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Salt responses of enzymes from species differing in salt tolerance.

Enzymes which are affected by the addition of inorganic salts during in vitro assay were extracted from salt-sensitive Phaseolus vulgaris, salt-tolerant Atriplex spongiosa, and Salicornia australis and tested for sensitivity to NaCl. In each case malate dehydrogenase, aspartate transaminase, glucose 6-phosphate dehydrogenase, and isocitrate dehydrogenase showed NaCl responses similar to those found for commercially available crystalline enzymes from other organisms. Enzymes extracted from plants grown in saline cultures showed no important changes in specific activity or salt sensitivity. Interaction of pH optima and NaCl concentrations suggests that enzymes may differ in the way they respond to salt treatment.

Journal Article↗

Glycerol metabolism and osmoregulation in the salt-tolerant yeast Debaryomyces hansenii.

A glycerol-nonutilizing mutant of the salt-tolerant yeast Debaryomyces hansenii was isolated. When subjected to salt stress the mutant produced glycerol, and the internal level of glycerol increased linearly in proportion to increases of external salinity as in the wild-type strain. However, at increased salinity the mutant showed a more pronounced decrease of growth rate and growth yield and lost more glycerol to the surrounding medium than did the wild type. Uptake experiments showed glycerol to be accumulated against a strong concentration gradient, and both strains displayed similar kinetic parameters for the uptake of glycerol. An examination of enzyme activities of the glycerol metabolism revealed that the apparent Km of the sn-glycerol 3-phosphate dehydrogenase (EC 1.1.99.5) was increased 330-fold for sn-glycerol 3-phosphate in the mutant. Based on the findings, a scheme for the pathways of glycerol metabolism is suggested.

Glycerol↗

Relation between Ion Accumulation of Salt-Sensitive and Isolated Stable Salt-Tolerant Cell Lines of Citrus aurantium.

Four selected NaCl-tolerant cell lines of Sour orange (Citrus aurantium) were compared with the nonselected cell line in their growth and internal ion content of Na(+), K(+), and Cl(-) when exposed to increasing NaCl concentrations. No difference was found among the various NaCl-tolerant cell lines in Na(+) and Cl(-) uptake, and all these cell lines took up similar or even larger amounts of Na(+) and Cl(-) than the NaCl-sensitive cell line. Exposure of cells of NaCl-sensitive and NaCl-tolerant lines to equal external concentrations of NaCl, resulted in a greater loss of K(+) from the NaCl-sensitive cell line. This observation leads to the conclusion that growth and ability to retain high levels of internal K(+) are correlated. Exposure of the NaCl-tolerant cell lines to salts other than NaCl resulted in even greater tolerance to Na(2)SO(4), but rather poor tolerance to K(+) introduced as either K(2)SO(4) or KCl; the latter has a stronger inhibitory effect. The NaCl-sensitive cell line proved to be more sensitive to replacement of Na(+) by K(+). Analyses of internal Na(+), K(+), and Cl(-) concentrations failed to identify any particular internal ion concentration which could serve as a reliable marker for salt tolerance.

Journal Article↗

Natural protein engineering: a uniquely salt-tolerant, but not halophilic, alpha-type carbonic anhydrase from algae proliferating in low- to hyper-saline environments.

Dunaliella salina is a unicellular green alga thriving in environments ranging from fresh water to hyper-saline lakes, such as the Dead Sea. An unusual, internally duplicated, 60 kDa alpha-type carbonic anhydrase (dCA I), located on the surface of this alga, is expected to function over a broad range of salinities. It would therefore differ from other carbonic anhydrases that already lose activity at low salinities and also from halophilic proteins that require high salinities for conformational stability. Enzymatic analyses indeed indicated that dCA I retained activity at salt concentrations ranging from low salt to at least 1.5 M NaCl or KCl for CO(2) hydration, 2.0 M NaCl for esterase activity and 0.5 M for bicarbonate dehydration. Although measurements at higher salinities were constrained by the interference of salt in the respective assayed reactions, activity was noticeable even at 4.0 M NaCl. Comparisons of the internally duplicated dCA I to single-domain derivatives indicated that inter-domain interactions played a decisive role in the stability, activity, salt tolerance and pH responses of dCA I. Hence dCA I is a uniquely salt- tolerant protein, retaining an active conformation over a large range of salinities and, as a Zn metalloenzyme, largely immune to the specific inhibitory effects of anions. Its unique features make dCA I a useful model to understand the physico-chemical basis of halotolerance and protein-salt interactions in general.

