NHX Genes Research Articles

Resistance genes of salinization in sugar beet

Aim of the investigations was to study genes of resistance to salinization in sugar beet breeding materials. The experiment was conducted using the sugar beet hybrids (Beta vulgaris L.): F119170 (VNIISS), Karioka, Mitika and Hamber (Lion Seeds), Lgovka (Lgovskaya OSS). Expression of genes coding ascorbate peroxidase and NHX-antiporter were studied. In the work, specific primers were used: APX, NHX1, NHX4.1, NHХ5.1 and NHX5. Studying of NHX1, NHX4 and NHX5 genes resulted in selection of the sugar beet genotypes: MS17070, Op18094, F119170 and Humber, the hybrid of Lgovskaya OSS. From the results of gene expression analysis, a main role of NHX5 gene in formation of resistance to salinization was determined. As for ascorbate peroxidase activity, the highest indices were also revealed in the domestic genotypes of MS17070 (115.01 U/mg when treated with 70 mM NaCl), Op18094 (42.3 U/mg when treated with 70 mM NaCl) and Humber (68.6 U/mg when treated with 70 mM NaCl). The sufficiently high level of APO activity was shown both in F119170 (18.8 U/mg when treated with 150 mМ NaCl) and the hybrid of Lgovskaya OSS (13.2 U/mg when treated with 150 mM NaCl). It was determined that BvNHX1 and BvNHX5 genes made a special contribution to formation of resistance to salinization, i.e. they could work as independent units. On the contrary, BvNHX4 was supposed to work only together with them and could not provide stable resistance when BvNHX1 and BvNHX5 were inhibited.

Salinity alleviator bacteria in rice (Oryza sativa L.), their colonization efficacy, and synergism with melatonin.

In this study, rhizospheric and endophytic bacteria were tested for the alleviation of salinity stress in rice. Endophytic isolates were taken from previous studies based on their salt stress-alleviating traits. The rhizospheric bacteria were isolated from rice and screened based on salt tolerance and plant growth-promoting traits. Molecular identification indicated the presence of class Gammaproteobacteria, Bacillota, and Actinomycetia. Two-two most potential isolates each from rhizospheric and endophytic bacteria were selected for in planta trials. Results showed that microbial inoculation significantly improved germination and seedling vigor under elevated salinity. The confocal scanning laser microscopy showed higher bacterial colonization in inoculated rice roots than in control. Based on this experiment, rhizospheric bacteria Brevibacterium frigoritolerans W19 and endophytic Bacillus safensis BTL5 were selected for pot trial along with a growth-inducing compound melatonin 20 ppm. Inoculation of these two bacteria improved the levels of chlorophyll, proline, phenylalanine ammonia-lyase, catalase, superoxide dismutase, polyphenol oxidase, root-shoot length, and dry weight under elevated salt concentration. The gene expression studies showed modulation of SOD1, CATa, NHX1, and PAL1 genes by the bacterial strains and melatonin application. The inoculation was found to have additive effects with 20 ppm melatonin. This enhancement in dry matter accumulation, compatible solute production, and oxidative stress regulation could help plants in mitigating the ill effects of high salinity. Exploring such a combination of microbes and inducer molecules could be potentially useful in developing stress-alleviating bioformulations.

A Na+/H+ antiporter localized on the Golgi-to-vacuole transport system from Camellia sinensis, CsNHX6, plays a positive role in salt tolerance

Tea plant (Camellia sinensis) is an important traditional horticultural plant known for the tea products processed from its leaves, which often faces many different adverse conditions, including saline environments. Na+/H+ antiporters (NHXs) are extensively involved in the process of plant response to salt stress and resistance acquisition, but studies in tea plant are less common. In this study, a novel NHX gene named CsNHX6 was cloned from tea plant, which encodes 528 amino acids with 12 typical transmembrane domains. Our results showed that CsNHX6 had both Na+ and K+ dual transport function, and the transport activity depended on an appropriate H+ concentration. In addition, three conserved acidic residues, D164, E188 and D193 in CsNHX6, are essential for Na+ and K+ transport. Further, we found that CsNHX6 was significantly induced by salt stress, and its overexpression enhanced the tolerance of yeast and Arabidopsis to salt stress, this was closely related to the improvement of Na+ storage capacity of cells. Furthermore, subcellular localization assay revealed that CsNHX6 was localized in a Golgi-to-vacuole transport system, including Golgi, TGN, PVC and vacuole, and this localized distribution could be enhanced by salt stress. Taken together, these findings suggest that a potential Na+ transport network is dominated by CsNHX6 under salt stress, which directly or indirectly achieves the regionalization of excessive Na+, thus endowing organisms with salt tolerance.

