Monovalent cation transporters at the plasma membrane in yeasts

Maintenance of proper intracellular concentrations of monovalent cations, mainly sodium and potassium, is a requirement for survival of any cell. In the budding yeast Saccharomyces cerevisiae, monovalent cation homeostasis is determined by the active extrusion of protons through the Pma1 H⁺-ATPase (reviewed in another chapter of this issue), the influx and efflux of these cations through the plasma membrane transporters (reviewed in this chapter), and the sequestration of toxic cations into the vacuoles. Here, we will describe the structure, function, and regulation of the plasma membrane transporters Trk1, Trk2, Tok1, Nha1, and Ena1, which play a key role in maintaining physiological intracellular concentrations of Na⁺, K⁺, and H⁺, both under normal growth conditions and in response to stress.     

吡哆醇对鲁氏接合酵母高盐胁迫耐受的影响

为了提高鲁氏接合酵母抗高盐胁迫能力,作者探索了添加吡哆醇对鲁氏接合酵母高盐胁迫耐受的影响。通过测量甘油、海藻糖含量以及Na⁺/K⁺-ATP酶活性等一系列生理表征来验证鲁氏接合酵母高盐适应性的变化。实验结果表明,鲁氏接合酵母在高盐胁迫条件下外源添加吡哆醇,在延滞期时,菌株提前24 h积累细胞内甘油,并且加快了细胞内海藻糖分解代谢速率;在对数期时,细胞内钠钾比率（Na⁺/K⁺）降低82.3%,细胞膜上Na⁺/K⁺-ATP酶活性增加16.9%,2-苯乙醇产量（质量浓度）提高6.42倍;在稳定期时,生物量提高10.6%,乙醇产量（质量浓度）提高5%,2-苯乙醇产量（质量浓度）提高1.26倍。综上所述,吡哆醇的添加能有效提高鲁氏接合酵母的高盐胁迫耐受能力,进一步增强了鲁氏接合酵母在酱油等高盐发酵食品中的应用前景。     

Monovalent cation fluxes and physiological changes of Debaryomyces hansenii grown at high concentrations of KCl and NaCl

Debaryomyces hansenii showed an increased growth in the presence of either 1m KCl or 1m NaCl and a low acidification of the medium, higher for the cells grown in the presence of NaCl. These cells accumulated high concentrations of the cations, and showed a very fast capacity to exchange either Na⁺ or K⁺ for the opposite cation. They showed a rapid uptake of ⁸⁶ Rb⁺ and ²² Na⁺ . ⁸⁶ Rb⁺ transport was saturable, with K_m and V_max values higher for cells grown in 1m NaCl. ²² Na⁺ uptake showed a diffusion component, also higher for the cells grown with NaCl. Changes depended on growth conditions, and not on further incubation, which changed the internal ion concentration. K⁺ stimulated proton pumping produced a rapid extrusion of protons, and also a decrease of the membrane potential. Cells grown in 1m KCl showed a higher fermentation rate, but significantly lower respiratory capacity. ATP levels were higher in cells grown in the presence of NaCl; upon incubation with glucose, those grown in the presence of KCl reached values similar to the ones grown in the presence of NaCl. In both, the addition of KCl produced a transient decrease of the ATP levels. As to ion transport mechanisms, D. hansenii appears to have (a) an ATPase functioning as a proton pump, generating a membrane potential difference which drives K⁺ through a uniporter; (b) a K⁺ /H⁺ exchange system; and (c) a rapid cation/cation exchange system. Most interesting is that cells grown in different ionic environments change their studied capacities, which are not dependent on the cation content, but on differences in their genetic expression during growth. © 1998 John Wiley & Sons, Ltd.     

