Evidence that human α-thrombin is a monovalent cation-activated enzyme

The activity of human α-thrombin (EC 3.4.21.5) on small peptide substrates was enhanced by NaCl or KCl while tetramethylammonium chloride ((CH₃)₄NCl) or choline chloride (HO(CH₂)₂N(CH₃)₃Cl) which were used as ionic strength controls were without effect. The steady-state kinetic parameters of thrombin amidolysis of several peptidyl p-nitroanilide substrates were measured. Na⁺ enhanced thrombin activity by decreasing the K_m,app (0.2 to 0.7-fold) of all substrates, as well as increasing thombin turnover (3.4 to 4.5-fold) of some substrates. The average K_A for Na⁺for the four substrates examined was 3.5 × 10^?2m. A comparison of the effects of Na⁺ vs K⁺ on thrombin hydrolysis of a single substrate indicated that both cations similarly decreased the K_m,app (0.2 to 04.-fold) and increased thek_cat,app (3.1 to 3.4-fold) except that higher K⁺ concentrations (K_A = 2.8 × 10^?1M) were required. The rate of inactivation of thrombin by the active site-directed inhibitor N-p-tosyl-lysine chloromethyl ketone under pseudo-first-order conditions was enhanced 3-fold by saturating NaCl. Also, the fibrinogen clotting activity of thrombin was enhanced by NaCl compared to the choline chloride control. Spectral studies demonstrated that thrombin titration by Na⁺ caused a positive ultraviolet difference spectrum with maxima at 281.5 and 288.5 nm (Δ?_288.5 = +1067). The K_m for Na⁺ was 2.3 × 10^?2m which agrees with the kinetically determined K_A for Na⁺. The results are consistent with Na⁺ binding to thrombin causing a conformational change in the active site. It is concluded that human α-thrombin is a monovalent cation-activated enzyme.     

Biosorption of tributyltin and other organotin compounds by cyanobacteria and microalgae

Biosorption of triorganotin compounds by the cyanobacteria Synechocystis PCC 6803 and Plectonema boryanum and the microalga Chlorella emersonii, incubated in 2-(N-morpholino)ethanesulphonic acid (MES) buffer, pH 5.5, in the presence of 0.5 mm organotin (supplied as chlorides), increased with molecular mass of the organotins, the order being triphenyltin > tributylin (Bu₃SnCl) > tripropyltin >- trimethyltin >- triethylin. In the butylin series, monobutyltin biosorption was lowest, although levels of dibutyltin uptake were greater than for Bu₃SnCl. Cyanobacterial Bu₃SnCl biosorption was complete in 5 min with no subsequent accumulation. In contrast, a second phase of uptake in C. emersonii resulted in an approximate 2.4-fold increase in cellular Bu₃SnCl between 5 min and 2 h. The external pH had a marked influence on biosorption of Bu₃SnCl by Synechocystis PCC 6803 and P. boryanum, with maximal uptake at pH 5.5 and 6.5, respectively. Effects of pH were less evident in C. emersonii. In all the organisms examined, no inhibition of Bu₃SnCl biosorption was observed between 0.05 and 50 mm NaCl. However, an increase in the external NaCl concentration from 50 to 500 mm resulted in an approximate 55–65% reduction in Bu₃SnCl uptake. Biosorption increased at increasing Bu₃SnCl concentrations (0.25–3.0 mm). Saturation of Bu₃SnCl biosorption at the higher concentrations was most evident in the cyanobacteria, although uptake levels were greater in these organisms at <- 2 mm Bu₃SnCl. Theoretical maximum biosorption levels at complete cell saturation, derived from reciprocal Langmuir plots, were approximately 565, 525 and 1050 nmol Bu₃SnCl mg^–1 dry weight, for Synechocystis PCC 6803, P. boryanum and C. emersonii, respectively. Correspondence to: G. M. Gadd     

Mitochondria from the salt-tolerant yeast <Emphasis Type="Italic">Debaryomyces hansenii</Emphasis> (halophilic organelles?)