A soybean sodium/hydrogen exchanger GmNHX6 confers plant alkaline salt tolerance by regulating Na+/K+ homeostasis.

Alkaline soil has a high pH due to carbonate salts and usually causes more detrimental effects on crop growth than saline soil. Sodium hydrogen exchangers (NHXs) are pivotal regulators of cellular Na+/K+ and pH homeostasis, which is essential for salt tolerance; however, their role in alkaline salt tolerance is largely unknown. Therefore, in this study, we investigated the function of a soybean NHX gene, GmNHX6, in plant response to alkaline salt stress. GmNHX6 encodes a Golgi-localized sodium/hydrogen exchanger, and its transcript abundance is more upregulated in alkaline salt tolerant soybean variety in response to NaHCO3 stress. Ectopic expression of GmNHX6 in Arabidopsis enhanced alkaline salt tolerance by maintaining high K+ content and low Na+/K+ ratio. Overexpression of GmNHX6 also improved soybean tolerance to alkaline salt stress. A single nucleotide polymorphism in the promoter region of NHX6 is associated with the alkaline salt tolerance in soybean germplasm. A superior promoter of GmNHX6 was isolated from an alkaline salt tolerant soybean variety, which showed stronger activity than the promoter from an alkaline salt sensitive soybean variety in response to alkali stress, by luciferase transient expression assays. Our results suggested soybean NHX6 gene plays an important role in plant tolerance to alkaline salt stress.

Overexpression of MsRCI2D and MsRCI2E Enhances Salt Tolerance in Alfalfa (Medicago sativa L.) by Stabilizing Antioxidant Activity and Regulating Ion Homeostasis.

Rare cold-inducible 2 (RCI2) genes from alfalfa (Medicago sativa L.) are part of a multigene family whose members respond to a variety of abiotic stresses by regulating ion homeostasis and stabilizing membranes. In this study, salt, alkali, and ABA treatments were used to induce MsRCI2D and MsRCI2E expression in alfalfa, but the response time and the expression intensity of the MsRCI2D,-E genes were different under specific treatments. The expression intensity of the MsRCI2D gene was the highest in salt- and alkali-stressed leaves, while the MsRCI2E gene more rapidly responded to salt and ABA treatment. In addition to differences in gene expression, MsRCI2D and MsRCI2E differ in their subcellular localization. Akin to MtRCI2D from Medicago truncatula, MsRCI2D is also localized in the cell membrane, while MsRCI2E is different from MtRCI2E, localized in the cell membrane and the inner membrane. This difference might be related to an extra 20 amino acids in the C-terminal tail of MsRCI2E. We investigated the function of MsRCI2D and MsRCI2E proteins in alfalfa by generating transgenic alfalfa chimeras. Compared with the MsRCI2E-overexpressing chimera, under high-salinity stress (200 mmol·L−1 NaCl), the MsRCI2D-overexpressing chimera exhibited a better phenotype, manifested as a higher chlorophyll content and a lower MDA content. After salt treatment, the enzyme activities of SOD, POD, CAT, and GR in MsRCI2D- and -E-overexpressing roots were significantly higher than those in the control. In addition, after salt stress, the Na+ content in MsRCI2D- and -E-transformed roots was lower than that in the control; K+ was higher than that in the control; and the Na+/K+ ratio was lower than that in the control. Correspondingly, H+-ATPase, SOS1, and NHX1 genes were significantly up-regulated, and the HKT gene was significantly down-regulated after 6 h of salt treatment. MsRCI2D was also found to regulate the expression of the MsRCI2B and MsRCI2E genes, and the MsRCI2E gene could alter the expression of the MsRCI2A, MsRCI2B, and MsRCI2D genes. MsRCI2D- and -E-overexpressing alfalfa was found to have higher salt tolerance, manifested as improved activity of antioxidant enzymes, reduced content of reactive oxygen species, and sustained Na+ and K+ ion balance by regulating the expression of the H+-ATPase, SOS1, NHX1, HKT, and MsRCI2 genes.

