Intracellular Na and K distribution in Debaryomyces hansenii. Cloning and expression in Saccharomyces cerevisiae of DhNHX1

Debaryomyces hansenii is a salt-tolerant yeast that contains high amounts of internal Na(+). Debaryomyces hansenii kept more sodium than Saccharomyces cerevisiae in both the cytoplasm and vacuole when grown under a variety of NaCl concentrations. These results indicate a higher tolerance of Debaryomyces to high internal Na(+), and, in addition, suggest the existence of a transporter driving Na(+) into the vacuole. Moreover, a gene encoding a Na(+) (K(+))/H(+) antiporter from D. hansenii was cloned and sequenced. The gene, designated DhNHX1, exhibited significant homology with genes of the NHE/NHX family. DhNHX1 expression was induced neither at low pH nor by extracellular NaCl. A mutant of S. cerevisiae lacking its own Na(+) transporters (ena1-4Delta nha1 Delta nhx1 Delta), when transformed with DhNHX1, partially recovered cation tolerance as well as the ability to accumulate Na(+) and K(+) into the vacuole. Our analysis provides evidence that DhNhx1p transports Na(+) (and K(+)) into the vacuole and that it can play an important role in ion homeostasis and salt tolerance.     

Genes from Debaryomyces hansenii increase salt tolerance in Saccharomyces cerevisiae W303

The yeast Debaryomyces hansenii has been chosen as a model for molecular studies of tolerance to NaCl. A gene library was built and transformants of Saccharomyces cerevisiae W303 containing genes from D. hansenii were selected for their ability to grow in the presence of high concentrations of NaCl and/or low concentrations of KCl. In three of these transformants 500 mM NaCl improved growth at pH 7.6 like in D. hansenii but not in S. cerevisiae. One of the plasmids restored growth at 50 microM KCl and K(+) uptake in a mutant of S. cerevisiae lacking genes that encode K(+) transporters.     

Physiological characterization of two genes for Na+ exclusion in durum wheat, Nax1 and Nax2

Durum wheat (Triticum turgidum L. subsp. durum Desf.) Line 149 contains two novel major genes for excluding Na(+) from leaf blades, named Nax1 and Nax2. The genes were separated into families containing a single gene and near-isogenic homozygous lines were selected. Lines containing either Nax1 or Nax2 had lower rates of Na(+) transport from roots to shoots than their near-isogenic pairs due to lower rates of net loading of the xylem, not to lower rates of net uptake from the soil or higher rates of retranslocation in the phloem. Nax1 and Nax2 lines also had higher rates of K(+) transport from root to shoot, resulting in an enhanced discrimination of K(+) over Na(+). Lines containing Nax1 differed from those containing Nax2 by unloading Na(+) from the xylem as it entered the shoot so that Na(+) was retained in the base of the leaf, leading to a high sheath to blade ratio of Na(+) concentration. Gradients in tissue concentrations of Na(+) along the leaf suggested that Na(+) was continually removed from the xylem. The Nax2 line did not retain Na(+) in the base of the leaf, suggesting that it functioned only in the root. The Nax2 gene therefore has a similar function to Kna1 in bread wheat (Triticum aestivum).     

Regulation of the potassium to sodium ratio and of the osmotic potential in relation to salt tolerance in yeasts

By using the isotope pairs (22)Na-(24)Na and (42)K-(86)Rb, the uptake and retention of Na and K was studied in the salt-tolerant Debaryomyces hansenii and in the less tolerant Saccharomyces cerevisiae at NaCl levels of 4 mm and 0.68, 1.35, and 2.7 m in the medium. The ratio of K to Na is much higher in the cells than in the media, and higher in D. hansenii than in S. cerevisiae under comparable conditions. The difference between the two species is due to a better Na extrusion and a better uptake of K in D. hansenii. The kinetics of ion transport show that at about the time when extrusion of Na could be demonstrated in D. hansenii, K-Rb previously lost to an easily washable compartment of the cells was reabsorbed in both organisms. More H(+) was given off from S. cerevisiae than from D. hansenii in the course of these events. The findings fit the working hypothesis tested, which regards salt tolerance as partly dependent on the ability to mobilize energy to extrude Na from the cells and to take up K. The volume changes in S. cerevisiae are greater and are more slowly overcome than those in D. hansenii. The total salt level of the cells is not sufficient to counteract the osmotic potential of the medium, so that additional osmoregulatory mechanisms must be involved in determining halotolerance.     