The yeast Debaryomyces hansenii is considered a marine organism. Sea water contains 0.6 M Na⁺ and 10 mM K⁺; these cations permeate into the cytoplasm of D. hansenii where proteins and organelles have to adapt to high salt concentrations. The effect of high concentrations of monovalent and divalent cations on isolated mitochondria from D. hansenii was explored. As in S. cerevisiae, these mitochondria underwent a phosphate-sensitive permeability transition (PT) which was inhibited by Ca²⁺ or Mg²⁺. However, D. hansenii mitochondria require higher phosphate concentrations to inhibit PT. In regard to K⁺ and Na⁺, and at variance with mitochondria from all other sources known, these monovalent cations promoted closure of the putative mitochondrial unspecific channel. This was evidenced by the K⁺/Na⁺-promoted increase in: respiratory control, transmembrane potential and synthesis of ATP. PT was equally sensitive to either Na⁺ or K⁺. In the presence of propyl-gallate PT was still observed while in the presence of cyanide the alternative pathway was not active enough to generate a ΔΨ due to a low AOX activity. In D. hansenii mitochondria K⁺ and Na⁺ optimize oxidative phosphorylation, providing an explanation for the higher growth efficiency in saline environments exhibited by this yeast.     

Potassium‐induced decrease in cytosolic Na+ alleviates deleterious effects of salt stress on wheat (Triticum aestivum L.)

• Accumulation of NaCl in soil causes osmotic stress in plants, and sodium (Na⁺) and chloride (Cl^?) cause ion toxicity, but also reduce the potassium (K⁺) uptake by plant roots and stimulate the K⁺ efflux through the cell membrane. Thus, decreased K⁺/Na⁺ ratio in plant tissue lead us to hypothesise that elevated levels of K⁺ in nutrient medium enhance this ratio in plant tissue and cytosol to improve enzyme activation, osmoregulation and charge balance.
• In this study, wheat was cultivated at different concentrations of K⁺ (2.2, 4.4 or 8.8 mm ) with or without salinity (1, 60 or 120 mm NaCl) and the effects on growth, root and shoot Na⁺ and K⁺ distribution and grain yield were determined. Also, the cytosolic Na⁺ concentration was investigated, as well as photosynthesis rate and water potential.
• Salinity reduced fresh weight of both shoots and roots and dry weight of roots. The grain yield was significantly reduced under Na⁺ stress and improved with elevated K⁺ fertilisation. Elevated K⁺ level during cultivation prevented the accumulation of Na⁺ into the cytosol of both shoot and root protoplasts. Wheat growth at vegetative stage was transiently reduced at the highest K⁺ concentration, perhaps due to plants' efforts to overcome a high solute concentration in the plant tissue, nevertheless grain yield was increased at both K⁺ levels.
• In conclusion, a moderately elevated K⁺ application to wheat seedlings reduces tissue as well as cytosolic Na⁺ concentration and enhances wheat growth and grain yield by mitigating the deleterious effects of Na⁺ toxicity.

     

Characteristics of 3-O-methylfluorescein phosphate hydrolysis by the (Na+ + K+)-ATPase

With 3-O-methylfluorescein phosphate (3-OMFP) as substrate for the phosphatase reaction catalyzed by the (Na⁺ + K⁺)-ATPase, a number of properties of that reaction differ from those with the common substratep-nitrophenyl phosphate (NPP): theK _m is 2 orders of magnitude less and the V_max is two times greater, and dimethyl sulfoxide (Me₂SO) inhibits rather than stimulates. In addition, reducing the incubation pH decreases both theK _m and V_max for K⁺-activated 3-OMFP hydrolysis as well as theK _0.5 for K⁺ activation. However, reducing the incubation pH increases inhibition by P_i and the V_max for 3-OMFP hydrolysis in the absence of K⁺. When choline chloride is varied reciprocally with NaCl to maintain the ionic strength constant, NaCl inhibits K⁺-activated 3-OMFP hydrolysis modestly with 10 mM KCl, but stimulates (in the range 5–30 mM NaCl) with suboptimal (0.35 mM) KCl. In the absence of K⁺, however, NaCl stimulates increasingly over the range 5–100 mM when the ionic strength is held constant. These observations are interpreted in terms of (a) differential effects of the ligands on enzyme conformations; (b) alternative reaction pathways in the absence of Na⁺, with a faster, phosphorylating pathway more readily available to 3-OMFP than to NPP; and (c) a (Na⁺ + K⁺)-phosphatase pathway, most apparent at suboptimal K⁺ concentrations, that is also more readily available to 3-OMFP.Abbreviations Et₃N triethyl amine - FITC fluorescein isothiocyanate - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonate - MES 2-(N-morpholino)ethanesulfonate - Me₂SO dimethyl sulfoxide - NPP p-nitrophenyl phosphate - 3-OMFP 3-O-methylfluorescein phosphate - TNP-ATP 2, (or 3)-O-(2,4,6-trinitrophenyl)-ATP     