Calcium/calmodulin modulates salt responses by binding a novel interacting protein SAMS1 in peanut (Arachis hypogaea L.)

The Ca2+/CaM signal transduction pathway helps plants adapt to environmental stress. However, our knowledge on the functional proteins of Ca2+/CaM pathway in peanut (Arachis hypogeae L.) remains limited. In the present study, a novel calmodulin 4 (CaM4)-binding protein S-adenosyl-methionine synthetase 1 (SAMS1) in peanut was identified using a yeast two-hybrid assay. Expression of AhSAMS1 was induced by Ca2+, ABA, and salt stress. To elucidate the function of AhSAMS1, physiological and phenotypic analyses were performed with wild-type and transgenic materials. Overexpression of AhSAMS1 increased spermidine and spermidine synthesis while decreased the contents of ethylene, thereby eliminating excessive reactive oxygen species (ROS) in transgenic lines under salt stress. AhSAMS1 reduced uptake of Na+ and leakage of K+ from mesophyll cells, and was less sensitive to salt stress during early seedling growth, in agreement with the induction of SOS and NHX genes Transcriptomics combined with epigenetic regulation uncovered relationships between differentially expressed genes and differentially methylated regions, which raised the salt tolerance and plants growth. Our findings support a model in which the role of AhSAMS1 in the ROS-dependent regulation of ion homeostasis was enhanced by Ca2+/CaM while AhSAMS1-induced methylation was regulated by CaM, thus providing a new strategy for increasing the tolerance of plants to salt stress.

Agrobacterium rhizogenes-mediated genetic transformation of Musa acuminata cv. Vaibalhla (AAA)

Agrobacterium rhizogenes-mediated genetic transformation of Musa acuminata cv. Vaibalhla (AAA) was successfully carried out using A. rhizogenes strain A4 harboring the binary vector pCAMBIA2301with VrNHX1gene. In the study, male flowers of M. acuminata cv. Vaibalhla were used as explants to obtain white bud-like structures by culturing on MS basal medium supplemented with 2 mg/l 6-Benzylaminopurine (BAP) and 0.5 mg/l 1-Naphthaleneacetic acid (NAA). The subsequent shoot induction and plantlet regeneration were carried out in MS medium supplemented with Kinetin (2 mg/l) and NAA (0.5 mg/l). For the genetic transformation, in vitro raised plantlets were inoculated with A. rhizogenes strain A4 harboring pCAMBIA2301VrNHX1 for 30 min followed by 2 days of co-cultivation in the dark in a semi solid MS basal medium. The treated plants were then transferred and maintained in MS basal medium supplemented with ascorbic acid (75 mg/l), kanamycin (150 mg/l) and cefotaxime (400 mg/l). Initiation of hairy roots were observed within 2 days of transfer which were evaluated for GUS activity and the subsequent GUS+ roots were assayed for transient transformation by PCR using nptII and NHX1 gene specific primers. The transfer of the plasmid as well as the gene was confirmed with positive bands observed at 540 bp and 1.6 kb respectively.

Identification and verification of key salt-tolerant amino acid sites of banana MaNHX5

In order to improve the salt tolerance of banana NHX genes, we cloned a MaNHX5 gene from Musa acuminata L. AAA group and predicted the key salt-tolerant amino acid sites and mutant protein structure changes of MaNHX5 by using bioinformatics tools. The 276-position serine (S) of MaNHX5 protein was successfully mutated to aspartic acid (D) by site-directed mutagenesis, and the AXT3 salt-sensitive mutant yeast was used for a functional complementation test. The results showed that after the mutated MaNHX5 gene was transferred to AXT3 salt-sensitive mutant yeast, the salt tolerance of the mutant yeast was significantly improved under 200 mmol/L NaCl treatment. It is hypothesized that Ser276 of MaNHX5 protein plays an important role in the transport of Na+ across the tonoplast.

