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Genome-wide identification of the Glutathione Peroxidase (GPX) gene family in Taxodium distichum and functional characterization of TdGPX9 in enhancing salt tolerance.

This study systematically identified 10 TdGPX genes in Taxodium distichum, demonstrating that the nucleocytoplasmic-localized TdGPX9 plays a pivotal role in salt stress response. Overexpression of TdGPX9 significantly enhances salt tolerance by strengthening the antioxidant defense system and improving root system plasticity under stress. Taxodium distichum is a premier coniferous species renowned for its exceptional waterlogging and salinity tolerance, serving as a vital forest resource for coastal afforestation and wetland ecological restoration. Within the physiological framework of plant stress resistance, the glutathione peroxidase (GPX) family represents a cornerstone of the antioxidant enzymatic system, playing a critical role in scavenging reactive oxygen species and maintaining cellular redox homeostasis. In this study, 10 TdGPX genes were identified via a comprehensive genome-wide analysis and mapped across eight chromosomes. These genes possess a highly conserved Thioredoxin_like domain, with structural and motif analyses revealing a well-maintained arrangement of conserved motifs within each subgroup. The promoter analysis identified a sophisticated regulatory network enriched with cis-acting elements responsive to light, phytohormones, and abiotic stresses, suggesting their integration into diverse signaling pathways. Expression profiling across various tissues and embryonic developmental stages further highlighted the versatile roles of TdGPX members in plant growth and organogenesis. Notably, qRT-PCR analysis identified the nucleocytoplasmic-localized TdGPX9 as a primary respondent to salinity. Functional validation demonstrated that TdGPX9 overexpression significantly enhances salt tolerance in transgenic Arabidopsis and T. distichum callus by strengthening the antioxidant defense system. Furthermore, TdGPX9 promoted root system plasticity under stress, as evidenced by increased lateral root density. These findings provide a systematic basis for understanding the redox-regulatory mechanisms in baldcypress and offer vital genetic resources for improving forest resilience in coastal wetland ecosystems.

Salt Tolerance↗

Enhancement of cyanobacterial salt tolerance by combined nitrogen.

Presence of certain nitrogenous compounds in the growth medium significantly enhanced the salt tolerance of the fresh-water cyanobacterium Anabaena sp. strain L-31 as well as the brackish water cyanobacterium Anabaena torulosa. Among these, nitrate, ammonium, and glutamine were most effective followed by glutamate and aspartate. These nitrogenous compounds also inhibited Na(+) influx in both Anabaena spp. with the same order of effectiveness as that observed for protection against salt stress. The inhibition of Na(+) influx on addition of the nitrogenous substances was rapid; nitrate and ammonium inhibited Na(+) influx competitively. Proline and glycine did not affect Na(+) influx and also had no influence on the salt tolerance of either Anabaena sp. The observed protection was not consequent to a stimulatory effect of combined nitrogen on growth per se. Uptake of NO(3) (-) and NH(4) (+) increased during salt stress but was not correlated with growth. Intracellular levels of NO(3) (-) and NH(4) (+) were found to be inadequate to constitute a major component of the internal osmoticum. The results suggest that inhibition of Na(+) influx by combined nitrogen is a major mechanism for protection of cyanobacteria against salt stress.

Journal Article↗

Growth under alkaline conditions of the salt-tolerant yeast Debaryomyces hansenii IFO10939.

A salt-tolerant yeast Debaryomyces hansenii IFO 10939, which is able to grow at pH 10.0, was isolated and characterized. IFO 10939 had the ability of maintaining intracellular pH. The in vivo activation of plasma membrane ATPase was observed in cells grown at pH 6.2 during conditioning in buffer at pH 9.0. Alkalification of growth medium exhibited a significant increase in acetate and propionate production. The results suggested that the regulation of intracellular pH was involved in plasma membrane ATPase pumping protons out of the cells and weak acid formation for the source of the protons in cells growing at high pH.

Adenosine Triphosphatases↗

Nitrification and occurrence of salt-tolerant nitrifying bacteria in the Negev desert soils.

Ammonia oxidation potential, major ammonia oxidizers and occurrence of salt-tolerant nitrifying bacteria were studied in soil samples collected from diverse ecosystems along the northern Negev desert. Great diversity in ammonia oxidation potential was observed among the soil samples, and ammonia oxidizers were the rate-limiting step of nitrification. Denaturing gradient gel electrophoresis and partial 16S rRNA gene sequences indicate that members of the genus Nitrosospira are the major ammonia oxidizers in the natural desert soil samples. Upon enrichment with different salt concentrations, salt-tolerant nitrifying enrichments were established from several soil samples. In two enrichments, nitrification was not inhibited by 400 mM NaCl. Electrophoretic analysis and partial 16S rRNA gene sequences indicate that Nitrosomonas species were dominant in the 400 mM salt enrichment. The results point towards the potential of the desert ecosystem as a source of stress-tolerant nitrifying bacteria or other microorganisms with important properties.