Comparative analysis of trehalose production by Debaryomyces hansenii and Saccharomyces cerevisiae under saline stress

The comparative analysis of growth, intracellular content of Na⁺ and K⁺, and the production of trehalose in the halophilic Debaryomyces hansenii and Saccharomyces cerevisiae were determined under saline stress. The yeast species were studied based on their ability to grow in the absence or presence of 0.6 or 1.0 M NaCl and KCl. D. hansenii strains grew better and accumulated more Na⁺ than S. cerevisiae under saline stress (0.6 and 1.0 M of NaCl), compared to S. cerevisiae strains under similar conditions. By two methods, we found that D. hansenii showed a higher production of trehalose, compared to S. cerevisiae; S. cerevisiae active dry yeast contained more trehalose than a regular commercial strain (S. cerevisiae La Azteca) under all conditions, except when the cells were grown in the presence of 1.0 M NaCl. In our experiments, it was found that D. hansenii accumulates more glycerol than trehalose under saline stress (2.0 and 3.0 M salts). However, under moderate NaCl stress, the cells accumulated more trehalose than glycerol. We suggest that the elevated production of trehalose in D. hansenii plays a role as reserve carbohydrate, as reported for other microorganisms.     

披针叶黄华试管苗适应盐胁迫的渗透调节机制

以披针叶黄华(Thermopsis lanceolata)试管苗为材料,通过组培方法研究其在0、0.2%、0.4%、0.6%、0.8%和1.0%NaCl和Na2SO4胁迫30d后的生长、有机渗透调节物质和无机渗透调节物质(Na+、K+和Ca2+)含量的变化,以探讨其耐盐性机制。结果显示:(1)随NaCl和Na2SO4胁迫浓度的增加,披针叶黄华试管苗叶片脯氨酸和可溶性糖含量均显著持续增加,且NaCl胁迫下脯氨酸上升的幅度均大于相同浓度Na2SO4胁迫下的增幅,而可溶性糖上升的幅度却小于相同浓度Na2SO4胁迫下的幅度;可溶性蛋白含量随NaCl浓度的增大呈先升高后降低的趋势,但随Na2SO4浓度的增加呈持续上升的趋势。(2)随NaCl和Na2SO4浓度的增加,披针叶黄华试管苗Na+含量呈增加趋势且各处理均显著高于对照,Ca2+含量和叶片K+含量却呈逐渐减少趋势且各处理均显著低于对照,而根系K+含量呈先降后升的趋势;Na2SO4胁迫下披针叶黄华试管苗叶片Na+含量上升幅度以及K+和Ca2+含量下降幅度均明显低于相同浓度NaCl胁迫组;而Na+/K+和Na+/Ca2+比值随NaCl和Na2SO4浓度增加而升高;NaCl胁迫下,叶片Na+/K+和Na+/Ca2+高于相同浓度Na2SO4胁迫下的比值,而根系Na+/K+和Na+/Ca2+却低于相同浓度Na2SO4胁迫下的比值。研究表明,盐胁迫下,披针叶黄华试管苗通过抑制叶片中Na+积累并增加可溶性糖和可溶性蛋白含量,在根系中维持较高K+和Ca2+含量以及较低水平Na+/K+和Na+/Ca2+比,以降低披针叶黄华细胞渗透势来适应盐渍环境;披针叶黄华对NaCl胁迫的调节能力弱于Na2SO4。     

