Salinity Responsive Genes Research Articles

Overexpression of GmProT1 and GmProT2 increases tolerance to drought and salt stresses in transgenic Arabidopsis

The proline transporter protein (ProT) plays an important role in protective stress responses in various plants. However, its function in abiotic stress responses in soybean (Glycine max) remains obscure. In the present study, two soybean ProT genes, namely GmProT1 and GmProT2, were isolated by homologous cloning. GmProT1 and GmProT2 encode polypeptides of 435 and 433 amino acids, respectively. The GmProT1 and GmProT2 proteins showed high similarity to other ProT proteins. GmProT1 and GmProT2 transcripts were detected in different soybean tissues including roots, stems, leaves, flowers, and developmental seeds, and during diverse developmental stages. GmProT1 was strongly expressed in seeds 35 days after flowering. Quantitative real-time PCR analysis showed that the two genes were highly expressed in leaves and could be strongly induced in response to salt and drought conditions and ABA treatment. Transgenic Arabidopsis thaliana plants overexpressing the two genes were generated, which showed that GmProT genes attenuate damage from salt and drought stress. In addition, transgenic Arabidopsis plants accumulated proline in response to salt and osmotic stress. Transcription levels of salinity-responsive gene (RD29B and S0S3) and drought-induced gene (CDPK1) were higher in the transgenic lines than that of wild type plants. Our work provides evidence that GmProT genes function in the response to abiotic stresses and may affect the synthesis and response system of proline.

Isolation and Functional Validation of Salinity and Osmotic Stress Inducible Promoter from the Maize Type-II H+-Pyrophosphatase Gene by Deletion Analysis in Transgenic Tobacco Plants.

Salinity and drought severely affect both plant growth and productivity, making the isolation and characterization of salinity- or drought-inducible promoters suitable for genetic improvement of crop resistance highly desirable. In this study, a 1468-bp sequence upstream of the translation initiation codon ATG of the promoter for ZmGAPP (maize Type-II H+-pyrophosphatase gene) was cloned. Nine 5´ deletion fragments (D1–D9) of different lengths of the ZmGAPP promoter were fused with the GUS reporter and translocated into tobacco. The deletion analysis showed that fragments D1–D8 responded well to NaCl and PEG stresses, whereas fragment D9 and CaMV 35S did not. The D8 segment (219 bp; -219 to -1 bp) exhibited the highest promoter activity of all tissues, with the exception of petals among the D1–D9 transgenic tobacco, which corresponds to about 10% and 25% of CaMV 35S under normal and NaCl or PEG stress conditions, respectively. As such, the D8 segment may confer strong gene expression in a salinity and osmotic stress inducible manner. A 71-bp segment (-219 to -148 bp) was considered as the key region regulating ZmGAPP response to NaCl or PEG stress, as transient transformation assays demonstrated that the 71-bp sequence was sufficient for the salinity or osmotic stress response. These results enhance our understanding of the molecular mechanisms regulating ZmGAPP expression, and that the D8 promoter would be an ideal candidate for moderating expression of drought and salinity response genes in transgenic plants.

Identification of a 467 bp Promoter of Maize Phosphatidylinositol Synthase Gene (ZmPIS) Which Confers High-Level Gene Expression and Salinity or Osmotic Stress Inducibility in Transgenic Tobacco.

Salinity and drought often affect plant growth and crop yields. Cloning and identification of salinity and drought stress inducible promoters is of great significance for their use in the genetic improvement of crop resistance. Previous studies showed that phosphatidylinositol synthase is involved in plant salinity and drought stress responses but its promoter has not been characterized by far. In the study, the promoter (pZmPIS, 1834 bp upstream region of the translation initiation site) was isolated from maize genome. To functionally validate the promoter, eight 5′ deletion fragments of pZmPIS in different lengths were fused to GUS to produce pZmPIS::GUS constructs and transformed into tobacco, namely PZ1–PZ8. The transcription activity and expression pattern obviously changed when the promoter was truncated. Previous studies have demonstrated that NaCl and PEG treatments are usually used to simulate salinity and drought treatments. The results showed that PZ1–PZ7 can respond well upon NaCl and PEG treatments, while PZ8 not. PZ7 (467 bp) displayed the highest transcription activity in all tissues of transgenic tobacco amongst 5′ deleted promoter fragments, which corresponds to about 20 and 50% of CaMV35S under normal and NaCl or PEG treatment, respectively. This implied that PZ7 is the core region of pZmPIS which confers high-level gene expression and NaCl or PEG inducible nature. The 113 bp segment between PZ7 and PZ8 (-467 to -355 bp) was considered as the key sequence for ZmPIS responding to NaCl or PEG treatment. GUS transient assay in tobacco leaves showed that this segment was sufficient for the NaCl or PEG stress response. Bioinformatic analysis revealed that the 113 bp sequence may contain new elements that are crucial for ZmPIS response to NaCl or PEG stress. These results promote our understanding on transcriptional regulation mechanism of ZmPIS and the characterized PZ7 promoter fragment would be an ideal candidate for the overexpression of drought and salinity responsive gene to improve crop resistance.