Comparative study of physiological adaptation to salt stress in the genome shuffled Candida versatilis and a wild-type salt-tolerant yeast strain

Candida versatilis is a yeast with a complex salt-tolerant system. It can maintain normal physiological activities and metabolic fermentation under a high-salt environment. The cellular mechanisms of adaptation to salt stress in strains of a wild type of C. versatilis (WT) and S3–5, genome shuffling strains of C. versatilis with improved tolerance to salt, were investigated. The content of intra- and extra-cellular glycerol, intra-cellular Na⁺, as well as membrane fluidity and permeability, were determined under salt-stressed yeast growth conditions. The results showed that Na⁺/H⁺-antiporter played a primary role in Na⁺ extrusion and H⁺-ATPase has been associated with yeast survival under salt stress. Considerable amounts of glycerol were produced and secreted by the yeast to outside the cell under this salt stress. Changes in the portion of membrane saturated and unsaturated fatty acid composition of C. versatilis in response to osmotic stress lead to membrane permeability and fluidity decreases. They could restrict the influx of Na⁺, enhance H⁺-ATPase activity, and prevent leakage of glycerol across the cell membrane under osmotic stress. The salt tolerance of genome shuffled strain S3–5 was higher than WT. It could be correlated with a higher level of intra-cellular accumulation of glycerol and sodium ions in cells of S3–5 than WT as well as a higher portion of oleic fatty acid (C18: 1) and a lower level of linoleic acid (C18: 2) in cell membranes of the studied yeast mutant. It can be concluded that S3–5 improved physiological regulatory mechanisms of response to salt stress, such as decreased membrane fluidity and a permeability that rapidly adjusted to osmotic stress.     

Characterization of a glycerol/H+ symport in the halotolerant yeast Pichia sorbitophila

Pichia sorbitophila is a halotolerant yeast capable of surviving to extracellular NaCl concentrations up to 4 M in mineral medium when glucose or glycerol are the only carbon and energy sources. Evidence is presented here that glycerol, the main compatible solute this yeast accumulates so as to maintain osmotic balance, is actively co-transported with protons. This transport system was shown to be constitutive, not needing induction by either glycerol or salt, and was not repressible by glucose. In glucose- or glycerol-grown cells, a simple diffusion was detectable, and iterative calculations were performed to calculate kinetic parameters, in the presence and in the absence of NaCl. At 25°C, pH 5·0, in glucose-grown cells these were: K_m = 0·81 ± 0·11 mM and V_max = 634·2 ± 164·8 μmol h^?1 per g (glycerol); K_m = 1·28 ± 0·60 mM and V_max = 558·6 · 100·6 μmol h^?1 per g (protons). Correspondent stoichiometry was approximately 1, either for these conditions or in the presence of 1 M -NaCl. An increase in acumulation capacity was evident when different concentrations of NaCl were present. This capacity was shown to be dependent on ΔpH and membrane potential, consistently with an electrogenic character. We suggest that the main role of this system is in osmoregulation, by keeping glycerol accumulated inside the cells, compensating for leakage, due to its liposoluble character.     

The role of K+ and H+ transport systems during glucose‐ and H2O2‐induced cell death in Saccharomyces cerevisiae

Glucose, in the absence of additional nutrients, induces programmed cell death in yeast. This phenomenon is independent of yeast metacaspase (Mca1/Yca1) and of calcineurin, requires ROS production and it is concomitant with loss of cellular K⁺ and vacuolar collapse. K⁺ is a key nutrient protecting the cells and this effect depends on the Trk1 uptake system and is associated with reduced ROS production. Mutants with decreased activity of plasma membrane H⁺‐ATPase are more tolerant to glucose‐induced cell death and exhibit less ROS production. A triple mutant ena1‐4 tok1 nha1, devoid of K⁺ efflux systems, is more tolerant to both glucose‐ and H₂O₂‐induced cell death. We hypothesize that ROS production, activated by glucose and H⁺‐ATPase and inhibited by K⁺ uptake, triggers leakage of K⁺, a process favoured by K⁺ efflux systems. Loss of cytosolic K⁺ probably causes osmotic lysis of vacuoles. The nature of the ROS‐producing system sensitive to K⁺ and H⁺ transport is unknown. Copyright © 2010 John Wiley & Sons, Ltd.     