Effects of organotin and organolead compounds on yeasts

Summary Four methods were used to screen nine organotin and two organolead compounds for toxicity to 29 yeasts, representing 10 genera. Center well diffusion plates were useful in comparing the sensitivity of yeasts to the most toxic organometals but were not useful for comparisons between compounds because of differences in diffusion rates and lack of sensitivity. Two-layer diffusion plates (density gradient plates) were also of limited use for comparisons between compounds but provided quantitative information on toxicity and allowed comparisons between organisms. Two-dimensional diffusion plates were useful for estimating the effect of pH on organometal toxicity. Release of K⁺ from cell suspensions measured using a K⁺-electrode provided quantitative information and allowed comparisons between compounds and organisms. The presence of 3% NaCl in cell suspensions decreased the rates and extent of organotin-induced K⁺ release. Yeasts varied in their sensitivity from strain to strain, but tributyltin was the most toxic compound tested. Mono- and dimethyltins were the least toxic. Triphenyltin, dibutyltin, monobutyltin, trimethyltin, triethyltin, diethyllead, diethyltin, and dimethylleads showed intermediate toxicity, but triphenyltin and monobutyltin were the most toxic among the group.     

Differential effects of Na+, Mg2+, K+, Ca2+ and osmotic stress on the wild type and the NaCl-tolerant mutants stl1 and stl2 of Ceratopteris richardii

In order to identify physiological components that contribute to salinity tolerance, we compared the effects of Na⁺, Mg²⁺ and K⁺ salts (NaCl, Na₂SO₄, MgCl₂, MgSO₄, KCl and K₂SO₄), Ca₂₊ (CaSO₄), mannitol and melibiose on the wild type and the single-gene NaCl-tolerant mutants stl1 and stl2 of Ceratopteris richardii. Compared with gametophytic growth of the wild type, stl2 showed a low level of tolerance that was restricted to Na⁺ salts and osmotic stress. stl2 exhibited high tolerance to both Na⁺ and Mg²⁺ salts, as well as to osmotic stress. In response to short-term exposure (3 d) to NaCl, accumulation of K⁺ and Na⁺ was similar in the wild type and stl1. In contrast, stl2 accumulated higher levels of K⁺ and lower levels of Na⁺. Ca²⁺ supplementation (1.0 mol m^?3) ameliorated growth inhibition by Na⁺ and Mg²⁺ stress in wild type and stll, but not in stl2. In addition, under Na⁺ stress (175 mol m^?3) wild-type, stll and stl2 gametopbytes maintained higher tissue levels of K⁺ and lower levels of Na⁺ when supplemented with Ca²⁺ (1.0 mol m^?3). stl2 gametophytes were extremely sensitive to K⁺ supplementation. Growth of stl2 was greater than or equal to that of the wild type at trace concentrations of K⁺ but decreased substantially with increasing K⁺ concentration. Supplementation with K⁺ from 0 to 1.85 mol m^?3 alleviated some of the inhibition by 75 mol m^?3 NaCl in the wild type and in stl1. In stl2, growth at 75 mol m^?3 NaCl was similar at 0 and 1.85 mol m^?3 K⁺ supplementation. Although K⁺ supplementation above 1.85 mol m^?3 did not alleviate inhibition of growth by Na⁺ in any genotype, stl2 maintained greater relative tolerance to NaCl at all K⁺ concentrations tested.     