Comparative Analysis of Salinity Responsive Candidate Gene Expression in Selected Sri Lankan Rice Varieties

Studying of expression dynamics of salt responsive candidate genes under different genetic background of rice is important to develop resilient salt tolerant rice varieties. The present study was focused to assess the comparative expression of salinity responsive genes which are involved in Na+/K+ homeostasis (OsNHX1), signal transduction pathway (OsMAPK5, OsCDPK7) and synthesis of osmoprotectant (OsTPS1). Comparative expression of selected genes were assessed in At354 (improved), Godawee (traditional) and Kuruwi Perunel (traditional), salinity tolerant varieties and Suwandel, a salinity susceptible traditional variety. The assessment was done before salinization (at 0 h) and after salinization at 24 h and 72 h using semi-quantitative Reverse Transcriptase PCR (RT-PCR). RT-PCR products were analysed using 1.5% agarose gel electrophoresis. Resulted gel profiles were assessed and relative level of gene expression was measured based on the intensity of the PCR product in terms of average pixel values. Relative level of gene expression in four varieties was plotted against the exposure time to salinity. Accordingly, marked variation in expression pattern of the selected genes was detected in four varieties over the tested in Godawee and Suwandel compared to At354 and Kuruwi Perunel. At 24 h, all four genes were up-regulated in At354 and Godawee while they were down-regulated in Suwandel. In Kuruwi Perunel, OsNHX1, OsMAPK5 and OsCDPK7 genes were up-regulated at 24 h while OsTPS1 was up-regulated at 72 h. At 72 h, continuous expression of all genes was observed in Kuruwi Perunel and Godawee, whereas expression was comparatively down-regulated in At354 approximately to the same level under non-stress condition. It could be suggested that At354 might have been adapted to the stress relatively earlier compared to Godawee and Kuruwi Perunel, where expressions of genes were less pronounced. Further experiments with various time period of salinity exposure should be carried out to make detailed inference regarding the expression dynamics in tested rice varieties.

Physiological and gene expression analysis of extreme chickpea (Cicer arietinum L.) genotypes in response to salinity stress

Salinity is a major abiotic stress, which reduces productivity of a broad range of crops, especially legumes. This study provides a comparative overview of physiological responses and expression patterns of critical genes in chickpea genotypes during five time courses of NaCl treatment. Considering lipid peroxidation (MDA), electrolyte leakage, proline content and relative water content which are considered to be direct indicators of salinity tolerance, the Flip 97-43c (T1) and Flip 97-196c (S2) genotypes displayed, respectively, maximum and minimum maintenance of cell membrane integrity, osmolyte accumulation and water retention capacity during salinity stress. Relative gene expression analysis of extreme genotypes was carried out using semi-quantitative RT-PCR and the up- and down regulation of the genes was confirmed by real-time qPCR for nine putative salinity responsive genes. However, up-regulation of salinity responsive genes and sequences including late embryogenesis abundant (CapLEA-1), H1 and 219 cDNA sequences, Nonspecific LTP precursor (LTP), Cu/Zn superoxide dismutase (Cu/Zn SOD) and protein kinase (PK) in tolerant genotype was significantly more than control (no-salinity seedlings) and S2 genotype (p ≤ 0.05). Transcript accumulation of trehalose 6 phosphate synthase (T6PS) and NADPH: isoflavone oxide reductase (IFR) genes in T1 did not record significant differences with the control or S2 genotype. These results suggested that, faster activation of studied genes in T1 genotype and higher accumulation of transcripts, especially LEA and H1, could be possible reasons for its higher tolerance under salinity stress.