Ammonia↗

Thialkalivibrio halophilus sp. nov., a novel obligately chemolithoautotrophic, facultatively alkaliphilic, and extremely salt-tolerant, sulfur-oxidizing bacterium from a hypersaline alkaline lake.

A new chemolithoautotrophic, facultatively alkaliphilic, extremely salt-tolerant, sulfur-oxidizing bacterium was isolated from an alkaline hypersaline lake in the Altai Steppe (Siberia, Russia). According to 16S rDNA analysis and DNA-DNA hybridization, strain HL 17T was identified as a new species of the genus Thialkalivibrio belonging to the gamma subdivision of the Proteobacteria for which the name Thialkalivibrio halophilus is proposed. Strain HL 17T is an extremely salt-tolerant bacterium growing at sodium concentrations between 0.2 and 5 M, with an optimum of 2 M Na+. It grew at high concentrations of NaCl and of Na2CO3/NaHCO3 (soda). Strain HL 17T is a facultative alkaliphile growing at pH range 7.5-9.8, with a broad optimum between pH 8.0 and 9.0. It used reduced inorganic sulfur compounds (thiosulfate, sulfide, polysulfide, elemental sulfur, and tetrathionate) as energy sources and electron donors. In continuous culture under energy limitation, thiosulfate was stoichiometrically oxidized to sulfate. In sodium carbonate medium under alkaline conditions, the maximum growth rate was similar, while the biomass yield was lower as compared with the NaCl-grown culture. The maximum sulfur-oxidizing capacity measured in washed cells was higher in the soda buffer independent of the growth conditions. The compatible solute content of the biomass was higher in the sodium chloride-grown culture than in the sodium carbonate/bicarbonate-grown culture. The data suggest that the osmotic pressure differences between soda and NaCl solutions might be responsible for the difference observed in compatible solutes production. This may have important implications in overall energetic metabolism of high salt adaptation.

Chromatiaceae↗

A novel and conserved salt-induced protein is an important determinant of salt tolerance in yeast.

We have isolated a novel yeast gene, HAL1, which upon overexpression improves growth under salt stress. In addition, disruption of this gene decreases salt tolerance. Therefore HAL1 constitutes a rate-limiting determinant for halotolerance. It encodes a polar protein of 32 kDa located in the yeast cytoplasm and unrelated to sequences in data banks. The expression of this gene is increased by high concentrations of either NaCl, KCl or sorbitol. On the other hand, the growth advantage obtained by overexpression of HAL1 is specific for NaCl stress. In cells overexpressing HAL1, sodium toxicity seems to be counteracted by an increased accumulation of potassium. The HAL1 protein could interact with the transport systems which determine intracellular K+ homeostasis. The HAL1 gene and encoded protein are conserved in plants, being induced in these organisms by salt stress and abscisic acid. These results suggest that yeast serves as a convenient model system for the molecular biology of plant salt tolerance.

Amino Acid Sequence↗

Uptake of sodium in protoplasts of salt-sensitive and salt-tolerant cultivars of rice, Oryza sativa L. determined by the fluorescent dye SBFI.

In this study, the uptake of Na+ into the cytosol of rice (Oryza sativa L. cvs Pokkali and BRRI Dhan29) protoplasts was measured using the acetoxy methyl ester of the fluorescent sodium-binding benzofuran isopthalate, SBFI-AM, and fluorescence microscopy. By means of inhibitor analyses the mechanisms for uptake and sequestration of Na+ in the salt-sensitive indica rice cv. BRRI Dhan29 and in the salt-tolerant indica rice cv. Pokkali were detected. Less Na+ was taken up into the cytosol of Pokkali than into BRRI Dhan29. The results indicate that K+-selective channels do not contribute to the Na+ uptake in Pokkali, whereas they are the major pathways for Na+ uptake in BRRI Dhan29 along with non-selective cation channels. However, non-selective cation channels seem to be the main pathways for Na+ uptake in Pokkali. Protoplasts from Pokkali leaves took up Na+ only transiently in the presence of extracellular Na+ at 5-100 mM. Therefore, it is likely that the protoplasts have a mechanism for fast extrusion of Na+ out of the cytoplasm. Experiments with protoplasts pretreated with NH4NO3 and NH4VO3 suggest that the salt-tolerant Pokkali extrudes Na+ mainly into the vacuole. After cultivation of both cultivars in the presence of 10 or 50 mM NaCl for 72 h, the isolated protoplasts from Pokkali took up less Na+ than the control protoplasts. The results suggest that the salt-tolerance in Pokkali depends on reduced uptake through K+-selective channels and a fast extrusion of Na+ into the vacuoles.