Kinetic studies on the (Na+ + K+)-dependent ATPase. Evidence for coexisting sites for Na+, K+ and Mg2+

Na⁺-ATPase activity of a dog kidney (Na⁺ + K⁺)-ATPase enzyme preparation was inhibited by a high concentration of NaCl (100 mM) in the presence of 30 μM ATP and 50 μM MgCl₂, but stimulated by 100 mM NaCl in the presence of 30 μM ATP and 3 mM MgCl₂. The $\text{K}{}_{0.5}$ for the effect of MgCl₂ was near 0.5 mM. Treatment of the enzyme with the organic mercurial thimerosal had little effect on Na⁺-ATPase activity with 10 mM NaCl but lessened inhibition by 100 mM NaCl in the presence of 50 μM MgCl₂. Similar thimerosal treatment reduced (Na⁺ + K⁺)-ATPase activity by half but did not appreciably affect the $\text{K}{}_{0.5}$ for activation by either Na⁺ or K⁺, although it reduced inhibition by high Na⁺ concentrations. These data are interpreted in terms of two classes of extracellularly-available low-affinity sites for Na⁺: Na⁺-discharge sites at which Na⁺-binding can drive E₂-P back to E₁-P, thereby inhibiting Na⁺-ATPase activity, and sites activating E₂-P hydrolysis and thereby stimulating Na⁺-ATPase activity, corresponding to the K⁺-acceptance sites. Since these two classes of sites cannot be identical, the data favor co-existing Na⁺-discharge and K⁺-acceptance sites. Mg²⁺ may stimulate Na⁺-ATPase activity by favoring E₂-P over E₁-P, through occupying intracellular sites distinct from the phosphorylation site or Na⁺-acceptance sites, perhaps at a coexisting low-affinity substrate site. Among other effects, thimerosal treatment appears to stimulate the Na⁺-ATPase reaction and lessen Na⁺-inhibition of the (Na⁺ + K⁺)-ATPase reaction by increasing the efficacy of Na⁺ in activating E₂-P hydrolysis.     

Functional Characterization of a Na+-dependent Aspartate Transporter from Pyrococcus horikoshii

Excitatory amino acid transporters (EAATs) are crucial in maintaining extracellular levels of glutamate, the most abundant excitatory neurotransmitter, below toxic levels. The recent three-dimensional crystal structure of Glt_Ph, an archaeal homolog of the EAATs, provides elegant structural details of this family of proteins, yet we know little about the mechanism of the bacterial transporter. Conflicting reports in the literature have described Glt_Ph as an aspartate transporter driven by Na⁺ or a glutamate transporter driven by either Na⁺ or H⁺. Here we use purified protein reconstituted into liposomes to thoroughly characterize the ion and substrate dependence of the Glt_Ph transport. We confirm that Glt_Ph is a Na⁺-dependent transporter that is highly selective for aspartate over other amino acids, and we show that transport is coupled to at least two Na⁺ ions. In contrast to the EAATs, transport via Glt_Ph is independent of H⁺ and K⁺. We propose a kinetic model of transport in which at least two Na⁺ ions are coupled to the cotransport of each aspartate molecule by Glt_Ph, and where an ion- and substrate-free transporter reorients to complete the transport cycle.     

Sodium plays a more important role than potassium and chloride in growth of <Emphasis Type="Italic">Salicornia europaea</Emphasis>

Salicornia europaea is a succulent euhalophyte that belongs to the Chenopodiaceae family. It is found that moderate concentration of NaCl can dramatically stimulate the growth of S. europaea plants. To elucidate the mechanism underlying the phenomenon, morphological and physiological changes of S. europaea in response to different ions, including cations (Na⁺, K⁺, Li⁺, Cs⁺) and anions (Cl⁻, NO₃ ⁻, CH₃COO⁻) were investigated, and the effects of Na⁺, Cl⁻ and K⁺ on the growth of S. europaea were also studied. Na⁺ was more effective than K⁺ and Cl⁻ in stimulating shoot succulence, cell expansion, and stomatal opening. Plants treated with Na⁺ (including NaCl, Na⁺, NaNO₃) showed better plant growth, increased photosynthesis and less cell membrane damage than those untreated and treated with 200 mM of Cl⁻ and K⁺ (including KCl and KNO₃). Both SEM-X-Ray microanalysis and flame emission results revealed that well developed S. europaea plants had a higher content of sodium but lower potassium and chlorine. It is concluded that sodium plays a more important role in the growth and development of S. europaea than potassium and chloride.     