Overexpression of a NF-YB3 transcription factor from Picea wilsonii confers tolerance to salinity and drought stress in transformed Arabidopsis thaliana

Nuclear factor Y (NF-Y) is a highly conserved transcription factor comprising NF-YA, NF-YB and NF-YC subunits. To date, the roles of NF-Y subunit in plant still remain elusive. In this study, a subunit NF-YB (PwNF-YB3), was isolated from Picea wilsonii Mast. and its role was studied. PwNF-YB3 transcript was detected in all vegetative and reproductive tissues with higher levels in stem and root and was greatly induced by salinity, heat and PEG but not by cold and ABA treatment. Over-expression of PwNF-YB3 in Arabidopsis showed a significant acceleration in the onset of flowering and resulted in more vigorous seed germination and significant tolerance for seedlings under salinity, drought and osmotic stress compared with wild type plants. Transcription levels of salinity-responsive gene (SOS3) and drought-induced gene (CDPK1) were substantially higher in transgenic Arabidopsis than in wild-type plants. Importantly, CBF pathway markers (COR15B, KIN1, LEA76), but not ABA pathway markers CBF4, were greatly induced under condition of drought. The nuclear localization showed that NF-YB3 acted as a transcription factor. Taken together, the data provide evidence that PwNF-YB3 positively confers significant tolerance to salt, osmotic and drought stress in transformed Arabidopsis plants probably through modulating gene regulation in CBF-dependent pathway.

Comprehensive analysis of transcriptome response to salinity stress in the halophytic turf grass Sporobolus virginicus.

The turf grass Sporobolus virginicus is halophyte and has high salinity tolerance. To investigate the molecular basis of its remarkable tolerance, we performed Illumina high-throughput RNA sequencing on roots and shoots of a S. virginicus genotype under normal and saline conditions. The 130 million short reads were assembled into 444,242 unigenes. A comparative analysis of the transcriptome with rice and Arabidopsis transcriptome revealed six turf grass-specific unigenes encoding transcription factors. Interestingly, all of them showed root specific expression and five of them encode bZIP type transcription factors. Another remarkable transcriptional feature of S. virginicus was activation of specific pathways under salinity stress. Pathway enrichment analysis suggested transcriptional activation of amino acid, pyruvate, and phospholipid metabolism. Up-regulation of several unigenes, previously shown to respond to salt stress in other halophytes was also observed. Gene Ontology enrichment analysis revealed that unigenes assigned as proteins in response to water stress, such as dehydrin and aquaporin, and transporters such as cation, amino acid, and citrate transporters, and H+-ATPase, were up-regulated in both shoots and roots under salinity. A correspondence analysis of the enriched pathways in turf grass cells, but not in rice cells, revealed two groups of unigenes similarly up-regulated in the turf grass in response to salt stress; one of the groups, showing excessive up-regulation under salinity, included unigenes homologos to salinity responsive genes in other halophytes. Thus, the present study identified candidate genes involved in salt tolerance of S. virginicus. This genetic resource should be valuable for understanding the mechanisms underlying high salt tolerance in S. virginicus. This information can also provide insight into salt tolerance in other halophytes.

Paenibacillus yonginensis DCY84T induces changes in Arabidopsis thaliana gene expression against aluminum, drought, and salt stress

