NHX Genes Research Articles

Chitosan nanoparticles (CSNPs) conferred salinity tolerance in maize by upregulating E3 ubiquitin-protein ligase, P5CS1, HKT1, NHX1, and PMP3 genes.

This study explored the transcriptional behaviors of several candidate genes in response to the application of CSNPs (50 and 100 mgl-1) in maize seedlings grown under two salinity levels (NaCl of 0.07 and 0.14 gkg-1soil). Employing CSNPs at both concentrations mitigated the inhibitory role of salinity on the leaf and root fresh weights. The application of CSNPs enhanced the transcription of the E3 ubiquitin-protein ligase gene by an average of threefold, contrasted with the salinity controls. The Δ1-pyrroline-5-carboxylate synthetase (P5CS1) gene was upregulated in response to both individual and mixed treatments of CSNPs and salinity. The transcription of the high-affinity K+ transporter (HKT1) gene displayed an upward trend in response to the CSNPs and salinity treatments. The Na+/H+ exchangers (NHX1) gene exhibited a similar trend to that of the HKT1 gene. The utilization of CSNPs was accompanied by an upregulation in the plasma membrane proteolipid 3 (PMP3) gene, contrasted with the salinity controls. The phenylalanine ammonia-lyase (PAL) activity displayed an upward trend in response to the foliar application of CSNPs. The CSNPs at the 100 mgl-1 concentration were more capable of inducing the ascorbate peroxidase enzyme under both salinity conditions than the 50 mgl-1 dose. The simultaneous exposure of maize seedlings to CSNPs and salinity resulted in the drastic upregulation of the catalase activities. This study provides novel insights into the major mechanisms underlying the stress-mitigating effects of CSNPs, thereby providing a suitable platform for their application in sustainable agricultural practices.

THE ARABIDOPSIS NHX1 GENE: OVEREXPRESSION AND ITS IMPACT ON SALT TOLERANCE IN TRANSGENIC PLANTS

Abiotic stresses, such as salinity, negatively impact crop growth, development and overall production. The discovery and operational confirmation of new genes will provide the framework for successful genetic engineering techniques to increase crop plants' resistance to salinity stress. The most effective method for maintaining ionic balance in plants under stress due to saltiness is provided by the membrane and vacuolar Na+/H+ counter transporter. The function of AtNHX1, the initially identified vacuolar antiporter from more advanced crops, was extensively researched. In this article, we will review the possible function of the Arabidopsis NHX1 gene that is responsible for salt tolerance and its transformation into Wheat, Barley, Poplar, Fescue, Petunia hybrid, Alfalfa & Soybean. Reason to engineer higher plants' genomes using Arabidopsis NHX1 genes to provide food sustainably in salinity-affected locations.

Agro-Physiological and DNA Methylation Responses to Salinity Stress in Wheat (Triticum aestivum L.), Aegilops cylindrica Host, and Their Introgressed Lines

Bottlenecks, including limited genetic variation and the ongoing loss of genetic diversity, have hindered the development of modern wheat cultivars., making it crucial to use genetic diversity from wild relatives to improve wheat’s adaptation to abiotic stress, such as salinity. This study assessed the phenotypic and epigenetic variation of introgressed wheat lines (BC4F2) derived from hybridizing two wheat cultivars with Aegilops cylindrica (AC). This study assessed the phenotypic and epigenetic variation of 156 introgressed wheat lines (BC4F2) derived from hybridization between wheat cultivars “Chinese Spring” (CS) and “Roshan” (R) and Aegilops cylindrica (AC). These lines and their recurrent parents (total of 158) were evaluated under normal and saline field conditions for the agronomic traits and stress tolerance indices. The data were used to select the most tolerant and most sensitive lines. Then, the selected BC4F2 lines and their parents (AC, CS, and R) were subjected to physiological, DNA cytosine methylation, and expression analysis of HKT1;5, NHX1, and SOS1 genes under control and salt stress conditions. Agro-physiological, epigenetic, and gene expression analyses showed the significant effects of salt stress and genetic background, as well as the differential response of the BC4F2 lines to salt stress. The variations in leaf and root K, Na, and K/Na ratios, and leaf Chla, Chlb, Car, and MDA levels, unlike DPPH radical scavenging levels, between salt-tolerant and salt-sensitive BC4F2 lines under saline conditions indicated a substantial distinction in salinity tolerance responses. RT-qPCR indicated higher expression levels of NHX1 and SOS1 genes in the leaf and root tissues of tolerant lines than those of sensitive lines. Global leaf and root DNA methylation analysis revealed the significant effects of salinity on the methylation modifications and confirmed the successful introgression of the salt-tolerance epigenome from Ae. cylindrica into wheat. Exploiting the genetic diversity of wild wheat relatives is a crucial goal for increasing genetic and epigenetic variation to enhance plant adaptation to salt stress.