Free energy simulations of ligand binding to the aspartate transporter Glt(Ph)

Glutamate/Aspartate transporters cotransport three Na⁺ and one H⁺ ions with the substrate and countertransport one K⁺ ion. The binding sites for the substrate and two Na⁺ ions have been observed in the crystal structure of the archeal homolog Glt_Ph, while the binding site for the third Na⁺ ion has been proposed from computational studies and confirmed by experiments. Here we perform detailed free energy simulations of Glt_Ph, giving a comprehensive characterization of the substrate and ion binding sites, and calculating their binding free energies in various configurations. Our results show unequivocally that the substrate binds after the binding of two Na⁺ ions. They also shed light into Asp/Glu selectivity of Glt_Ph, which is not observed in eukaryotic glutamate transporters.     

Monovalent Cations Induce Microsporidian Spore Germination In Vitro

ABSTRACT The relative capacity of Na⁺, K⁺ and Cl^- to stimulate germination of spores of the microsporidian Nosema algerae, a pathogen of mosquitoes, was examined by ion substitution experiments. Sodium at 0.1 M was ineffective to produce the high percentage of germination that typically occurs with 0.1 M NaCl (the normal stimulation solution) if Cl^- was substituted with the usually impermeant anions SO₄^2-, HPO₄^2-, or the organic acids oxalate, cacodylate, EGTA, MES and HEPES. However, substantial concentration- and pH-dependent germination was seen with Na₂SO₄ in the 0.2-0.8 M Na⁺ range. Similar results were obtained with solutions of K⁺ accompanied by impermeant anions. In contrast, the chloride salts of usually impermeant cations, like choline and triethanolamine, failed to germinate spores even at 0.8 M unless Na⁺ or K⁺ was independently added. The presence of 0.5 M choline chloride in the medium reduced the levels of Na₂SO₄ required to produce germination down to equivalence with those of Na⁺ in the normal stimulation solution. Monensin, a Na⁺ ionophore, facilitated the germination induced by a medium-level stimulus (0.04 M NaCl) in sonicated samples. These findings indicate that N. algerae spores germinate in response to the alkali metal cations, while CI^- plays a passive role by diffusing to maintain internal electroneutrality during cation influx. A possible mechanism of cation action in spore germination is suggested on the basis of these results and observations on other systems of intracellular motility.     

Salt tolerance inNitellopsis obtusa

Summary The mechanism of salt tolerance was studied using isolated internodal cells of the charophyteNitellopsis obtusa grown in fresh water. When 100 mM NaCl was added to artificial pond water (0.1 mM each of NaCl, KC1, CaCl₂), no cell survived for more than one day. Within the first 30 minutes, membrane potential (E_m) depolarized and membrane resistance (R_m) decreased markedly. Simultaneously, cytoplasmic Na⁺ increased and K⁺ decreased greatly. At steady state the increase in Na⁺ content was roughly equal to the decrease in K⁺ content. The Cl^– content of the cytoplasm did not change. These results suggest that Na⁺ enters the cytoplasm by exchange with cytoplasmic K⁺. Both the entry of Na⁺ and the exit of K⁺ are assumed to be passive and the latter being caused by membrane depolarization. Vacuolar K⁺, Na⁺, and Cl^– remained virtually constant, suggesting that rapid influx of Na⁺ from the cytoplasm did not occur.In 100 mM NaCl containing 10 mM CaCl₂, membrane depolarization, membrane resistance decrease and changes in cytoplasmic [Na⁺] and [K⁺] did not occur, and cells survived for many days. When cells treated with 100 mM NaCl were transferred within 1 hour to 100 mM NaCl containing 10 mM CaCl₂, Em decreased, Rm increased, cytoplasmic Na⁺ and K⁺ returned to their initial levels, and cells survived. Two possible mechanisms for the role of Ca²⁺ in salt tolerance inNitellopsis are discussed; one a reduction in plasmalemma permeability to Na⁺ and the other a stimulation of active Na⁺-extrusion.     