Benzofurans↗

Regulation of salt tolerance in fission yeast by a protein-phosphatase-Z-like Ser/Thr protein phosphatase.

In the yeast Saccharomyces cerevisiae, Na+ efflux is mediated by the Ena1 ATPase, and the expression of the ENA1 gene is regulated by the Ppz1 and Ppz2 Ser/Thr protein phosphatases. On the contrary, in the fission yeast Schizosaccharomyces pombe, effective output of Na+ is attributed to the H+/Na+ antiporter encoded by the sod2 gene. We have isolated a S. pombe gene (pzh1) that encodes a 515-amino-acid protein that is 78% identical, from residue 193 to the COOH terminus, to the PPZ1 and PPZ2 gene products. Bacterially expressed Pzh1p shows enzymatic characteristics virtually identical to those of recombinant Ppz1p. When expressed in high-copy number from the PPZ1 promoter, the pzh1 ORF rescues the caffeine-induced lytic defect and slightly decreases the high salt tolerance of S. cerevisiae ppz1delta mutants. Disruption of pzh1 yields viable S. pombe cells and has virtually no effect on tolerance to caffeine or osmotic stress, but it renders the cells highly tolerant to Na+ and Li+, and hypersensitive to K+. Although lack of pzh1 results in a 2-3-fold increase in sod2 mRNA, the pzh1 mutation significantly increases salt tolerance in the absence of the sod2 gene, suggesting that the phosphatase also regulates a Sod2-independent mechanism. Therefore, the finding of a PPZ-like protein phosphatase involved in the regulation of salt tolerance in fission yeast reveals unexpected aspects of cation homeostasis in this organism.

Amino Acid Sequence↗

Identification of BoRR gene family in cauliflower: roles in curd development and salt tolerance.

BACKGROUND: Cauliflower, as an important vegetable crop, the research on its curd formation mechanism and stress-responsive gene networks is of great significance for improving its quality, yield and abiotic stress tolerance. The response regulator (RR) gene family plays a crucial role in the regulation of various life processes of many organisms. In this research, a comprehensive analysis of the BoRR gene family in cauliflower was carried out. RESULTS: A Total of 57 BoRR genes were identified in cauliflower and classified into seven subtypes (type A/B-I/B-II/B-IV/C/B-PRR/Clock PRR) based on sequence homology. Chromosomal mapping showed even distribution across genomes, while physicochemical analysis revealed diverse protein properties (134-915 amino acids, pI 4.51-9.19) with predominant nuclear localization. Structural analyses found all BoRR proteins contain REC-type domains, with subtype-specific features: type A has REC_typeA_ARR, type B harbors REC_typeB_ARR domains, and Clock PRR shows circadian-related psREC_RR domains. Exon numbers range from 2 to 10, with type A BoRR genes having shorter CDS lengths. Collinearity analysis identified 28 pairs of gene duplicates (26 inter-chromosomal). Comparative analysis showed 133 collinear pairs with Brassica napus, 96 with Brassica. rapa, and only 1 with monocots specie (rice and maize). Promoter analysis identified hormone-responsive motifs (ABRE, TGACG), development-related elements (ARE), and stress-responsive sequences (e.g., MBS for drought tolerance) in the promoters of BoRR genes. GO enrichment linked BoRR genes to phosphorelay signaling, cytokinin/ethylene response, and developmental processes like meristem maintenance. Expression profiling during curd development showed type A genes (BoRR23/27/34/38/45) up-regulated in vegetative-reproductive transition, BoRR3/6/12/32/54 in curd enlargement, and several genes like BoRR49 in flower bud differentiation. Salt stress (1.5% NaCl) induced transient expression in 8 of 9 selected BoRR genes at day 1 after treatment. qRT-PCR validated their roles in developmental regulation and salt tolerance. CONCLUSION: This study provides valuable insights into the BoRR gene family in cauliflower, laying a foundation for further understanding its genetic mechanisms and potentially guiding efforts to enhance curd quality and salt tolerance in cauliflower.

Salt Tolerance↗

Addition of NaCl to MS-2 system clinical cuvettes for combined salt tolerance and susceptibility testing for group D streptococci.