Amino Acid Sequence↗

Studies on high salt tolerance of transgenic tobacco.

With E. coli mtlD gene (encoding mannitol 1-phosphate dehydrogenase) and gutD gene (encoding glucitol 6-phosphate dehydrogenase) cloned, plant expression vector pBIGM had been obtained by inserting mtlD and gutD genes into binary vector pBin438. Tobacco was transformed with A. tumefaciens LBA4404 containing pBIGM. Results of molecular hybridization of transformed plants indicated that mtlD and gutD genes had integrated into the genomic DNA of tobacco plants. Experiments of salt tolerance and analysis of sugar alcohols showed that the accumulation of different sugar alcohols in transgenic tobacco plants had increased salt tolerance of tobacco.

Blotting, Southern↗

GhDMT7-mediated DNA methylation dynamics enhance starch and sucrose metabolism pathways to confer salt tolerance in cotton.

This study provides a comprehensive analysis of the impact of DNA methylation in cotton under salt stress conditions, elucidating its effects on gene expression and biological processes. Here, we determined the structures of the DNA methylation landscape across the cotton genome subjected to salt stress using whole-genome bisulfite sequencing (WGBS) and RNA-seq methodologies. We identified 4938 differentially methylated regions (DMRs) correlated with alterations in gene expression. Salt stress induced significant shifts in DNA methylation patterns, particularly in CHH contexts, suggesting context-dependent epigenetic regulation. DMRs were found to be implicated in diverse biological processes and pathways, encompassing protein metabolism, cellular homeostasis, starch and sucrose metabolism, and plant hormone signaling, all pivotal for cotton's adaptation to salt stress. Furthermore, RNA-seq analysis confirmed the impact of DNA methylation on gene expression, uncovering 9642 salt stress-responsive differentially expressed genes (DEGs). These DEGs exhibited enrichment in pathways such as carbohydrate metabolism, cell wall synthesis, and defense response, underscoring the intricate interplay between methylation and gene regulation in stress response. Moreover, the study investigated the role of the key DNA methyltransferase gene GhDMT7 in modulating cotton's response to salt stress, revealing that its downregulation enhanced cotton's salt tolerance, potentially attributed to decreased DNA methylation levels, reduced membrane damage, and enhanced antioxidant capacity. These findings elucidate the role of DNA methylation in abiotic stress resilience and provide insights for crop improvement.

Gossypium↗

Plant biotechnology--genetic engineering to enhance plant salt tolerance.

Plants not only provide food to humans and animals, but also provide a large number of non-food products of industrial and chemical importance. Moreover, they have the ability to purify the air, soil and water on the earth. Various trials to genetically improve the potential of plants are actively in progress. Salt-tolerance would be an especially important ability to bestow upon plants for agricultural and industrial purposes, because high salinity conditions are ubiquitous on earth and represent major barriers to growth. Enhancement of resistance against both hyper-osmotic stress and Na+ toxity is necessary for successful molecular breeding of salt tolerant plants. Introduction of genes for osmolyte bio-synthesis is useful to increase hyperosmotic tolerance of plant cells. It is introduced in this review that genetically engineered ectoine synthesis results in increased hyperosmotic tolerance of tobacco cells. High concentrations of Na+ reduce cellular activity by interfering with vital Na+-sensitive enzymes and by affecting K+ transport. Understanding the regulation of K+ and Na+ homeostasis is thus indispensable for enhancement of plant Na+ tolerance. My research group is investigating the Na+ efflux activity of the yeast Na+-ATPase (Ena1) when installed in the plasma membrane of plant cells, and the rice K+-Na+ co-transporters (HKT) that contribute to the regulation of K+ and Na+ uptake in root cells.

Journal Article↗

Expression of Arabidopsis SR-like splicing proteins confers salt tolerance to yeast and transgenic plants.

Searching for novel targets of salt toxicity in eukaryotic cells, we have screened an Arabidopsis thaliana cDNA library to isolate genes conferring increased tolerance to salt stress when expressed in the yeast Saccharomyces cerevisiae. Here we show that expression of the 'alternating arginine-rich' (or RS) domains of two different SR-like, putative splicing proteins from Arabidopsis allows yeast cells to tolerate higher lithium and sodium concentrations. Protection against salt stress appears to require the in vivo phosphorylation of these plant polypeptides, since the yeast SR protein kinase Sky1p, which was able to phosphorylate in vitro at least one of them, also proved to be essential for the observed salt tolerance phenotype. In addition, a clone encoding the U1A protein, a previously characterised Arabidopsis splicing factor, was also isolated in the screening. No significant decrease in the intracellular concentration of lithium was observed in yeast cells incubated in the presence of LiCl upon expression of any of the Arabidopsis proteins, suggesting that their effects are not mediated by the stimulation of ion transport. In support of the general significance of these data, we also show that the expression of the RS domain of one of the SR-like proteins in transgenic Arabidopsis plants increases their tolerance to LiCl and NaCl. These results point to an important role of pre-mRNA splicing and SR-like proteins in the salt tolerance of eukaryotic cells, offering a novel route to improve this important trait in crop plants.