Ecosystem changes in Galápagos highlands by the invasive tree Cinchona pubescens

Background and aims

Salinity is an increasing problem for agricultural production worldwide. Understanding how Na⁺ enters plants is important if reducing Na⁺ influx, a key component of the regulation of Na⁺ accumulation in plants and improving salt tolerance of crop plants, is to be achieved. Our previous work indicated that two distinct low-affinity Na⁺ uptake pathways exist in the halophyte Suaeda maritima. Here, we report the external NaCl concentration at which uptake switches from pathway 1 to pathway 2 and the kinetics of the interaction between external K⁺ concentration and Na⁺ uptake and accumulation in S. maritima in order to determine the roles of K⁺ transporters or channels in low-affinity Na⁺ uptake.

Methods

Na⁺ influx, Na⁺ and K⁺ accumulations in S. maritima exposed to various concentrations of NaCl (0–200 mM) were analyzed in the absence and presence of the inhibitors TEA and Ba⁺ (5 mM TEA or 3 mM Ba²⁺) or KCl (0, 10 or 50 mM).

Results

Our earlier proposal was confirmed and extended that there are two distinct low-affinity Na⁺ uptake pathways in S. maritima: pathway 1 might be mediated by a HKT-type transporter under low salinity conditions and pathway 2 by an AKT1-type channel or a KUP/HAK/KT type transporter under high salinity conditions. The external NaCl concentration at which two distinct low-affinity Na⁺ uptake switches from pathway 1 to pathway 2, the ‘turning point’, is between 90 and 95 mM. Over a short period (12 h) of Na⁺ and K⁺ treatments, a low concentration of K⁺ (10 mM) facilitated Na⁺ uptake by S. maritima under high salinity (100–200 mM NaCl), whether or not the plants had been subjected to a longer (3 d) period of K⁺ starvation. The kinetics suggests that low concentration of K⁺ (10 mM) might activate AKT1-type channels or KUP/HAK/KT-type transporters under high salinity (100–200 mM NaCl).

Conclusions

The turning-point of external NaCl concentrations for the two low-affinity Na⁺ uptake pathways in Suaeda maritima is between 90 and 95 mM. A low concentration of K⁺ (10 mM) might activate AKT1 or KUP/HAK/KT and facilitate Na⁺ uptake under high salinity (100–200 mM NaCl). The kinetics of K⁺ on Na⁺ uptake and accumulation in S maritima are also consistent with there being two low-affinity Na⁺ uptake pathways.     

Glucose-6-phosphate dehydrogenase-dependent hydrogen peroxide production is involved in the regulation of plasma membrane H+-ATPase and Na+/H+ antiporter protein in salt-stressed callus from Carex moorcroftii

Glucose‐6‐phosphate dehydrogenase (G6PDH) is important for the activation of plant resistance to environmental stresses, and ion homeostasis is the physiological foundation for living cells. In this study, we investigated G6PDH roles in modulating ion homeostasis under salt stress in Carex moorcroftii callus. G6PDH activity increased to its maximum in 100 mM NaCl treatment and decreased with further increased NaCl concentrations. K⁺/Na⁺ ratio in 100 mM NaCl treatment did not exhibit significant difference compared with the control; however, in 300 mM NaCl treatment, it decreased. Low‐concentration NaCl (100 mM) stimulated plasma membrane (PM) H⁺‐ATPase and NADPH oxidase activities as well as Na⁺/H⁺ antiporter protein expression, whereas high‐concentration NaCl (300 mM) decreased their activity and expression. When G6PDH activity and expression were reduced by glycerol treatments, PM H⁺‐ATPase and NADPH oxidase activities, Na⁺/H⁺ antiporter protein level and K⁺/Na⁺ ratio dramatically decreased. Simultaneously, NaCl‐induced hydrogen peroxide (H₂O₂) accumulation was abolished. Exogenous application of H₂O₂ increased G6PDH, PM H⁺‐ATPase and NADPH oxidase activities, Na⁺/H⁺ antiporter protein expression and K⁺/Na⁺ ratio in the control and glycerol treatments. Diphenylene iodonium (DPI), the NADPH oxidase inhibitor, which counteracted NaCl‐induced H₂O₂ accumulation, decreased G6PDH, PM H⁺‐ATPase and NADPH oxidase activities, Na⁺/H⁺ antiporter protein level and K⁺/Na⁺ ratio. Western blot result showed that G6PDH expression was stimulated by NaCl and H₂O₂, and blocked by DPI. Taken together, G6PDH is involved in H₂O₂ accumulation under salt stress. H₂O₂, as a signal, upregulated PM H⁺‐ATPase activity and Na⁺/H⁺ antiporter protein level, which subsequently resulted in the enhanced K⁺/Na⁺ ratio. G6PDH played a central role in the process.     