Salt stress triggers augmented levels of Na+, K+ and ROS alters salt-related gene expression in leaves and roots of tall wheatgrass (Agropyron elongatum)

In turfgrass breeding, competent grass ecotypes are preferably identified for their resistance to salinity condition. This research was designed to explore genes that induce salt resistance (NHX1, NHX2, HKT1;4, SnRK2.4 and NAC9) and their role in physiological modifications of six tall wheatgrass ecotypes (Agropyron elongatum L.). The sites of sample collection were characterized by different levels of salinity, i.e. low (EC: 4 dS m−1 and pH: 6.5), moderate (EC: 7 dS m−1 and pH: 6.5) and high (EC: 12 dS m−1 and pH: 7.5). This study was designed as a split-plot in a randomized complete block where salinity treatments served as the whole-plot factor and ecotypes served as the subplot factor. The ecotypes were screened for their resistance to salinity, based on visual symptoms, salt injury index, physiological features and biochemical parameters. The results revealed that ecotype ‘AE5’ was most resistant to salinity than other ecotypes, whereas ‘AE3’ was the most susceptible. To understand why these differences occurred, measurements were aimed at revealing mRNA levels that resulted from genes responsible for salt resistance. Our results demonstrated that salinity-resistant ecotypes showed high expression levels of several genes, i.e. NHX1, NHX2, HKT1;4, SnRK2.4 and NAC9 in the leaves and roots. These results were corroborated by a decrease (by 1.5–2.5 times) in stress markers, namely, superoxide anion (O2−), hydrogen peroxide (H2O2) and malondialdehyde (MDA), as well as an increase (by 0.5–7 times) in enzymatic and non-enzymatic antioxidant activity in salinity-resistant ecotypes when the plants were exposed to salinity. We observed higher values of initial root length and lateral root density (21% and 18%, respectively) in salinity-resistant ecotypes under salinity condition, compared to other ecotypes. There were lower expression levels of NHX1 and NHX2 in the roots, which were 3.2 and 2.1 times less, respectively, compared to the leaves. This implied that NHX1 and NHX2 expressions can lead to the sequestration of Na+ in the leaves during salinity condition. The current research revealed that HKT1;4 was more able to restrict Na + accumulation, compared to the actions of NHX1 and NHX2 genes. The over-expression of HKT1;4 in ‘AE5’ allowed a better maintenance of root growth during salinity condition. The expression of NAC9 had an increase of 2.1-fold which correlated with an increase in the amount of antioxidant enzymes. In general, the location of sample collection explained the differences in gene expression, especially regarding the extent to which plants respond to salinity condition. Ultimately, these differences can define physiological features in salinity-resistant and salinity-susceptible ecotypes of tall wheatgrass.

Genome-wide identification, molecular characterization, and gene expression analyses of honeysuckle NHX antiporters suggest their involvement in salt stress adaptation.

BackgroundIon homeostasis is an essential process for the survival of plants under salt stress. Na+/H+ antiporters (NHXs) are secondary ion transporters that regulate Na+ compartmentalization or efflux reduce Na+ toxicity and play a critical role during plant development and stress responses.Methods and ResultsTo gain insight into the functional divergence of NHX genes in honeysuckle, a total of seven LjNHX genes were identified on the whole genome level and were renamed according to their chromosomal positions. All LjNHXs possessed the Na+/H+ exchanger domain and the amiloride-binding site was presented in all NHX proteins except LjNHX4. The phylogenetic analysis divided the seven NHX genes into Vac-clade (LjNHX1/2/3/4/5/7) and PM-clade (LjNHX6) based on their subcellular localization and validated by the distribution of conserved protein motifs and exon/intron organization analysis. The protein-protein interaction network showed that LjNHX4/5/6/7 shared the same putatively interactive proteins, including SOS2, SOS3, HKT1, and AVP1. Cis-acting elements and gene ontology (GO) analysis suggested that most LjNHXs involve in the response to salt stress through ion transmembrane transport. The expression profile analysis revealed that the expression levels of LjNHX3/7 were remarkably affected by salinity. These results suggested that LjNHXs play significant roles in honeysuckle development and response to salt stresses.ConclusionsThe theoretical foundation was established in the present study for the further functional characterization of the NHX gene family in honeysuckle.