Major genes for Na+ exclusion, Nax1 and Nax2 (wheat HKT1;4 and HKT1;5), decrease Na+ accumulation in bread wheat leaves under saline and waterlogged conditions

Two major genes for Na(+) exclusion in durum wheat, Nax1 and Nax2, that were previously identified as the Na(+) transporters TmHKT1;4-A2 and TmHKT1;5-A, were transferred into bread wheat in order to increase its capacity to restrict the accumulation of Na(+) in leaves. The genes were crossed from tetraploid durum wheat (Triticum turgidum ssp. durum) into hexaploid bread wheat (Triticum aestivum) by interspecific crossing and marker-assisted selection for hexaploid plants containing one or both genes. Nax1 decreased the leaf blade Na(+) concentration by 50%, Nax2 decreased it by 30%, and both genes together decreased it by 60%. The signature phenotype of Nax1, the retention of Na(+) in leaf sheaths resulting in a high Na(+) sheath:blade ratio, was found in the Nax1 lines. This conferred an extra advantage under a combination of waterlogged and saline conditions. The effect of Nax2 on lowering the Na(+) concentration in bread wheat was surprising as this gene is very similar to the TaHKT1;5-D Na(+) transporter already present in bread wheat, putatively at the Kna1 locus. The results indicate that both Nax genes have the potential to improve the salt tolerance of bread wheat.     

Functional expression in Escherichia coli of low-affinity and high-affinity Na(+)(Li(+))/H(+) antiporters of Synechocystis

Synechocystis sp. strain PCC 6803 has five genes for putative Na(+)/H(+) antiporters (designated nhaS1, nhaS2, nhaS3, nhaS4, and nhaS5). The deduced amino acid sequences of NhaS1 and NhaS2 are similar to that of NhaP, the Na(+)/H(+) antiporter of Pseudomonas aeruginosa, whereas those of NhaS3, NhaS4, and NhaS5 resemble that of NapA, the Na(+)/H(+) antiporter of Enterococcus hirae. We successfully induced the expression of nhaS1, nhaS3, and nhaS4 under control of an Na(+)-dependent promoter in Escherichia coli TO114, a strain that is deficient in Na(+)/H(+) antiport activity. Inverted membrane vesicles prepared from TO114 nhaS1 and TO114 nhaS3 cells exhibited Na(+)(Li(+))/H(+) antiport activity. Kinetic analysis of this activity revealed that nhaS1 encodes a low-affinity Na(+)/H(+) antiporter with a K(m) of 7.7 mM for Na(+) ions and a K(m) of 2.5 mM for Li(+) ions, while nhaS3 encodes a high-affinity Na(+)/H(+) antiporter with a K(m) of 0.7 mM for Na(+) ions and a K(m) of 0.01 mM for Li(+) ions. Transformation of E. coli TO114 with the nhaS1 and nhaS3 genes increased cellular tolerance to high concentrations of Na(+) and Li(+) ions, as well as to depletion of K(+) ions during cell growth. To our knowledge, this is the first functional characterization of Na(+)/H(+) antiporters from a cyanobacterium. Inverted membrane vesicles prepared from TO114 nhaS4 cells did not have Na(+)/H(+) antiport activity, and the cells themselves were as sensitive to Na(+) and Li(+) ions as the original TO114 cells. However, the TO114 nhaS4 cells were tolerant to depletion of K(+) ions. Taking into account these results and the growth characteristics of Synechocystis mutants in which nhaS genes had been inactivated by targeted disruption, we discuss possible roles of NhaS1, NhaS3, and NhaS4 in Synechocystis.     