Identification and characterization of NHX gene family for their role under salt stress in Vigna mungo.

In the current study, we have performed a comprehensive analysis of the Sodium Hydrogen Exchanger (NHX) gene family in Vigna mungo, and a total of 44 NHX genes were identified. A bimodal distribution based on domains, gene structure and phylogenetic analysis was evident. All intronpoor and intron-rich genes were clustered in clades I and II, respectively. Interestingly, all genes of subclade IIb were localized to vacuoles and possess only the NHX domain. The isoelectric point and trans-membrane domain analysis reflect the wide distribution of the NHX genes. Interestingly, Vm_NHX2 and Vm_NHX3 lacked trans-membrane domain but were found to interact with other NHX genes as well as vital salinity pathway genes, including calcium-mediated salt-responsive genes. The comparison of the mRNA sequences with that of V. marina, a halophytic species, reflects their independent evolution, majorly supporting the convergent evolution. The Ka/Ks ratio reflects the abundance of purifying selection supporting their conserved function during evolution. In our analysis, several abiotic stress and hormone-responsive elements and transcription factor binding sites were present in the promoter of the NHX genes. Further, the ion partitioning of a tolerant (K90) and a susceptible (K49) variety of V. mungo suggested that K90 managed the Na+/K+ ratio more affluently, which was also supported by profiling of superoxide radicals, hydrogen peroxide, phenol, peroxidase activity and superoxide dismutase activity. From the expression, we identified five candidate Vm_NHX genes, four of which, i.e. Vm_NHX16, Vm_NHX17, Vm_NHX29 and Vm_NHX33, were localized to the vacuolar and lysosomal membrane.

Relative Expression of a Salinity Stress‐Responsive Na+/H+ Exchanger (NHX) in Root and Leaf Tissues of the African Leafy Vegetable, Amaranthus dubius

ABSTRACTAmaranthus dubius, an African leafy vegetable (ALV), is an easy‐to‐grow, annual shrub and a highly nutritious food source, containing elevated levels of essential nutrients in the leaves. Many ALVs, including A. dubius, can tolerate salinity stress, enabling their cultivation on marginal land. However, the widespread propagation of A. dubius as a stable food source has thus far not been realised due partially to the high frequency at which hybridisation occurs, resulting in high genotypic and phenotypic variability. Therefore, to increase the agricultural output capacity of this species on salt‐affected marginal lands, it is important to screen, select and then clonally propagate the identified salinity‐tolerant genotypes to ensure true‐to‐type fidelity in the regenerated population. It is also important, thereafter, to elucidate their underlying gene expression of the stress response. The present study exposed 4‐week‐old A. dubius seedlings to 100, 200 and 400 mM NaCl to determine their degree of salt tolerance. Genotypes were then screened, selected and clonally propagated through cuttings, based on high growth rates and biomass, and salt tolerance. Generally, growth and physiological parameters decreased as substrate salinity increased. However, individual salt‐stressed genotypes demonstrated similar vigour to nonstressed plants and were able to maintain total protein and chlorophyll concentrations despite increasing salinity. The relative expression of an NHX1‐like transcript was quantified in 15 genotypes using degenerately primed real‐time qPCR. The relative expression of the putative NHX1 gene was 6.7 times greater in root tissues of seedlings treated with 400 mM NaCl (10.7 ± 1.8) compared to the roots of untreated seedlings (1.6 ± 1.3), and 2.8‐fold more than leaf tissues harvested from seedlings treated with 400 mM NaCl. Furthermore, the relative electrical conductivity (EC) of root tissues was 10 times greater than the EC of leaf tissues from the same 400 mM NaCl treatment. Numerous genotypes yielded similar chlorophyll content between 200 and 400 mM NaCl treatments, with genotypes salinity‐1 (S1) (3.5 ± 0.2 μg/cm2) and S34 (4.0 ± 0.4 μg/cm2) having the highest concentrations of chlorophyll in the 400 mM group, which was positively correlated with total protein content. Following micropropagation through direct organogenesis, selected clones maintained true‐to‐type traits such as similar chlorophyll, protein and NHX1‐like expression as their parent plants when exposed to 400 mM NaCl. This study revealed that some genotypes demonstrated salt stress tolerance capabilities rivalling established halophytes by regulating the constitutive or inducible expression of an NHX1‐like protein in roots and leaves. The correlation between protein content and NHX1‐like expression was nonlinear and nonproportional, demonstrating the complexity of this response and necessitating further exploration of specific protein families or functional groups conferring salinity tolerance in this species.