Current agricultural production methods, for example the improper use of chemical fertilizers and pesticides, create many health and environmental problems. Use of plant growth-promoting bacteria (PGPB) for agricultural benefits is increasing worldwide and also appears to be a trend for the future. There is possibility to develop microbial inoculants for use in agricultural biotechnology, based on these beneficial plant–microbe interactions. For this study, ten bacterial strains were isolated from Yongin forest soil for which in vitro plant-growth promoting trait screenings, such as indole acetic acid (IAA) production, a phosphate solubilization test, and a siderophore production test were used to select two PGPB candidates. Arabidopsis thaliana plants were inoculated with Paenibacillus yonginensis DCY84T and Micrococcus yunnanensis PGPB7. Salt stress, drought stress and heavy metal (aluminum) stress challenges indicated that P. yonginensis DCY84T-inoculated plants were more resistant than control plants. AtRSA1, AtVQ9 and AtWRKY8 were used as the salinity responsive genes. The AtERD15, AtRAB18, and AtLT178 were selected to check A. thaliana responses to drought stress. Aluminum stress response was checked using AtAIP, AtALS3 and AtALMT1. The qRT-PCR results indicated that P. yonginensis DCY84T can promote plant tolerance against salt, drought, and aluminum stress. P. yonginensis DCY84T also showed positive results during in vitro compatibility testing and virulence assay against X. oryzae pv. oryzae Philippine race 6 (PXO99). Better germination rates and growth parameters were also recorded for the P. yonginensis DCY84T Chuchung cultivar rice seed which was grown on coastal soil collected from Suncheon. Based on these results, P. yonginensis DCY84T can be used as a promising PGPB isolate for crop improvement.

Differential expression of TLP, ERF1, and R2R3MYB in annual Medicago species under salinity conditions.

The present study was conducted to evaluate the responses of three annual Medicago species (M. truncatula, M. laciniata, and M. polymorpha) to salinity. We analyzed publicly available microarray data in NCBI pertaining to salinity-response genes in M. truncatula. Our data search identified Tubby C2 (TLP) and ethylene responsive transcription factor 1 (ERF1) as genes that potentially respond to salinity. We evaluated morpho-physiological traits and the expression of the genes in three Medicago species that had been maintained under control and saline conditions. The analysis of morpho-physiological traits showed that M. polymorpha and M. laciniata were more tolerant to salinity than M. truncatula, as they had lower reductions in biomass and dry root weight and lower increases in anthocyanin concentration. The saline conditions caused a significant increase (P < 0.01) in the expression of TLP in all Medicago species, but caused a significant decrease in the expression of ERF1. Considerable variation in anthocyanin concentrations was found among the three Medicago species. To investigate the cause of this variation, we examined the expression of R2R3MYB, a gene involved in the biosynthesis of anthocyanins. Our analysis showed that saline conditions induced high over-expression of R2R3MYB in all three Medicago spp. The high efficiency of the primer pairs used in qRT-PCR enabled us to compare the expression levels of each gene in the three species. We concluded that the more salt tolerant species showed higher expression of TLP and R2R3MYB under both control and salinity conditions.

Overexpression of a Lotus corniculatus AP2/ERF transcription factor gene, LcERF080, enhances tolerance to salt stress in transgenic Arabidopsis

The APETALA2/ethylene-responsive element binding factors (AP2/ERF) play central roles in the stress response in plants. In this study, we identified and isolated a novel salt stress-related gene, LcERF080, that encodes an AP2/ERF protein in Lotus corniculatus cultivar Leo. LcERF080 was classified into the B-4 group of the ERF subfamily based on multiple sequence alignment and phylogenetic characterization. Expression of LcERF080 was strongly induced by salt, abscisic acid, 1-aminocyclopropane-1-carboxylic acid, methyl jasmonate, and salicylic acid stresses. Subcellular localization assay confirmed that LcERF080 is a nuclear protein. LcERF080 overexpression in Arabidopsis resulted in pleiotropic phenotypes with a higher seed germination rate and transgenic plants with enhanced tolerance to salt stress. Further, under stress conditions, the transgenic lines exhibited elevated levels of soluble sugars and proline as well as relative moisture contents but a lower malondialdehyde content than in control plants. The expression levels of hyperosmotic salinity response genes COR15A, RD22, and P5CS1 were found to be elevated in the LcERF080-overexpressing Arabidopsis plants compared to the wild-type plants. These results reveal that LcERF080 is involved in the responses of plants to salt stress.

Transcriptome analysis of salinity responsiveness in contrasting genotypes of finger millet (Eleusine coracana L.) through RNA-sequencing.