The Wheat cDNA LCT1 Generates Hypersensitivity to Sodium in a Salt-Sensitive Yeast Strain

Salinity affects large areas of agricultural land, and all major crop species are intolerant to high levels of sodium ions. The principal route for Na(+) uptake into plant cells remains to be identified. Non-selective ion channels and high-affinity potassium transporters have emerged as potential pathways for Na(+) entry. A third candidate for Na(+) transport into plant cells is a low-affinity cation transporter represented by the wheat protein LCT1, which is known to be permeable for a wide range of cations when expressed in yeast (Saccharomyces cerevisiae). To investigate the role of LCT1 in salt tolerance we have used the yeast strain G19, which is disrupted in the genes encoding Na(+) export pumps and as a result displays salt sensitivity comparable with wheat. After transformation with LCT1, G19 cells became hypersensitive to NaCl. We show that LCT1 expression results in a strong decrease of intracellular K(+)/Na(+) ratio in G19 cells due to the combined effect of enhanced Na(+) accumulation and loss of intracellular K(+). Na(+) uptake through LCT1 was inhibited by K(+) and Ca(2+) at high concentrations and the addition of these ions rescued growth of LCT1-transformed G19 on saline medium. LCT1 was also shown to mediate the uptake of Li(+) and Cs(+). Expression of two mutant LCT1 cDNAs with N-terminal truncations resulted in decreased Ca(2+) uptake and increased Na(+) tolerance compared with expression of the full-length LCT1. Our findings strongly suggest that LCT1 represents a molecular link between Ca(2+) and Na(+) uptake into plant cells.     

Conserved Ser/Arg-rich motif in PPZ orthologs from fungi is important for its role in cation tolerance

PPZ1 orthologs, novel members of a phosphoprotein phosphatase family of phosphatases, are found only in fungi. They regulate diverse physiological processes in fungi e.g. ion homeostasis, cell size, cell integrity, etc. Although they are an important determinant of salt tolerance in fungi, their physiological role remained unexplored in any halotolerant species. In this context we report here molecular and functional characterization of DhPPZ1 from Debaryomyces hansenii, which is one of the most halotolerant and osmotolerant species of yeast. Our results showed that DhPPZ1 knock-out strain displayed higher tolerance to toxic cations, and unlike in Saccharomyces cerevisiae, Na(+)/H(+) antiporter appeared to have an important role in this process. Besides salt tolerance, DhPPZ1 also had role in cell wall integrity and growth in D. hansenii. We have also identified a short, serine-arginine-rich sequence motif in DhPpz1p that is essential for its role in salt tolerance but not in other physiological processes. Taken together, these results underscore a distinct role of DhPpz1p in D. hansenii and illustrate an example of how organisms utilize the same molecular tool box differently to garner adaptive fitness for their respective ecological niches.     

Extracellular Zn2+ activates epithelial Na+ channels by eliminating Na+ self-inhibition

Inhibition of epithelial Na(+) channel (ENaC) activity by high concentrations of extracellular Na(+) is referred to as Na(+) self-inhibition. We investigated the effects of external Zn(2+) on whole cell Na(+) currents and on the Na(+) self-inhibition response in Xenopus oocytes expressing mouse alphabetagamma ENaC. Na(+) self-inhibition was examined by analyzing inward current decay from a peak current to a steady-state current following a fast switching of a low Na(+) (1 mm) bath solution to a high Na(+) (110 mm) solution. Our results indicate that external Zn(2+) rapidly and reversibly activates ENaC in a dose-dependent manner with an estimated EC(50) of 2 microm. External Zn(2+) in the high Na(+) bath also prevents or reverses Na(+) self-inhibition with similar affinity. Zn(2+) activation is dependent on extracellular Na(+) concentration and is absent in ENaCs containing gammaH239 mutations that eliminate Na(+) self-inhibition and in alphaS580Cbetagamma following covalent modification by a sulfhydryl-reactive reagent that locks the channels in a fully open state. In contrast, external Ni(2+) inhibition of ENaC currents appears to be additive to Na(+) self-inhibition when Ni(2+) is present in the high Na(+) bath. Pretreatment of oocytes with Ni(2+) in a low Na(+) bath also prevents the current decay following a switch to a high Na(+) bath but rendered the currents below the control steady-state level measured in the absence of Ni(2+) pretreatment. Our results suggest that external Zn(2+) activates ENaC by relieving the channel from Na(+) self-inhibition, and that external Ni(2+) mimics or masks Na(+) self-inhibition.     