Amino Acid Sequence↗

Plastid-expressed betaine aldehyde dehydrogenase gene in carrot cultured cells, roots, and leaves confers enhanced salt tolerance.

Salinity is one of the major factors that limits geographical distribution of plants and adversely affects crop productivity and quality. We report here high-level expression of betaine aldehyde dehydrogenase (BADH) in cultured cells, roots, and leaves of carrot (Daucus carota) via plastid genetic engineering. Homoplasmic transgenic plants exhibiting high levels of salt tolerance were regenerated from bombarded cell cultures via somatic embryogenesis. Transformation efficiency of carrot somatic embryos was very high, with one transgenic event per approximately seven bombarded plates under optimal conditions. In vitro transgenic carrot cells transformed with the badh transgene were visually green in color when compared to untransformed carrot cells, and this offered a visual selection for transgenic lines. BADH enzyme activity was enhanced 8-fold in transgenic carrot cell cultures, grew 7-fold more, and accumulated 50- to 54-fold more betaine (93-101 micromol g(-1) dry weight of beta-Ala betaine and Gly betaine) than untransformed cells grown in liquid medium containing 100 mm NaCl. Transgenic carrot plants expressing BADH grew in the presence of high concentrations of NaCl (up to 400 mm), the highest level of salt tolerance reported so far among genetically modified crop plants. BADH expression was 74.8% in non-green edible parts (carrots) containing chromoplasts, and 53% in proplastids of cultured cells when compared to chloroplasts (100%) in leaves. Demonstration of plastid transformation via somatic embryogenesis utilizing non-green tissues as recipients of foreign DNA for the first time overcomes two of the major obstacles in extending this technology to important crop plants.

Aldehyde Oxidoreductases↗

[Cloning of DNA fragment related to salt tolerance in Sinorhizobium meliloti 042B].

Total DNA partially digested by EcoR I was prepared for S. meliloti 042B, in which 15-25 kb DNA fragments were collected. Vector pLAFR I was purified and digested by EcoR I, and then the various DNA fragments of 042B were ligated with pLAFR I by T4DNA ligase. Gene library of S. meliloti 042B was constructed with pLAFR I using E. coli S17-1 as recipient. The number of bacterial recombinants obtained was about 8,000 and 95% of them contained foreign DNA fragments. Using NTG, 042B was mutated on FY plates and 12 sensitive strains were screened at 0.5 mol/L NaCl from 2,000 colonies. One of them was named GZ17 and selected as a recipient strain. By biparental mating the foreign DNA fragments were introduced from gene library of strain 042B into recipient strain GZ17 which is sensitive to 0.5 mol/L NaCl. Then the transconjugants were grown on FY plates containing tetracycline (20 micrograms/mL) and 0.5 mol/L NaCl. A 7 kb inserted DNA fragment related to salt tolerance was obtained. In subcloning experiment, a 4 kb DNA fragment related to salt tolerance was obtained.

Cloning, Molecular↗

The expression of the Saccharomyces cerevisiae HAL1 gene increases salt tolerance in transgenic watermelon [Citrullus lanatus (Thunb.) Matsun. & Nakai.].