Sodium chloride was added directly to individual wells of MS-2 system clinical cuvettes for evaluating the feasibility of simultaneous susceptibility and salt tolerance testing for group D streptococcal isolates. Salt tolerance was correctly detected for all 177 enterococci tested; there was no false-positive results for 29 group D nonenterococcal or 45 viridans streptococcal isolates.

Enterococcus faecalis↗

Overexpression of mtlD gene in transgenic Populus tomentosa improves salt tolerance through accumulation of mannitol.

The mtlD gene encoding mannitol-1-phosphate dehydrogenase, which catalyzes the biosynthesis of mannitol from fructose, was cloned from Escherichia coli and transferred to poplar (Populus tomentosa Carr.) through Agrobacterium-mediated transformation. The transgenic plants were screened and selected on Murashige and Skoog (MS) medium containing 30-50 mg l(-1) kanamycin and verified by polymerase chain reaction (PCR) and Southern blotting. Expression of the gene led to synthesis and accumulation of mannitol in the transgenic plants. Gas chromatography and mass spectrometry (GC/MS) and capillary gas chromatography (GC) showed that transgenic plants accumulated much more mannitol in their tissues than the wild-type plants, whether cultured in vitro, or grown hydroponically or in the field. Increased salt tolerance of transgenic plants was observed both in vitro and in hydroponic culture. The transgenic buds rooted normally on MS medium containing 50 mM NaCl, whereas wild-type buds did not. In the 40-day hydroponic experiments, transgenic poplar plants survived in a 75-mM NaCl treatment, whereas the wild-type poplar plants tolerated only 25 mM NaCl. Under the same NaCl stress, stomatal conductance, transpiration rates and photosynthetic rates were all higher in transgenic plants than in wild-type plants, whereas cellular relative conductivity was lower. We demonstrated that the mtlD gene was expressed in transgenic poplar plants, resulting either directly or indirectly in mannitol accumulation and improved salt tolerance. The constant mannitol concentrations in transgenic plants during the NaCl treatments indicated that mannitol accumulation caused by the mtlD gene was not a consequence of NaCl stress. Height growth was reduced by about 50% in the transgenic plants compared with the wild-type plants in the absence of salt; however, relative growth rate was much less influenced by salt stress in transgenic plants than in wild-type plants. The stunted growth of the transgenic plants may in part explain their improved salt tolerance.

Escherichia coli↗

Crystallization and preliminary X-ray crystallographic studies of salt-tolerant glutaminase from Micrococcus luteus K-3.

Glutaminase from the marine bacterium Micrococcus luteus K-3 (Micrococcus glutaminase) is a salt-tolerant protein which shows equivalent activities both in the absence and the presence of 3 M sodium chloride and is distinct from halophilic proteins, which are inactivated in the absence of salt. To investigate the mechanisms of the salt-tolerant adaptation of Micrococcus glutaminase, the glutaminase and its major fragment containing about 80% of the protein were crystallized using the hanging-drop vapour-diffusion method. The glutaminase crystals belong to space group P622, with unit-cell parameters a = b = 111.4, c = 210.9 A, alpha = beta = 90, gamma = 120 degrees, and diffract to 2.6 A resolution. The fragment crystals belong to space group F222, with unit-cell parameters a = 115.7, b = 116.4, c = 144.9 A, alpha = beta = gamma = 90 degrees, and diffract to 2.4 A resolution. Data from selenomethionine (SeMet) substituted glutaminase crystals and from SeMet-substituted fragment crystals were collected to 2.6 and 2.4 A resolution, respectively. Structural analyses of the glutaminase and its fragment are currently being attempted using the multiwavelength anomalous diffraction (MAD) phasing method.

Crystallization↗

[The expression of gene related to salt tolerance from Sinorhizobium meliloti 042BM in Escherichia coli and purification of its fusion protein].

A 1.9kb DNA fragment related to salt tolerance of S. meliloti strain 042BM containing two open reading frames were obtained by PCR amplification and ligated into shuttle vector pBBR1-MCS2. The complementation experiment showed that ORF2 is related to salt tolerance and named as rstA gene. Then the gene was cloned into the expression vector pThio-HisA, B and C, respectively, and recombinant expression vectors pGSA, pGB and pGC were constructed, and transformed into E. coli Top10. Inducing by IPTG and analyzing with SDS-PAGE, the fusion protein encoded by pGSA was obtained,and it is 36% content of whole cell protein. It was isolated and purified by affinity chromography on ProBond, and the inclusion body precipitated by saturated sulfate ammonium, and 95% purity of fusion protein was obtained. The final product displayed a single band with a corresponding molecular weight 43kD in SDS-PAGE, and was verified by the Western blot.