KCI, CaCI2, Na+ Binding, and Salt Taste of Gum Systems

Aqueous nonionic (0.3% w/v) and ionic (0.1% and 0.3% w/v) gum systems containing NaCl, or equal weights of NaCl plus KCl, or NaCl plus CaCl, were examined. At equivalent molar concentrations of added ions, ²³Na NMR transverse relaxation rates (R₂, set^?1) showed an increase in average Na⁺ mobility with the addition of K⁺ or Ca²⁺ to ionic gum systems. Correspondingly, salt taste increased with addition of KCl as determined by Decision Boundary modeling of subject identification data. Viscosity did not affect saltiness. Na⁺ was free to induce salt taste when K⁺ was bound to the gum. Enhancement of salt taste by KCl is due, in part, to competitive binding of Na⁺ and K⁺ in a system.     

Deletion of the N-terminal domain of the yeast vacuolar (Na+,K+)/H+ antiporter Vnx1p improves salt tolerance in yeast and transgenic Arabidopsis

Cation/proton antiporters play a major role in the control of cytosolic ion concentrations in prokaryotes and eukaryotes organisms. In yeast, we previously demonstrated that Vnx1p is a vacuolar monovalent cation/H⁺ exchanger showing Na⁺/H⁺ and K⁺/H⁺ antiporter activity. We have also shown that disruption of VNX1 results in an almost complete abolishment of vacuolar Na⁺/H⁺ exchange, but yeast cells overexpressing the complete protein do not show improved salinity tolerance. In this study, we have identified an autoinhibitory N-terminal domain and have engineered a constitutively activated version of Vnx1p, by removing this domain. Contrary to the wild type protein, the activated protein has a pronounced effect on yeast salt tolerance and vacuolar pH. Expression of this truncated VNX1 gene also improves Arabidopsis salt tolerance and increases Na⁺ and K⁺ accumulation of salt grown plants thus suggesting a biotechnological potential of activated Vnx1p to improve salt tolerance of crop plants.     

Salt-induced modulation in inorganic nutrients, antioxidant enzymes, proline content and seed oil composition in safflower (Carthamus tinctorius L.)

BACKGROUND: Safflower (Carthamus tinctorius L.) has gained considerable ground as a potential oil‐seed crop. However, its yield and oil production are adversely affected under saline conditions. The present study was conducted to appraise the influence of salt (NaCl) stress on yield, accumulation of different inorganic elements, free proline and activities of some key antioxidant enzymes in plant tissues as well as seed oil components in safflower. Two safflower accessions differing in salt tolerance (Safflower‐33 (salt sensitive) and Safflower‐39 (salt tolerant)) were grown under saline (150 mmol L^?1) conditions and salt‐induced changes in the earlier‐mentioned physiological attributes were determined. RESULTS: Salt stress enhanced leaf and root Na⁺, Cl^? and proline accumulation and activities of leaf superoxide dismutase, catalase and peroxidase, while it decreased K⁺, Ca²⁺ and K⁺/Ca²⁺ and Ca²⁺/Na⁺ ratios and seed yield, 100‐seed weight, number of seeds, as well as capitula, seed oil contents and oil palmitic acid. No significant effect of salt stress was observed on seed oil α‐tocopherols, stearic acid, oleic acid or linoleic acid contents. Of the two safflower lines, salt‐sensitive Safflower‐33 was higher in leaf and root Na⁺ and Cl^?, while Safflower‐39 was higher in leaf and root K⁺, K⁺/Ca²⁺ and Ca²⁺/Na⁺ and seed yield, 100‐seed weight, catalase activity, seed oil contents, seed oil α‐tocopherol and palmitic acid. Other attributes remained almost unaffected in both accessions. CONCLUSION: Overall, high salt tolerance of Safflower‐39 could be attributed to Na⁺ and Cl^? exclusion, high accumulation of K⁺ and free proline, enhanced CAT activity, seed oil α‐tocopherols and palmitic acid contents. Copyright © 2011 Society of Chemical Industry     

Effects of Cation Properties on Sol-gel Transition and Gel Properties of κ-carrageenan

The phase transition temperatures, rheological properties and gel‐network characteristics for gelation of κ‐carrageenan‐salt (NaCl, KCl and CaCl₂) solutions and their aged gels were investigated. The effectiveness of increasing gelling and gel‐melting temperatures at the salt concentrations examined followed the sequence of K⁺ > Ca2⁺ > Na⁺. This sequence was also true for the gel strength and the melting enthalpy (DH) of the most crosslinked junction zones of aged gels at low salt concentrations. Nonetheless, a different order (Ca⁺⁺ > K⁺ and Na⁺) was found for increasing storage modulus and gelation rate during early‐stage gelation, thermal hysteresis and the DH of aged gels in some salt‐carrageenan systems.     