Finger millet (Eleusine coracana L.) is a hardy cereal known for its superior level of tolerance against drought, salinity, diseases and its nutritional properties. In this study, attempts were made to unravel the physiological and molecular basis of salinity tolerance in two contrasting finger millet genotypes viz., CO 12 and Trichy 1. Physiological studies revealed that the tolerant genotype Trichy 1 had lower Na(+) to K(+) ratio in leaves and shoots, higher growth rate (osmotic tolerance) and ability to accumulate higher amount of total soluble sugar in leaves under salinity stress. We sequenced the salinity responsive leaf transcriptome of contrasting finger millet genotypes using IonProton platform and generated 27.91 million reads. Mapping and annotation of finger millet transcripts against rice gene models led to the identification of salinity responsive genes and genotype specific responses. Several functional groups of genes like transporters, transcription factors, genes involved in cell signaling, osmotic homeostasis and biosynthesis of compatible solutes were found to be highly up-regulated in the tolerant Trichy 1. Salinity stress inhibited photosynthetic capacity and photosynthesis related genes in the susceptible genotype CO 12. Several genes involved in cell growth and differentiation were found to be up-regulated in both the genotypes but more specifically in tolerant genotype. Genes involved in flavonoid biosynthesis were found to be down-regulated specifically in the salinity tolerant Trichy 1. This study provides a genome-wide transcriptional analysis of two finger millet genotypes differing in their level of salinity tolerance during a gradually progressing salinity stress under greenhouse conditions.

Induced and constitutive DNA methylation in a salinity-tolerant wheat introgression line.

Cytosine methylation is a well recognized epigenetic mark. Here, the methylation status of a salinity-tolerant wheat cultivar (cv. SR3, derived from a somatic hybridization event) and its progenitor parent (cv. JN177) was explored both globally and within a set of 24 genes responsive to salinity stress. A further comparison was made between DNA extracted from plants grown under control conditions and when challenged by salinity stress. The SR3 and JN177 genomes differed with respect to their global methylation level, and methylation levels were reduced by exposure to salinity stress. We found the genetic stress- (triggered by a combination of different genomes in somatic hybridization) induced methylation pattern of 13 loci in non-stressed SR3; the same 13 loci were found to undergo methylation in salinity-stressed JN177. For the salinity-responsive genes, SR3 and JN177 also showed different methylation modifications. C methylation polymorphisms induced by salinity stress were present in both the promoter and coding regions of some of the 24 selected genes, but only the former were associated with changes in transcript abundance. The expression of both TaFLS1 (encoding a flavonol synthase) and TaWRSI5 (encoding a Bowman-Birk-type protease inhibitor), which showed both a different expression and a different DNA methylation level between SR3 and JN177, enhanced the salinity tolerance of Arabidopsis thaliana. C methylation changes appear to be a common component of the plant response to stress, and methylation changes triggered by somatic hybridization may contribute to the superior salinity tolerance of SR3.

Genome-wide analysis of AP2/ERF family genes from Lotus corniculatus shows LcERF054 enhances salt tolerance.

Lotus corniculatus is used in agriculture as a main forage plant. Members of the Apetala2/ethylene response factor (AP2/ERF) family play important roles in regulating gene expression in response to many forms of stress, including drought and salt. Here, starting from database of the L. corniculatus var. japonicus genome, we identified 127 AP2/ERF genes by insilico cloning method. The phylogeny, gene structures, and putative conserved motifs in L. corniculatus var. japonicus ERF proteins were analyzed. Based on the number of AP2/ERF domains and the function of the genes, 127 AP2/ERF genes from L. corniculatus var. japonicus were classified into five subfamilies named the AP2, dehydration-responsive element binding factor (DREB), ERF, RAV, and a soloist. Outside the AP2/ERF domain, many L. corniculatus var. japonicus-specific conserved motifs were detected. Expression profile analysis of AP2/ERF genes by quantitative real-time PCR revealed that 19 LcERF genes, including LcERF054 (KJ004728), were significantly induced by salt stress. The results showed that the LcERF054 gene encodes a nuclear transcription activator. Overexpression of LcERF054 in Arabidopsis enhanced the tolerances to salt stress, showed higher germination ratio of seeds, and had elevated levels of relative moisture contents, soluble sugars, proline, and lower levels of malondialdehyde under stress conditions compared to wild-type plants. The expression of hyperosmotic salinity response genes COR15A, LEA4-5, P5CS1, and RD29A was found to be elevated in the LcERF054-overexpressing Arabidopsis plants compared to wild type. These results revealed that the LcERF genes play important roles in L. corniculatus cv Leo under salt stress and that LcERFs are attractive engineering targets in applied efforts to improve abiotic stress tolerances in L. corniculatus cv Leo or other crops.