Comparative Transcriptome Profiling of Salinity-Induced Genes in Citrus Rootstocks with Contrasted Salt Tolerance

Salinity is one of the most destructive environmental challenges for citriculture worldwide, and all climate change scenarios are predicting an increased impact of salinity on citrus orchards. Citrus cultivars are grown as grafts on various rootstocks to provide specific adaptation to abiotic stress and tolerance to major diseases such as citrus tristeza virus. To understand rootstock–scion interactions with regard to salinity, transcriptome profiling of mRNA expression was analyzed for 12 candidate genes in leaves, shoots, and roots of five Hernandina clementine scions grafted on Rangpur lime (LR), Volkamer lemon (CV), Carrizo citrange (CC), sour orange (Big), and Cleopatra mandarin (MC) rootstocks in response to moderate and severe salinity. qRT-PCR analysis revealed differential gene expression that varied by rootstock, salinity level, and tissue. The majority of induced genes were those involved in ion transporter proteins (mainly NHX1 and HKT1 genes), Cl− homeostasis (CCC1 gene), biosynthesis and accumulation of compatible osmolytes, proline (P5CS gene) and glycine betaine (CMO gene), accumulation of proteins (LEA2 gene), and ROS scavenging antioxidant activity (mainly APX). We show that these expression patterns could explain the relative tolerance of the used rootstocks and report new insights on the main salt tolerance mechanisms activated by these rootstocks.

Genome-Wide Identification, Primary Functional Characterization of the NHX Gene Family in Canavalia rosea, and Their Possible Roles for Adaptation to Tropical Coral Reefs.

Canavalia rosea, distributed in the coastal areas of tropical and subtropical regions, is an extremophile halophyte with good adaptability to high salinity/alkaline and drought tolerance. Plant sodium/hydrogen (Na+/H+) exchanger (NHX) genes encode membrane transporters involved in sodium ion (Na+), potassium ion (K+), and lithium ion (Li+) transport and pH homeostasis, thereby playing key roles in salinity tolerance. However, the NHX family has not been reported in this leguminous halophyte. In the present study, a genome-wide comprehensive analysis was conducted and finally eight CrNHXs were identified in C. rosea genome. Based on the bioinformatics analysis about the chromosomal location, protein domain, motif organization, and phylogenetic relationships of CrNHXs and their coding proteins, as well as the comparison with plant NHXs from other species, the CrNHXs were grouped into three major subfamilies (Vac-, Endo-, and PM-NHX). Promoter analyses of cis-regulatory elements indicated that the expression of different CrNHXs was affected by a series of stress challenges. Six CrNHXs showed high expression levels in five tested tissues of C. rosea in different levels, while CrNHX1 and CrNHX3 were expressed at extremely low levels, indicating that CrNHXs might be involved in regulating the development of C. rosea plant. The expression analysis based on RNA-seq showed that the transcripts of most CrNHXs were obviously decreased in mature leaves of C. rosea plant growing on tropical coral reefs, which suggested their involvement in this species’ adaptation to reefs and specialized islands habitats. Furthermore, in the single-factor stress treatments mimicking the extreme environments of tropical coral reefs, the RNA-seq data also implied CrNHXs holding possible gene-specific regulatory roles in the environmental adaptation. The qRT-PCR based expression profiling exhibited that CrNHXs responded to different stresses to varying degrees, which further confirmed the specificity of CrNHXs’ in responding to abiotic stresses. Moreover, the yeast functional complementation test proved that some CrNHXs could partially restore the salt tolerance of the salt-sensitive yeast mutant AXT3. This study provides comprehensive bio-information and primary functional identification of NHXs in C. rosea, which could help improve the salt/alkaline tolerance of genetically modified plants for further studies. This research also contributes to our understanding of the possible molecular mechanism whereby NHXs maintain the ion balance in the natural ecological adaptability of C. rosea to tropical coral islands and reefs.

Enhanced Flavonoid Accumulation Reduces Combined Salt and Heat Stress Through Regulation of Transcriptional and Hormonal Mechanisms.

