T. viridis cells could actively grow and maintain osmoregulation and cytoplasmic ion homeostasis in the wide range of external salt concentrations, from 0.01 to 1.2 M NaCl. Using the plasma membrane vesicles isolated from T. viridis cells grown at various NaCl concentrations (0.01, 0.05, 0.5, 0.9, and 1.2 M), we studied the formation of the phosphorylated intermediate of Na⁺-ATPase, the enzyme responsible for Na⁺ export from the cells with a mol wt of ca. 100 kD. Na⁺-ATPase was shown to function in the plasma membrane even in the cells growing at an extremely low NaCl concentration (0.01 M). When alga was grown in high-salt media, the synthesis of several proteins with molecular weights close to 100 kD was induced. The data obtained argue for the hypothesis, which was put forward earlier, that a novel Na⁺-ATPase isoform is induced by T. viridis growing at high NaCl concentrations.     

Effects of salt-adaptation and salt-stress on extracellular acidification and microsome phosphohydrolase activities in tomato cell suspensions

The effects of NaCl-adaptation and NaCl-stress on in vivo H⁺ extrusion and microsomal vanadate- and bafilomycin-sensitive ATPase and PPase activities were studied in tomato cell suspensions. Acidification of the external medium by 50 mM NaCl-adapted and non-adapted (control) tomato cells was similar. Extracellular acidification by both types of cells during the first hour of incubation with 2 μM fusicoccin (FC) in the presence of 100 mM NaCl was lightly increased while in the presence of 100 mM KCl it was increased by 3 (control)- and 6.5 (adapted)-fold. Extracellular alkalinization after 2 h of cell incubation in 100 mM NaCl indicated the possibility that a Na⁺/H⁺ exchange activity could be operating in both types of cells. Moreover, acidification induced by adding 100 mM NaCl + FC to non-adapted cells was relatively less affected by vanadate than that induced by 5 mM KCl + FC, which suggested that salt stress could induce some component other than H⁺ extrusion by H⁺-ATPase. In addition, no differences were observed in microsomal vanadate-sensitive ATPase activity among control, NaCl-adapted and NaCl-stressed cells, while K⁺-stimulated H⁺-PPase and bafilomycin-sensitive H⁺-ATPase activities were higher in microsomes from NaCl-adapted than in those from control cells. Likewise, the stimulation of in vivo H⁺ extrusion in NaCl adapted cells under NaCl or KCl stress in the presence of FC occurred with an inhibition of H⁺-PPase and bafilomycin-sensitive H⁺-ATPase activities and without changes in the vanadate-sensitive H⁺-ATPase activity. These results suggest that the stimulation of tonoplast proton pumps in NaCl-adapted cells, without changes in plasmalemma H⁺-ATPase, could serve to energize Na⁺ efflux across the plasmalemma and Na⁺ fluxes into vacuoles catalyzed by the Na⁺/H⁺ antiports. This revised version was published online in June 2006 with corrections to the Cover Date.     

Organ-Specific Responses of Vacuolar H~+-ATPase in the Shoots and Roots of C_3 Halophyte Suaeda salsa to NaCl

Suaeda salsa L. is a halophytic species that is well adapted to high salinity. In order to understand its salt tolerance mechanism, we examined the growth and vacuolar H⁺-ATPase (V-ATPase) response to NaCl within the shoots and roots. The growth of shoots, but not roots, was dramatically stimulated by NaCl. Cl⁻ and Na⁺ were mainly accumulated in shoots. V-ATPase activity was significantly increased by NaCl in roots and especially in shoots. Interestingly, antisera ATP95 and ATP88b detected three V₁ subunits (66, 55 and 36 KDa) of V-ATPase only in shoots, while an 18 kDa V₀ subunit of V-ATPase was detected by both antisera in shoots and roots. It suggested that the tissue-specific characteristics of V-ATPase were related to the different patterns of growth and ion accumulation in shoots and roots of S. salsa.     

Abscisic acid does not mediate NaCl-induced accumulation of 70-kDa subunit tonoplast H+ -ATPase message in tomato

There is an increased accumulation of message for the catalytic (70-kDa) subunit of the tonoplast H⁺-ATPase in leaves of tomato (Lycopersicon esculentum L.) plants responding to NaCl. To determine if abscisic acid (ABA) mediates this response, message accumulation was examined in treatments designed to separate exposure to NaCl from increases in endogenous ABA. Under three different experimental conditions, salt-induced changes in the accumulation of 70-kDa message were unrelated to any change in endogenous ABA. The results were as follows: (i) under drought stress, plants accumulated levels of ABA similar to those measured in salt-treated plants; however, no increase in 70-kDa subunit message was observed; (ii) the ABA-deficient mutant sitiens exhibited an increased accumulation of message despite the absence of NaCl-induced accumulation of ABA; and (iii) the inhibitor of general isoprenoid biosynthesis, Lovastatin, blocked NaCl-induced accumulation of ABA but did not alter NaCl-induced accumulation of message. In addition to these three experimental responses, application of exogenous ABA increased endogenous ABA levels without any comparable increase in message accumulation. Based on these results, it is concluded that ABA does not mediate the NaCl-induced accumulation of 70-kDa subunit tonoplast H⁺ -ATPase message accumulation in tomato.     

Boric acid stimulates the plasma membrane H+-ATPase of ungerminated lily pollen grains

The stimulation of the plasma membrane (PM) H⁺-ATPase by boric acid was studied on a microsomal fraction (MF) obtained from ungerminated, boron-dependent pollen grains of Lilium longiflorum Thunb. which usually need boron for germination and tube growth. ATP hydrolysis and H+ transport activity increased by 14 and 18%, respectively, after addition of 2-4 mM boric acid. The optimum of boron stimulation was at pH 6.5-8.5 for ATP hydrolysis and at pH 6.5-7.5 for H⁺ transport. No boron stimulation was detected when vanadate was added to the MF, whereas an increase of 10-20% in ATP hydrolysis and H⁺ transport was still measured in the presence of inhibitors specific for V -type ATPase (nitrate and bafilomycin) and F-type ATPase (azide), respectively. A vanadate-sensitive increase in ATP hydrolysis activity was also observed in partially permeabilized vesicles (0.001%[w/v] Triton X-100) suggesting a direct interaction between borate and the PM H⁺-ATPase rather than a weak acid-induced stimulation. Additionally, we measured the effect of boron on membrane voltage (V_m) of ungerminated pollen grains and observed small hyperpolarizations in 48% of all experiments. Exposing pollen grains to a more acidic pH of 4 caused a depolarization, followed in some experiments by a repolarization (21%). In the presence of 2 mM boron such hyperpolarizations, perhaps caused by an enhanced activity of the H⁺-ATPase, were measured in 58% of all tested pollen grains. The effects of boron on V_m may be reduced by additional stimulation of a K⁺ inward current of opposite direction to the H⁺-ATPase. All experiments indicate that boron stimulates an electrogenic transport system in the plasma membrane which is sensitive to vanadate and has a pH optimum around 7, i.e. the plasma membrane H⁺-ATPase. A boron-increased PM H⁺-ATPase activity in turn may stimulate germination and growth of pollen tubes.