An optimised Agrobacterium-mediated gene transfer protocol was developed in order to obtain watermelon transgenic plants [Citrullus lanatus (Thunb.) Matsun. & Nakai.]. Transformation efficiencies ranged from 2.8% to 5.3%, depending on the cultivar. The method was applied to obtain genetically engineered watermelon plants expressing the Saccharomyces cerevisiae HAL1 gene related to salt tolerance. In order to enhance its constitutive expression in plants, the HAL1 gene was cloned in a pBiN19 plasmid under control of the 35S promoter with a double enhancer sequence from the cauliflower mosaic virus and the RNA4 leader sequence of the alfalfa mosaic virus. This vector was introduced into Agrobacterium tumefaciens strain LBA4404 for further inoculation of watermelon half-cotyledon explants. The introduction of both the neomycin phosphotransferase II and HAL1 genes was assessed in primary transformants (TG1) by polymerase chain reaction analysis and Southern hybridisation. The expression of the HAL1 gene was determined by Northern analysis, and the diploid level of transgenic plants was confirmed by flow cytometry. The presence of the selectable marker gene in the expected Mendelian ratios was demonstrated in TG2 progenies. The TG2 kanamycin-resistant plantlets elongated better and produced new roots and leaves in culture media supplemented with NaCl compared with the control. Salt tolerance was confirmed in a semi-hydroponic system (EC=6 dS m(-1)) on the basis of the higher growth performance of homozygous TG3 lines with respect to their respective azygous control lines without the transgene. The halotolerance observed confirmed the inheritance of the trait and supports the potential usefulness of the HAL1 gene of S. cerevisiae as a molecular tool for genetic engineering of salt-stress protection in other crop species.

Blotting, Northern↗

Expression of Choline Oxidase Gene (codA) Enhances Salt Tolerance of the Tobacco.

The codA gene for choline oxidase, which converts choline into betaine. This enzyme, cloned from a soil bacterium Arthrobactor globiformis, has been transferred and expressed in tobacco by the Agrobatcterium-method transformation through the binary plasmid pGAH/codA. The pGAH/codA carried with Km(R) and Hyg(R),and coding sequence for a transit peptide from Rubisco small subunit gene (rbcS) was inserted between 35 S promoter and codA, so that the COD could be introduced into the chloroplast by this transit peptide. The transformed plants were screened on the medium containing the Km and Hyg. PCR, Western and gold immunolocalization tests showed that the codA gene has integrated into the tobacco DNA genome and its protein was expressed and the mature peptide has gone into the chloroplasts by the transit peptide. The results of salt-tolerance measuring for transgenic plants showed that the transgenic plants were more tolerance to the salt than the control plants. The young transgenic plants (1.0--1.5 cm) could survive at 400 mmol/L NaCl MS medium for more than 30 days. From them the higher tolerance plant T4-400 was obtained, which could grow at the 300 mmol/L NaCl MS medium well. The transgenic plants (6--8cm) could grow normally at the 400 mmol/L NaCl MS medium while wild plants failed to do so. So the transferred plant with codA enhanced its tolerance to the salt stress.

Journal Article↗

[Cloning of HAL1 gene and characterization for salt tolerance tomato].

The HAL1 gene was cloned by PCR strategy and confirmed by sequencing. Its open read frame is 879 bp, encoding a peptide of 294 amino acids (32 kD Protein). A chimaeric construct of HAL1 and Npt II (neomycin phosphotransferase II) was constructed and introduced into commercial cultivars of tomato (Zhong SU No. 5: Lycopersicon escullentum) by Agrobacterium tumefacien-mediated gene transformation. Transformants were selected for their ability to grow and root on media containing kanamycin. Transformation was confirmed by analysis of PCR, Southern blot and RT-PCR. The salt tolerance of transgenic tomato is evaluated by comparing the fresh weight, dry weight, Na+, K+ content of transgenic tomato and control tomato. It is concluded that the over-expressing of HAL1 in tomato could enhance the salt tolerance of the transgenic tomato.

Cloning, Molecular↗

Mutations in the listerial proB gene leading to proline overproduction: effects on salt tolerance and murine infection.

The observed sensitivity of Listeria monocytogenes to the toxic proline analogue L-azetidine-2-carboxylic acid (AZ) suggested that proline synthesis in Listeria may be regulated by feedback inhibition of gamma-glutamyl kinase (GK), the first enzyme of the proline biosynthesis pathway, encoded by the proB gene. Taking advantage of the Epicurian coli mutator strain XL1-Red, we performed random mutagenesis of the recently described proBA operon and generated three independent mutations in the listerial proB homologue, leading to proline overproduction and salt tolerance when expressed in an E. coli (DeltaproBA) background. While each of the mutations (located within a conserved 26-amino-acid region of GK) was shown to confer AZ resistance (AZ(r)) on an L. monocytogenes proBA mutant, listerial transformants failed to exhibit the salt-tolerant phenotype observed in E. coli. Since proline accumulation has previously been linked to the virulence potential of a number of pathogenic bacteria, we analyzed the effect of proline overproduction on Listeria pathogenesis. However, our results suggest that as previously described for proline auxotrophy, proline hyperproduction has no apparent impact on the virulence potential of Listeria.

Amino Acid Sequence↗

Characterization of plasma membrane H(+)-ATPase from salt-tolerant yeast Candida versatilis.