Abiotic stresses, such as salt and heat stress, coexist in some regions of the world and can have a significant impact on agricultural plant biomass and production. Rice is a valuable crop that is susceptible to salt and high temperatures. Here, we studied the role of flavanol 3-hydroxylase in response to combined salt and heat stress with the aim of better understanding the defensive mechanism of rice. We found that, compared with wild-type plants, the growth and development of transgenic plants were improved due to higher biosynthesis of kaempferol and quercetin. Furthermore, we observed that oxidative stress was decreased in transgenic plants compared with that in wild-type plants due to the reactive oxygen species scavenging activity of kaempferol and quercetin as well as the modulation of glutathione peroxidase and lipid peroxidase activity. The expression of high-affinity potassium transporter (HKT) and salt overly sensitive (SOS) genes was significantly increased in transgenic plants compared with in control plants after 12 and 24 h, whereas sodium-hydrogen exchanger (NHX) gene expression was significantly reduced in transgenic plants compared with in control plants. The expression of heat stress transcription factors (HSFs) and heat shock proteins (HSPs) in the transgenic line increased significantly after 6 and 12 h, although our understanding of the mechanisms by which the F3H gene regulates HKT, SOS, NHX, HSF, and HSP genes is limited. In addition, transgenic plants showed higher levels of abscisic acid (ABA) and lower levels of salicylic acid (SA) than were found in control plants. However, antagonistic cross talk was identified between these hormones when the duration of stress increased; SA accumulation increased, whereas ABA levels decreased. Although transgenic lines showed significantly increased Na+ ion accumulation, K+ ion accumulation was similar in transgenic and control plants, suggesting that increased flavonoid accumulation is crucial for balancing Na+/K+ ions. Overall, this study suggests that flavonoid accumulation increases the tolerance of rice plants to combined salt and heat stress by regulating physiological, biochemical, and molecular mechanisms.

NHX Gene Family in Camellia sinensis: In-silico Genome-Wide Identification, Expression Profiles, and Regulatory Network Analysis.

Salt stress affects the plant growth and productivity worldwide and NHX is one of those genes that are well known to improve salt tolerance in transgenic plants. It is well characterized in several plants, such as Arabidopsis thaliana and cotton; however, not much is known about NHXs in tea plant. In the present study, NHX genes of tea were obtained through a genome-wide search using A. thaliana as reference genome. Out of the 9 NHX genes in tea, 7 genes were localized in vacuole while the remaining 2 genes were localized in the endoplasmic reticulum (ER; CsNHX8) and plasma membrane (PM; CsNHX9), respectively. Furthermore, phylogenetic relationships along with structural analysis which includes gene structure, location, and protein-conserved motifs and domains were systematically examined and further, predictions were validated by the expression analysis. The dN/dS values show that the majority of tea NHX genes is subjected to strong purifying selection under the course of evolution. Also, functional interaction was carried out in Camellia sinensis based on the orthologous genes in A. thaliana. The expression profiles linked to various stress treatments revealed wide involvement of NHX genes from tea in response to various abiotic factors. This study provides the targets for further comprehensive identification, functional study, and also contributed for a better understanding of the NHX regulatory network in C. sinensis.

Exogenous Myo-Inositol Alleviates Salt Stress by Enhancing Antioxidants and Membrane Stability via the Upregulation of Stress Responsive Genes in Chenopodium quinoa L.

Myo-inositol has gained a central position in plants due to its vital role in physiology and biochemistry. This experimental work assessed the effects of salinity stress and foliar application of myo-inositol (MYO) on growth, chlorophyll content, photosynthesis, antioxidant system, osmolyte accumulation, and gene expression in quinoa (Chenopodium quinoa L. var. Giza1). Our results show that salinity stress significantly decreased growth parameters such as plant height, fresh and dry weights of shoot and root, leaf area, number of leaves, chlorophyll content, net photosynthesis, stomatal conductance, transpiration, and Fv/Fm, with a more pronounced effect at higher NaCl concentrations. However, the exogenous application of MYO increased the growth and photosynthesis traits and alleviated the stress to a considerable extent. Salinity also significantly reduced the water potential and water use efficiency in plants under saline regime; however, exogenous application of myo-inositol coped with this issue. MYO significantly reduced the accumulation of hydrogen peroxide, superoxide, reduced lipid peroxidation, and electrolyte leakage concomitant with an increase in the membrane stability index. Exogenous application of MYO up-regulated the antioxidant enzymes’ activities and the contents of ascorbate and glutathione, contributing to membrane stability and reduced oxidative damage. The damaging effects of salinity stress on quinoa were further mitigated by increased accumulation of osmolytes such as proline, glycine betaine, free amino acids, and soluble sugars in MYO-treated seedlings. The expression pattern of OSM34, NHX1, SOS1A, SOS1B, BADH, TIP2, NSY, and SDR genes increased significantly due to the application of MYO under both stressed and non-stressed conditions. Our results support the conclusion that exogenous MYO alleviates salt stress by involving antioxidants, enhancing plant growth attributes and membrane stability, and reducing oxidative damage to plants.