Physiological basis for the high salt tolerance of Debaryomyces hansenii.

The effects of KCl, NaCl, and LiCl on the growth of Debaryomyces hansenii, usually considered a halotolerant yeast, and Saccharomyces cerevisiae were compared. KCl and NaCl had similar effects on D. hansenii, indicating that NaCl created only osmotic stress, while LiCl had a specific inhibitory effect, although relatively weaker than in S. cerevisiae. In media with low K+, Na+ was able to substitute for K+, restoring the specific growth rate and the final biomass of the culture. The intracellular concentration of Na+ reached values up to 800 mM, suggesting that metabolism is not affected by rather high concentrations of salt. The ability of D. hansenii to extrude Na+ and Li+ was similar to that described for S. cerevisiae, suggesting that this mechanism is not responsible for the increased halotolerance. Also, the kinetic parameters of Rb+ uptake in D. hansenii (Vmax, 4.2 nmol mg [dry weight]-1 min-1; K(m), 7.4 mM) indicate that the transport system was not more efficient than in S. cerevisiae. Sodium (50 mM) activated the transport of Rb+ by increasing the affinity for the substrate in D. hansenii, while the effect was opposite in S. cerevisiae. Lithium inhibited Rb+ uptake in D. hansenii. We propose that the metabolism of D. hansenii is less sensitive to intracellular Na+ than is that of S. cerevisiae, that Na+ substitutes for K+ when K+ is scarce, and that the transport of K+ is favored by the presence of Na+. In low K+ environments, D. hansenii behaved as a halophilic yeast.     

Halotolerant cyanobacterium Aphanothece halophytica contains an Na(+)/H(+) antiporter, homologous to eukaryotic ones, with novel ion specificity affected by C-terminal tail

Recently, a cyanobacterium Synechocystis sp. PCC 6803 has been shown to contain an Na(+)/H(+) antiporter gene homologous to plants (SOS1 and AtNHX1 from Arabidopsis) and mammalians (NHEs from human) but not to Escherichia coli (nhaA and nhaB). Here, we examined whether a halotolerant cyanobacterium Aphanothece halophytica has homologous genes. It turned out that A. halophytica contains an Na(+)/H(+) antiporter homologous to plants, mammalians, and some bacteria (nhaP from Pseudomonas and synnhaP from Synechocystis) but with novel ion specificity. Its gene product, ApNhaP (Na(+)/H(+) antiporter from Aphanothece halophytica), exhibited the Na(+)/H(+) antiporter activity over a wide pH range between 5 and 9 and complemented the Na(+)-sensitive phenotype of the antiporter-deficient E. coli mutant. The ApNhaP had virtually no activity for the Li(+)/H(+) antiporter but showed high Ca(2+)/H(+) antiporter activity at alkaline pH. The ApNhaP complemented the Ca(2+)-sensitive phenotype of the E. coli mutant but not the Li(+)-sensitive phenotype. The replacement of a long C-terminal tail of ApNhaP with that of Synechocystis altered the ion specificity of the antiporter. These results suggest that the ion specificity of an Na(+)/H(+) antiporter is partly determined by the structural properties of the C-terminal tail, which was well exemplified in the case of A. halophytica.     

The Euryhaline Yeast Debaryomyces hansenii has Two Catalase Genes Encoding Enzymes with Differential Activity Profile

Debaryomyces hansenii is a spoilage yeast able to grow in a variety of ecological niches, from seawater to dairy products. Results presented in this article show that (i) D. hansenii has an inherent resistance to H2O2 which could be attributed to the fact that this yeast has a basal catalase activity which is several-fold higher than that observed in Saccharomyces cerevisiae under the same culture conditions, (ii) D. hansenii has two genes (DhCTA1 and DhCTT1) encoding two catalase isozymes with a differential enzymatic activity profile which is not strictly correlated with a differential expression profile of the encoding genes.     