Physiological changes, phenolic content and antioxidant activity of Salvia officinalis L. grown under saline conditions

BACKGROUND: Hydroponic culture was used to investigate the effect of NaCl concentrations on the growth, nutrient uptake, phenolic content and antioxidant activity of Salvia officinalis L. leaves. The antioxidant capacity of the methanolic extract of S. officinalis was evaluated by using 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) radical scavenging test and β‐carotene‐linoleic acid bleaching assay. Physiological and biochemical parameters of S. officinalis were assessed after 4 weeks of salt treatment with 0, 25, 50, 75 and 100 mmol L^?1 NaCl. RESULTS: Plant growth exhibited a reduction of 61% at 100 mmol L^?1 NaCl. Assessment of Na⁺, K⁺ and Ca²⁺ and water contents of shoots and roots showed that S. officinalis is able to regulate Na⁺ concentration by active compartmentation in vacuoles. Salvia officinalis phenolics were increased in response to salinity at the threshold of 75 mmol L^?1 NaCl. This herb was also found to be able to achieve important DPPH^? quenching activity and to inhibit the β‐carotene‐linoleic acid bleaching notably enhanced by salt treatment. It is interesting to highlight the correlation between the phenolic and antioxidant activity, suggesting the involvement of these compounds in this activity. CONCLUSION: Salvia officinalis treated with 75 mmol L^?1 NaCl constitutes a potential source for production of secondary metabolites useful in several applications. Copyright © 2011 Society of Chemical Industry     

The Osmotic Hypersensitivity of the Yeast Saccharomyces cerevisiae is Strain and Growth Media Dependent: Quantitative Aspects of the Phenomenon

Osmotic hypersensitivity is manifested as cellular death at magnitudes of osmotic stress that can support growth. Cellular capacity for survival when plated onto high NaCl media was examined for a number of laboratory and industrial strains of Saccharomyces cerevisiae. During respiro-fermentative growth in rich medium with glucose as energy and carbon source, the hypersensitivity phenomenon was fairly strain invariant with a threshold value of about 1 m-NaCl; most strains fell within a 300 mm range in LD₁₀ values (lethal dose yielding 10% survival). Furthermore, all but one of the strains displayed similar differential death responses above the threshold value, i.e. ten-fold decreased viability for every 250 mm increase in salinity. Addition of small amounts of salt to the growth medium drastically improved tolerance and shifted the hypersensitivity threshold to higher NaCl concentrations. This salt-instigated tolerance could partly be reversed by washing in water. The washing procedure depleted cells of the glycerol that they had accumulated under saline growth, and the contribution from glycerol to the improved tolerance was about 50% in the two strains examined. Growth on derepressing carbon sources like galactose, ethanol or glycerol gave strain-dependent responses. The laboratory strain X2180–1A drastically improved tolerance while the bakers' yeast strain Y41 did so only marginally. It was concluded that all strains of S. cerevisiae display the osmotic hypersensitivity phenomenon in qualitative terms while the quantitative values differ. It was also proposed that growth rate does not dictate the level of osmotic hypersensitivity of S. cerevisiae. © 1997 John Wiley & Sons, Ltd.     

Enhanced Sweetness in Sweetener-NaCI-Gum Systems

Enhancement of sweetness in aqueous gum (0.03%, w/v) sweetener systems by added NaCl (0.05%, w/v) was evaluated by a sensory panel. ²³Na NMR spectroscopy was used to determine Na⁺ binding and its relationship to sweetness elicited by glucose, lactose, maltose, sucrose and aspartame. Sweetness intensity differed due to gum (p = 0.0001) and sweetener (p = 0.0001), but was not affected by NaCl (p = 0.0774). Sweetness increased with added NaCl in xanthan, guar and locust bean gum solutions. However, sweetness decreased in k-carrageenan systems possibly due to endogenous cation (Ca²⁺, K⁺ and Na⁺) content, which influences Na⁺ mobility. The sweetest systems containing lactose and/ or xanthan, showed the greatest enhancement by NaCl.     