Bacterial Proteins↗

Chloroplast fructose-1,6-bisphosphatase from Oryza differs in salt tolerance property from the Porteresia enzyme and is protected by osmolytes.

Salinity exerted a distinctly differential effect on fructose-1,6-bisphosphatase (EC. 3.1.3.11) isolated from salt-sensitive and salt-tolerant rice (Oryza sativa) varieties. Cytosolic and chloroplastic isoforms of the enzyme from salt-sensitive rice seedlings exhibited decreased catalytic activity during growth in the presence of salt. Furthermore, chloroplastic fructose 1,6-bisphosphatase purified from salt-sensitive (O. sativa cv. IR26) and from the wild halophytic rice Porteresia coarctata differed in their in vitro salt tolerance property although they exhibited otherwise identical biochemical and immunological properties. This decline in enzyme activity was not correlated with de novo synthesis of the chloroplastic fructose-1,6-bisphosphatase protein in the presence of salt. The inhibitory effect of increasing concentration of NaCl on in vitro enzymatic activity could be prevented by preincubation of the enzyme with a number of osmolytes with an effectiveness in the order polyol>sugars. Further, the intrinsic tryptophan fluorescence of the purified rice enzyme is altered in vitro with increasing NaCl concentration which could be prevented by preincubation with inositol. Purified chloroplastic fructose-1.6-bisphosphatase from P. coarctata however, exhibits no such inhibition of enzyme activity in vitro or alteration in tryptophan fluorescence with increasing NaCl concentration.

Journal Article↗

The Ppz protein phosphatases are key regulators of K+ and pH homeostasis: implications for salt tolerance, cell wall integrity and cell cycle progression.

The yeast Ppz protein phosphatases and the Hal3p inhibitory subunit are important determinants of salt tolerance, cell wall integrity and cell cycle progression. We present several lines of evidence showing that these disparate phenotypes are connected by the fact that Ppz regulates K+ transport. First, salt tolerance, cell wall integrity and cell cycle phenotypes of Ppz mutants are dependent on the Trk K+ transporters. Secondly, Ppz mutants exhibit altered activity of the Trk system, as measured by rubidium uptake. Thirdly, Ppz mutants exhibit altered intracellular K+ and pH, as expected from H+ efflux providing electrical balance during K+ uptake. Our unifying picture of Ppz phenotypes contends that activation of Trk by decreased Ppz activity results in plasma membrane depolarization (reducing uptake of toxic cations), increased intracellular K+ and turgor (compromising cell integrity), and increased intracellular pH (augmenting the expression of pH-regulated genes and facilitating alpha-factor recovery). In addition to providing a coherent explanation for all Ppz-dependent phenotypes, our results provide evidence for a causal relationship between intracellular cation homeostasis and a potential cell cycle checkpoint.

Adenosine Triphosphatases↗

Identification of Candida tropicalis HSR1, a gene of the heat-shock factor-related family, which confers salt tolerance in Saccharomyces cerevisiae.

A genomic library of Candida tropicalis in a yeast multicopy plasmid has been screened for clones conferring salt tolerance upon transformation into S. cerevisiae. The best halotolerance clone contained an open reading frame encoding a predicted protein of 728 amino acids with homology to transcription factors of the heat-shock family. This novel gene was named HSR1 and is present in single copy in the C. tropicalis genome. Upon transformation into S. cerevisiae it increases the expression of ENA1, a major determinant of salt tolerance encoding a cation-extrusion pump. The sequence of CtHSR1 has been deposited in the EMBL data library under Accession No. AJ296093.

Adenosine Triphosphatases↗

Comparative physiology of salt tolerance in Candida tropicalis and Saccharomyces cerevisiae.

The salt tolerance of the respiratory yeast Candida tropicalis and the fermentative yeast Saccharomyces cerevisiae have been compared in glucose media. C. tropicalis showed a better adaptation to Na+ and Li+ and maintained higher intracellular K+:Na+ and K+:Li+ ratios than S. cerevisiae. However, C. tropicalis showed a poorer adaptation to osmotic stress (produced by KCl and sorbitol) and exhibited reduced glycerol production as compared to S. cerevisiae. In media with the non-repressing sugar galactose as carbon source, S. cerevisiae exhibited reduced glycerol production and increased sensitivity to osmotic stress. Under these conditions, S. cerevisiae, but not C. tropicalis, utilized trehalose as a more important osmolyte than glycerol. These results suggest that the relative tolerance of yeast to the osmotic and cation toxicities of NaCl, and the underlying relative capabilities for osmolyte synthesis and cation transport, are modulated by the general catabolite control exerted by glucose.

Adaptation, Physiological↗