Sodium uptake and transport regulation, and photosynthetic efficiency maintenance as the basis of differential salt tolerance in rice cultivars

Rice (Oryza sativa L.) is among the most consumed cereals in the world. Its growth is severely affected by excessive salinity, leading to considerable negative economic impacts. Thus, BRS Esmeralda and São Francisco rice cultivars, presenting antagonist cultivation recommendations and differential salt tolerance, were selected to examine how salt stress influences ionic homeostasis and photosynthetic capacity. Phenotypic, physiological, molecular, and morphological results indicated that São Francisco had a better potential to withstand salt stress than BRS Esmeralda. Although salinity promoted a significant increase in Na+ content, particularly in BRS Esmeralda, the harmful effects were less severe in São Francisco. The upregulation of SOS and NHX gene expressions revealed that São Francisco used these mechanisms to control Na+ accumulation in cytosol. Besides, São Francisco plants were efficient in reducing the adverse effects of salinity on photosynthesis. Under salt stress, São Francisco leaves exhibited better effective quantum efficiency of PSII, photochemical extinction coefficient, and electron transport rate. Besides, the relative energy excess in PSII and non-photochemical quenching were both smaller compared to BRS Esmeralda. Na+ cytotoxic effects damaged the chloroplast ultrastructure in BRS Esmeralda, reducing photosynthetic capacity. In contrast, the São Francisco cultivar's better performance was followed by an efficient Na+ exclusion and photosynthetic capacity maintenance, leading to lower growth losses. Overall, the findings are suitable for understanding salt responses and developing functional markers associated with salt stress tolerance improvement in rice.

Can the transcriptional regulation of NHX1, SOS1 and HKT1 genes handle the response of two pomegranate cultivars to moderate salt stress?: Salt-tolerance of two pomegranate cultivars

Molecular mechanisms underlying plant functioning under salt conditions have not been completely elucidated, especially in a recalcitrant and less studied fruit trees such as pomegranate (Punica granatum L.). Here, we identified and characterized the expression of NHX1, HKT1 and SOS1 to understand their role in mediating Na+ and K+ transport, translocation and intracellular compartmentation in two pomegranate cultivars (Wonderful and Parfianka) during the first hours of a moderate salt stress (100 mM). In Wonderful, salt treatment significantly increased the Na+ content only in mature leaves (ML) at 3 h after the beginning of the irrigation (2-fold higher than controls), however a concomitant decrease of K+ content was observed (-33%). A significant decrease of NHX1 and SOS1 levels was observed in ML of Wonderful starting from 10 h. Salt irrigation significantly increased expression levels of these genes at all time points in young leaves of Wonderful (YL; with the exception of NHX1 at 24 h) and led to a 7-fold induction of HKT1 in roots. In Parfianka, salt treatment did not affect the Na+ content, irrespective of leaf age. A significant increase of K+ content was observed only in ML at 3 h (+46%). However, NHX1 gene expression was downregulated at the same time in ML of Parfianka, while it was upregulated in YL. An opposite trend was observed in relation to SOS1 expression. Our finding reinforces the idea that difference between cultivars in ion homeostasis and salt tolerance is associated with transcriptional regulation of NHX1, HKT1 and SOS1 genes, these being members of three major Na+ transporters gene families.