Stability and Activity of β-Galactosidase in Sonicated Cultures of Lactobacillus delbrueckii ssp. bulgaricus 11842 as Affected by Temperature and Ionic Environments

Stability of β‐galactosidase and related lactose hydrolyzing activity in sonicated cultures of Lactobacillus bulgaricus 11842 was investigated in the presence of Na⁺ or K⁺ ions. After sonication in Na⁺ or K⁺ buffers at various pH levels, cultures were held at various temperatures, before adding test lactose solutions. Hydrolysis was monitored by cryoscopy. Cultures sonicated in K⁺ buffer had higher activity and stability than those in Na⁺ buffer; both were highest at pH 6 and 7. Stability was unaffected at pH 6 and 7 below 56 °C. Holding at 61 °C for 60 min caused 70% activity loss with K⁺ ions at pH 6 and 7, while with Na⁺, activity loss was almost complete.     

Evidence for a selective and electroneutral K+/H+-exchange in Saccharomyces cerevisiae using plasma membrane vesicles

The existence of a K⁺/H⁺ transport system in plasma membrane vesicles from Saccharomyces cerevisiae is demonstrated using fluorimetric monitoring of proton fluxes across vesicles (ACMA fluorescence quenching). Plasma membrane vesicles used for this study were obtained by a purification/reconstitution protocol based on differential and discontinuous sucrose gradient centrifugations followed by an octylglucoside dilution/gel filtration procedure. This method produces a high percentage of tightly-sealed inside-out plasma membrane vesicles. In these vesicles, the K⁺/H⁺ transport system, which is able to catalyse both K⁺ influx and efflux, is mainly driven by the K⁺ transmembrane gradient and can function even if the plasma membrane H⁺-ATPase is not active. Using the anionic oxonol VI and the cationic DISC₂(5) probes, it was shown that a membrane potential is not created during K⁺ fluxes. Such a dye response argues for the presence of a K⁺/H⁺ exchange system in S. cerevisiae plasma membrane and established the non-electrogenic character of the transport. The maximal rate of exchange is obtained at pH 6·8. This reversible transport system presents a high selectivity for K⁺ among other monovalent cations and a higher affinity for the K⁺ influx into the vesicles (exit from cells). The possible role of this K⁺/H⁺ exchange system in regulation of internal potassium concentration in S. cerevisiae is discussed.     

Softening Effects of Monovalent Cations in Acidified Cucumber Mesocarp Tissue

Due to the importance of salt as an ingredient in cucumber pickle products, the effect of salt concentration on first-order softening rates during acid storage was determined. Softening rates of unfermented tissue increased as the NaCl increased from 0 to 1.5M, whether or not the tissue was blanched. For unheated, fermented tissue, softening rates increased between 0 and 0.2M NaCl but did not increase above 0.2M NaCl. Changes in the degree of pectin methylation were not highly correlated with changes in softening rates. The ions Li⁺, K⁺, Rb⁺, and Cs⁺ had softening effects similar to Na⁺. An enthalpy of activation of 145 kJ/mole was determined for softening of blanched tissue stored in 1.5M NaCl. This is the first demonstration of a softening effect by monovalent cations in cucumber tissue.     

Nutrient digestibility response to graded dietary levels of sodium chloride in weanling pigs

BACKGROUND: Sodium (Na⁺), chloride (Cl^?) and phosphorus (P_i) are involved in a number of metabolic and physiological processes in the body, and these mineral elements must be supplied to the animal via the diet. The intention of this study was to evaluate the effect of dietary supplementation with different levels of sodium chloride (0.1, 0.2, 0.3, 0.4, 0.5 and 0.6% NaCl) on apparent and true P digestibility (APD and TPD) and dry matter (DM), crude protein (CP) and calcium (Ca) digestibility in weanling pigs. RESULTS: Dietary NaCl had a quadratic effect on both APD and TPD (P < 0.05) but not on DM, CP and Ca digestibility (P > 0.05). At an NaCl concentration of 0.41% the APD and TPD values were 41.5 and 53.3% respectively. CONCLUSION: These results suggest that a high level of dietary Na⁺ may enhance P absorption and improve its digestibility by coupling Na⁺ transportation to P_i absorption via the energy‐requiring Na⁺/P_i co‐transporter. Copyright © 2008 Society of Chemical Industry     