Structural and functional characteristics of two sodium-coupled dicarboxylate transporters (ceNaDC1 and ceNaDC2) from Caenorhabditis elegans and their relevance to life span

We have cloned and functionally characterized two Na(+)-coupled dicarboxylate transporters, namely ceNaDC1 and ceNaDC2, from Caenorhabditis elegans. These two transporters show significant sequence homology with the product of the Indy gene identified in Drosophila melanogaster and with the Na(+)-coupled dicarboxylate transporters NaDC1 and NaDC3 identified in mammals. In a mammalian cell heterologous expression system, the cloned ceNaDC1 and ceNaDC2 mediate Na(+)-coupled transport of various dicarboxylates. With succinate as the substrate, ceNaDC1 exhibits much lower affinity compared with ceNaDC2. Thus, ceNaDC1 and ceNaDC2 correspond at the functional level to the mammalian NaDC1 and NaDC3, respectively. The nadc1 and nadc2 genes are not expressed at the embryonic stage, but the expression is detectable all through the early larva stage to the adult stage. Tissue-specific expression pattern studies using a reporter gene fusion approach in transgenic C. elegans show that both genes are coexpressed in the intestinal tract, an organ responsible for not only the digestion and absorption of nutrients but also for the storage of energy in this organism. Independent knockdown of the function of these two transporters in C. elegans using the strategy of RNA interference suggests that NaDC1 is not associated with the regulation of average life span in this organism, whereas the knockdown of NaDC2 function leads to a significant increase in the average life span. Disruption of the function of the high affinity Na(+)-coupled dicarboxylate transporter NaDC2 in C. elegans may lead to decreased availability of dicarboxylates for cellular production of metabolic energy, thus creating a biological state similar to that of caloric restriction, and consequently leading to life span extension.     

The Rice Monovalent Cation Transporter OsHKT2;4: Revisited Ionic Selectivity

The family of plant membrane transporters named HKT (for high-affinity K(+) transporters) can be subdivided into subfamilies 1 and 2, which, respectively, comprise Na(+)-selective transporters and transporters able to function as Na(+)-K(+) symporters, at least when expressed in yeast (Saccharomyces cerevisiae) or Xenopus oocytes. Surprisingly, a subfamily 2 member from rice (Oryza sativa), OsHKT2;4, has been proposed to form cation/K(+) channels or transporters permeable to Ca(2+) when expressed in Xenopus oocytes. Here, OsHKT2;4 functional properties were reassessed in Xenopus oocytes. A Ca(2+) permeability through OsHKT2;4 was not detected, even at very low external K(+) concentration, as shown by highly negative OsHKT2;4 zero-current potential in high Ca(2+) conditions and lack of sensitivity of OsHKT2;4 zero-current potential and conductance to external Ca(2+). The Ca(2+) permeability previously attributed to OsHKT2;4 probably resulted from activation of an endogenous oocyte conductance. OsHKT2;4 displayed a high permeability to K(+) compared with that to Na(+) (permeability sequence: K(+) > Rb(+) ≈ Cs(+) > Na(+) ≈ Li(+) ≈ NH(4)(+)). Examination of OsHKT2;4 current sensitivity to external pH suggested that H(+) is not significantly permeant through OsHKT2;4 in most physiological ionic conditions. Further analyses in media containing both Na(+) and K(+) indicated that OsHKT2;4 functions as K(+)-selective transporter at low external Na(+), but transports also Na(+) at high (>10 mm) Na(+) concentrations. These data identify OsHKT2;4 as a new functional type in the K(+) and Na(+)-permeable HKT transporter subfamily. Furthermore, the high permeability to K(+) in OsHKT2;4 supports the hypothesis that this system is dedicated to K(+) transport in the plant.