Endophytic bacteria associated with the enhanced cadmium resistance in NHX1- overexpressing tobacco plants

Human industrial activities have left large area of land polluted with cadmium (Cd), which poses tremendous threat to ecosystem. Na+/H+ antiporter 1 (NHX1) was a transporter located on vacuoles, which mediated the compartmentation of Na+ into vacuole and transportation of H+ into the cytoplasm. And the overexpressing of its code gene (NHX1 gene) was previously identified to enhance Cd resistance in tobacco (Nicotiana tabacum) plants possibly by maintaining the change of ion content. However, the mechanisms of Cd resistance in NHX1-overexpressing plants have not been well elucidated yet. In this study, the endophytic bacteria communities, the activities of soil enzymes and the antioxidant enzymes were characterized to explore possible mechanisms of the enhanced Cd resistance in NHX1-overeexpressing tobacco plants. Some differences in the endophytic bacterial community structure in roots of transgenic and WT tobaccos under Cd stress were observed, in which, Sphingomonas, Steroidobacter and Actinoplanes were proposed as vital endophytic bacteria for enhancing Cd resistance of transgenic tobacco plants. In addition, the soil enzymes were increased in the rhizospheric soil around the transgenic tobacco plants compared to WT plants under Cd stress. Under Cd exposure, the SOD and CAT activities were 1.65 times and 1.88 times of that in WT plants, respectively. The content of MDA and H2O2 were also observed to be 35 % and 28 % lower in transgenic tobacco roots compared to WT plants under Cd stress. Moreover, the Cd content of transgenic plants decreased by 38 % under Cd stress compared to WT plants. These findings suggested that the endophytic bacteria communities associated with the NHX1-overexpressing plants might lead to the increased Cd resistance by enhancing the rhizosphere soil enzyme activities, strengthening the antioxidative defense system, and better maintaining of the ion changes. Taken together, this study suggested that the enhanced Cd resistance was observed in transgenic tobacco plants associated with the increase of soil enzyme activities and changes of endophytic bacteria community, and the possible mechanisms involved in this process were also discussed.

Genome-Wide Identification of the NHX Gene Family in Punica granatum L. and Their Expressional Patterns under Salt Stress

Most cultivated lands are suffering from soil salinization, which is a global problem affecting agricultural development and economy. High NaCl concentrations in the soil result in the accumulation of toxic Cl− and Na+ in plants. Na+/H+ antiporter (NHX) can regulate Na+ compartmentalization or efflux to reduce Na+ toxicity. This study aims to identify the NHX genes in pomegranate (Punica granatum L.) from the genome sequences and investigate their expression patterns under different concentrations of NaCl stress. In this study, we used the sequences of PgNHXs to analyze the physicochemical properties, phylogenetic evolution, conserved motifs, gene structures, cis-acting elements, protein tertiary structure and expression pattern. A total of 10 PgNHX genes were identified, and divided into three clades. Conserved motifs and gene structures showed that most of them had an amiloride-binding site (FFI/LY/FLLPPI), except for the members of clade III. There were multiple cis-acting elements involved in abiotic stress in PgNHX genes. Additionally, protein-protein interaction network analysis suggested that PgNHXs might play crucial roles in keeping a balance of Na+ in cells. The qRT-PCR analysis suggested that PgNHXs had tissue-specific expressional patterns under salt stress. Overall, our findings indicated that the PgNHXs could play significant roles in response to salt stress. The theoretical foundation was established in the present study for the further functional characterization of the NHX gene family in pomegranate.

Characterization of trehalose-6-phosphate synthase and Na+/H+ antiporter genes in Vuralia turcica and expression analysis under salt and cadmium stresses.

Vuralia turcica (Fabaceae; Papilionoideae) is a critically endangered endemic plant species in Turkey. This plant grows naturally in saline environments, although the photosynthesis and physiological functions of many plants are affected by salt stress. Molecular control mechanisms and identification of genes involved in these mechanisms constitute the critical field of study in plant science. Trehalose-6-phosphate synthase (TPS) is one of the essential enzyme genes involved in trehalose biosynthesis, which is protective against salt stress. Also, the vacuolar Na+/H+ antiporter gene (NHX) is known to be useful in salt tolerance. In this study, the TPS and NHX-like genes in V. turcica were partially sequenced using degenerate primers for the first time and submitted to the NCBI database (accession numbers MK120983 and MH757417, respectively). Also, the expression levels of the genes encoding TPS and NHX were investigated. The results indicate that the increase in both the level of applied salt and cadmium is coupled with the increase in the expression level of NHX and TPS genes. However, salt exposure significantly affected the expression level of the NHX gene. The findings suggest that the NHX gene might play a crucial role in the salt tolerance ability of V. turcica.