Plasma membrane was isolated from the salt-tolerant yeast Candida versatilis and the ATPase in plasma membrane was characterized. The ATPase was a typical H(+)-ATPase with similar properties to the Saccharomyces cerevisiae and Zygosaccharomyces rouxii enzymes. It was reacted with antibody (IgG) raised against S. cerevisiae plasma membrane H(+)-ATPase. The ATPase activity was not changed by adding NaCl and KCl to the assay solutions, but was increased by NH4+, especially by ammonium sulfate. In vivo stimulation of ATPase activity was observed by the addition of NaCl into the culture medium, as observed in Z. rouxii. No in vivo activation of H(+)-ATPase by glucose metabolism was observed in C. versatilis cells and the activity was independent of the growth phase, like Z. rouxii and unlike S. cerevisiae cells.

Azides↗

Molecular characterization of a salt-tolerant bacterial community in the rice rhizosphere.

The diversity of salt-tolerant bacteria present in the rhizosphere of Oryza sativa was investigated. Fourteen bacterial strains, isolated after enrichment in nitrogen-free, semi-solid medium and showing tolerance to 3% NaCl, were analyzed by restriction patterns produced by amplified DNA coding for 16S rDNA (ARDRA) with enzymes Sau3AI, AluI and RsaI which showed that they were represented by 4 ARDRA types. Biodiversity among the 14 strains was also analyzed by the random amplified polymorphic DNA (RAPD) technique with a 10-mer primer. Partial nucleotide sequence of 16S rDNA assigned these clusters to Serratia marcescens, Pseudomonas aeruginosa, Alcaligenes xylosoxidans and Ochrobactrum anthropi. Notably, all four bacterial species are potential human pathogens that infect immunocompromised patients.

Alcaligenes↗

Function, intracellular localization and the importance in salt tolerance of a vacuolar Na(+)/H(+) antiporter from rice.

We examined the function and intracellular localization of the product of the Na(+)/H(+) antiporter gene (OsNHX1) cloned from rice (Oryza sativa). OsNHX1 has the ability to suppress Na(+), Li(+) and hygromycin sensitivity of yeast nhx1 mutants and sensitivity to a high K(+) concentration, a novel phenotype of the nhx1 mutants. Analysis using rice cells expressing a fusion protein of OsNHX1 and green fluorescent protein and Western blot analysis using antibodies specific for OsNHX1 confirmed the localization of OsNHX1 on the tonoplasts. These results indicate that the OsNHX1 gene encodes a vacuolar (Na(+), K(+))/H(+) antiporter. Treatment with high concentrations of NaCl and KCl increased the transcript levels of OsNHX1 in rice roots and shoots. In addition, overexpression of OsNHX1 improved the salt tolerance of transgenic rice cells and plants. These results suggest that OsNHX1 on the tonoplasts plays important roles in the compartmentation of Na(+) and K(+) highly accumulated in the cytoplasm into the vacuoles, and the amount of the antiporter is one of the most important factors determining salt tolerance in rice.

Cation Transport Proteins↗

PM2, a group 3 LEA protein from soybean, and its 22-mer repeating region confer salt tolerance in Escherichia coli.

To have knowledge of the effect of soybean PM2 protein in protecting dehydrated cells and its functional region, PM2 cDNA was isolated from soybean immature seeds. The recombinants expressing full-length PM2, truncated polypeptides of PM2A (aa 1-262) or PM2B (aa 129-262, 22-mer repeating region), or artificial polypeptide PM2C (duplication of 22-mer repeating region) were constructed. By using SDS-PAGE and mass spectrometry approaches, these fusion polypeptides were identified and proved to be hydrophilic and heat-stable. Spot assays of BL/PM2 and BL/pET28 (as control) showed that protein PM2 increased salt tolerance (500 mM NaCl or 500 mM KCl) of Escherichia coli, rather than osmotic tolerance (1100 mM sorbitol). In addition, comparing the survival ratios of the transformants under 500 mM NaCl or 500 mM KCl stresses, the results showed that: (1) the survival ratios of BL/PM2 and BL/PM2B were quite similar, both showing much higher values than those of BL/pET28. (2) The survival ratios of BL/PM2C were much higher than those of BL/PM2, BL/PM2A, and BL/PM2B. This provides the first experimental evidence that PM2 polypeptide enhances salt tolerance of E. coli cells, and the 22-mer repeat region is an important functional region.

Electrophoresis, Polyacrylamide Gel↗