Developmental and ripening-related effects on the cell wall of apricot (Prunus armeniaca) fruit

Alcohol-insoluble residues (AIRs) were prepared from apricots at six stages during development/ripening on the tree. To investigate the changes in cell wall polymers, and in particular those affecting pectic polysaccharides, the AIR preparations were sequentially extracted with water, cyclohexane-trans-1,2-diamine-N,N,N′,N′-tetraacetate (CDTA) and Na₂CO₃. A significant proportion of initially Na₂CO₃-soluble pectic polysaccharides became water- and CDTA-soluble during the ripening process. In terms of composition, a significant decrease in galactose and uronic acid content was detected in all the extractions, whereas the percentage of arabinose increased in both water and CDTA-soluble polymers but decreased in the Na₂CO₃-extracted polysaccharides. The ability of pectic polysaccharides to cross-link was diminished during ripening due to an overall increase in the concentration of Na⁺ or K⁺ associated with the AIRs. This was accompanied by a decrease in the amounts of Ca²⁺ and Mg²⁺. The decrease in pectic galactans and the inhibition of pectin cross-linking detected within the pectic backbone are probably linked to the softening process observed during apricot ripening. © 1998 SCI.     

Factors influencing permeability of the cell membrane of the osmotolerant yeast Saccharomyces rouxii grown in the presence and absence of 18% NaCl. I. Na+/K+-activated Mg2+-dependent ATPase

It was found that cells of Saccharomyces rouxii contain an ouabain-inhibited ATPase, assumed to be an Na⁺/K⁺-activated Mg²⁺-dependent ATPase, which could serve as a sodium pump protecting the cells in a high salt environment. Twenty-two cell homogenates or supernatants (centrifuged at 3000 × g) grown without added salt in the medium contained sufficient total ATPase activity to liberate (on average) 0.225 μM P_i min^?1 mg^?1 protein. The percentage of total ATPase inhibited by the addition of ouabain (1 × 10^?4 M) varied from 7 to 100%. Cell homogenates or supernatants from cells grown in the presence of 18% NaCl in the media contained sufficient ATPase activity to liberate (on average) 0.114 μM P_i min^?1 mg^?1 protein, about 50% of the total ATPase activity found in the non-salt grown cells. The percentage of total ATPase activity inhibited by ouabain ranged from 16 to 100%. Although the non-salt-grown cells contained approximately double the total ATPase activity of the salt-grown cells. there was evidence that the percentage of total ATPase that is ouabain sensitive (Na⁺/K⁺-activated ATPase) is higher in the salt-grown cells. Also, cells of S. rouxii grown in media without added NaCl, recovered by centrifugation and transferred to media containing 18% NaCl for 16 h and again recovered by centrifugation, homogenized and centrifuged at 10 000 × g contained 61.2% ouabain-sensitive ATPase compared with 21.3% ouabain-sensitive ATPase in the cells before adaptation to the high salt environment.     

Effects of Salts Added After Cooking on the Texture of Canned Snap Beans

Soaking bean pods in NaCl solutions caused decreases in firmness and increases in Ca⁺⁺ solubilization as the NaCl concentration increased. CaCl₂ soak solutions increased firmness of pods previously softened by soaking in KCl solutions. Chlorides of Na⁺, K⁺, Li⁺, NH₄⁺, and Mg⁺⁺, and Na acetate caused softening. LiCl caused the most softening, while MgCl₂ caused the greatest solubilization of Ca⁺⁺. Salt-induced softening was accompanied by Ca⁺⁺ displacement. Firmness of salt-softened pods was further decreased by subsequent removal of the salt, indicating an electrostatic component as a minor factor in pod texture.