Natural variation of salinity response, population structure and candidate genes associated with salinity tolerance in perennial ryegrass accessions

Natural variation in salinity response, effects of population structure on growth and physiological traits and gene-trait association were examined in 56 global collections of diverse perennial ryegrass (Lolium perenne L.) accessions. Three population structure groups were identified with 66 simple sequence repeat markers, which on average accounted for 9 and 11% of phenotypic variation for the control and salinity treatment at 300 mm NaCl. Group 1 (10 accessions) had greater plant height, leaf dry weight and water content, chlorophyll index, K(+) concentration and K(+) /Na(+) than group 2 (39 accessions) and group 3 (7 accessions) under salinity stress, while group 3 had higher Na(+) than groups 1 and 2. Eighty-seven single nucleotide polymorphisms were detected from four partial candidate genes encoding aquaporin and Na(+) /H(+) antiporter in both plasma and tonoplast membranes. Overall, rapid decay of linkage disequilibrium was observed within 500 bp. Significant associations were found between the putative LpTIP1 and Na(+) for the control and between the putative LpNHX1 and K(+) /Na(+) under the control and salinity treatments after controlling population structure. These results indicate that population structure influenced phenotypic traits, and allelic variation in LpNHX1 may affect salinity tolerance of perennial ryegrass.

OscR, a New Osmolarity-Responsive Regulator in Vibrio cholerae

Cholera, a debilitating enteric disease of humans, is characterized by massive diarrhea that is frequently lethal when untreated. Typically arising from ingestion of contaminated drinking water, the disease is caused by toxigenic strains of the gram-negative bacterium Vibrio cholerae through their colonization of the small intestine and production of cholera toxin (CT), an enterotoxin (5, 15). The bacterium occurs in coastal, bay, and estuarine waters and in association with aquatic plankton as an autochthonous member of aquatic microbial communities. Along with other environmental factors, salinity is thought to govern the distribution of V. cholerae in aquatic environments and consequently might play a key role in the incidence and seasonal occurrence of the disease (2, 3, 7-11, 16, 17, 22, 25, 26, 30, 31, 34). Understanding how the bacterium adapts to changes in salinity in its transitions between aquatic habitats, sources of drinking water, and the human intestine may therefore provide critical insight into the interrelated issues of the environmental dynamics of the bacterium and its pathogenic interactions with humans. In this issue, Shikuma and Yildiz (29) report a transcript profiling approach leading to identification of V. cholerae genes differentially regulated by salinity (osmolarity). Their identification and analysis of salinity-responsive genes, and in particular the identification of a new osmolarity-responsive regulator, OscR, open up new territory on how V. cholerae copes with and responds adaptively to changes in salinity during its transitions between the environment and the human intestine.

Salinity Stress Affects Assimilate Metabolism at the Gene-expression Level during Fruit Development and Improves Fruit Quality in Tomato (Solanum lycopersicum L.)

Irrigation with moderate salinity stress in a hydroponic system improves the inner and outer fruit quality of tomato (Solanum lycopersicon Mill. ‘House Momotaro’). We investigated the effects of 50 mM NaCl in a hydroponic solution on the levels of various metabolites, including soluble sugars, amino acids, and organic acids, and on the expression levels of salinity-responsive genes during fruit development. Under salinity, Brix (%), surface color density, and flesh firmness of the fruit were significantly enhanced, whereas fruit enlargement was suppressed. Salinity stress strongly promoted the accumulation of sucrose, citrate, malate, and glutamate, and slightly promoted glucose and γ-amino butyric acid in red fruit. At the transcriptional level, up-regulation of ethylene-synthetic 1-aminocyclopropane-1-carboxylate oxidase and down-regulation of photosynthetic chlorophyll a/b binding protein Cab-1B occurred earlier in stressed fruit than in control fruit. Additionally, the carotenoid-biosynthesis regulatory gene, Phytoene synthase 1, and phosphoenolpyruvate carboxykinase (PEPCK) were up-regulated under moderate salinity in the red stage. The expression profiles of these genes in stress-treated fruit were consistent with the changes in fruit quality, including earlier ripening and a deeper red color. Furthermore, the up-regulation of PEPCK suggested that gluconeogenesis is involved in the accumulation of sugars in salinity-stressed fruit.