Low Affinity and Slow Na+ Binding Precedes High Affinity Aspartate Binding in the Secondary-active Transporter GltPh

Glt_Ph from Pyrococcus horikoshii is a homotrimeric Na⁺-coupled aspartate transporter. It belongs to the widespread family of glutamate transporters, which also includes the mammalian excitatory amino acid transporters that take up the neurotransmitter glutamate. Each protomer in Glt_Ph consists of a trimerization domain involved in subunit interactions and a transport domain containing the substrate binding site. Here, we have studied the dynamics of Na⁺ and aspartate binding to Glt_Ph. Tryptophan fluorescence measurements on the fully active single tryptophan mutant F273W revealed that Na⁺ binds with low affinity to the apoprotein (K_d 120 mm), with a particularly low k_on value (5.1 m⁻¹s⁻¹). At least two sodium ions bind before aspartate. The binding of Na⁺ requires a very high activation energy (E_a 106.8 kJ mol⁻¹) and consequently has a large Q₁₀ value of 4.5, indicative of substantial conformational changes before or after the initial binding event. The apparent affinity for aspartate binding depended on the Na⁺ concentration present. Binding of aspartate was not observed in the absence of Na⁺, whereas in the presence of high Na⁺ concentrations (above the K_d for Na⁺) the dissociation constants for aspartate were in the nanomolar range, and the aspartate binding was fast (k_on of 1.4 × 10⁵ m⁻¹s⁻¹), with low E_a and Q₁₀ values (42.6 kJ mol⁻¹ and 1.8, respectively). We conclude that Na⁺ binding is most likely the rate-limiting step for substrate binding.     

Differential growth and yield responses of salt-tolerant and susceptible rice cultivars to individual (Na<Superscript>+</Superscript> and Cl<Superscript>−</Superscript>) and additive stress effects of NaCl

Negative impacts exerted by sodium (Na⁺) and chloride (Cl^?) ions individually as well their possible additive effects (under NaCl) were evaluated on growth and yield reductions in rice, besides investigating whether salt-tolerant genotypes respond differentially than their sensitive counterparts. Though both Na⁺ and Cl^? ions get accumulated in plant tissues under NaCl stress, most research has historically been aimed to decipher harmful effects induced by Na⁺ ions. Accordingly, physiological and molecular mechanisms involved in Cl^? toxicity are not clearly understood in crop plants. To address these issues, 65-day-old plants of two rice cultivars, Panvel-3 (tolerant) and Sahyadri-3 (sensitive) were subjected to Cl^?, Na⁺ and NaCl (each with 100 mM concentration and electrical conductivity of ≈10 dS m^?1) stress using soil-based systems. Stress conditions were maintained till harvesting of mature (128-day-old) plants. All three treatments induced substantial antagonistic effects on growth, dry mass, yield components (number of grains per panicle, length, width, thickness and weight of grain, along with the percentage of grains filled) and overall crop yield, with greater impacts under NaCl than its constituent ions. Salinity treatments caused an imbalance in reducing sugars, protein, starch and proline contents, with the greatest magnitude under NaCl. A negative correlation between Cl^?/Na⁺ accumulation and crop yield was witnessed, with higher severity on the sensitive cultivar. The overall magnitude of toxicity was observed highest in NaCl followed by Na⁺ and Cl^?, respectively, suggesting additive effects of constituent ions under NaCl. Both cultivars responded similarly; however, the tolerant cultivar, unlike the sensitive one, kept Na⁺:K⁺ ratio <1.0 and accumulated proline in response to salinity treatments used in this study.