Genome-wide identification, characterization and evolutionary analysis of betaine aldehyde dehydrogenase (BADH), mitogen-activated protein kinase (MAPK) and sodium/hydrogen exchanger (NHX) genes in maize (Zea mays) under salt stress

Betaine aldehyde dehydrogenase (BADH), mitogen-activated protein kinase (MAPK) and sodium/hydrogen exchanger (NHX) play important roles in the response to salt stress. This is the first study to identify the BADH and NHX genes in maize (Zea mays) via genome-wide analysis. The qRT‒PCR results indicated that ZmNHX was upregulated by 4.38-fold, while a significant difference was not observed in ZmBADH or ZmMAPK, with fold changes of 0.96 and 1.06, respectively, under salinity stress. Genome-wide analysis revealed 8 ZmBADH, 19 ZmMAPK and 11 ZmNHX proteins in Z. mays. Domain analysis confirmed the presence of the aldehyde dehydrogenase superfamily (ALDH-SF), protein kinase and Na_H_Exchanger domains in the ZmBADH, ZmMAPK and ZmNHX proteins, respectively. Motif analysis indicated that the phylogenetic relationships were similar to the conserved motif distributions within the clade. The Ka/Ks ratio indicated that the ZmBADH, ZmMAPK and ZmNHX genes were influenced primarily by purifying selection. This study provides comprehensive identification, characterization, and evolutionary analysis for a better understanding of the ZmBADH, ZmMAPK and ZmNHX genes in maize.

In silico genome-wide analysis of Citrus sinensis (L.) Osbeck NHX and KEA genes and their roles in abiotic stress

In silico genome-wide analysis of Citrus sinensis (L.) Osbeck NHX and KEA genes and their roles in abiotic stress

Genome-wide identification and drought stress-induced expression analysis of the NHX gene family in potato.

NHX proteins are transmembrane antiporters belonging to the cation/proton antiporter gene family, with a conserved Na+ (K+)/H+ exchange (PF00999) protein domain. NHXs play a prominent role in plant growth, development, and defense. However, the role of NHX gene family in potato (Solanum tuberosum L.) is yet to be known. In this study, we conducted a genome-wide analysis of the potato NHX gene family. A total of 25 StNHX family members were identified to be unevenly distributed on 10 chromosomes. The proteins ranged in length from 252 to 1,153 amino acids, with molecular masses ranging from 27516.32 to 127860.87 kD, and isoelectric points (pI) ranging from 4.96 to 9.3. Analyses of gene structures and conserved motifs indicated that StNHX genes in the same phylogenetic cluster are conserved. Phylogenetic analysis divided the StNHX genes into three subfamilies (Classes I, II, and III). Synteny analysis indicated that StNHX gene family Class III of NHX and all Arabidopsis thaliana NHXs shared a close evolutionary relationship. Analysis of cis-acting elements in the upstream 1,500bp promoter region of potato NHX genes showed that these genes could be regulated by light, stress, and hormones such as abscisic acid and gibberellic acid. Protein-protein interaction network analysis indicated that StNHX proteins may participate in the regulation of potato growth and stress response. Besides, To determine a potential role of these genes in tissue development and drought response, we analyzed the RNA-seq data of different DM potato tissues. The results showed that NHX genes exhibited distinct tissue-specific expression patterns. We further examined the expression patterns of StNHX in different tissues (leaves, roots, shoots, tubers, stolons, and flowers) during the flowering stage in 'Jizhangshu NO.8.' potato. The qRT-PCR results further confirmed the importance of StNHX genes in potato plant growth and development. We further analyzed the RNA-seq data (DM potato) under different abiotic stresses (salt, drought, and heat), and found that the expression of StNHX genes was induced under abiotic stress. qRT-PCR analysis of shoots and roots of 'Jizhangshu NO.8.' potato treated for 0, 6, 12, and 24h with 15% PEG6000 confirmed that the 25 StNHX genes are involved in the response to drought stress in potato. The results of this study may be useful for selecting appropriate candidate genes for the breeding of new drought-tolerant potato varieties. Furthermore, this study lays a foundation for prospective analysis of StNHX gene functions.

Genome-wide identification and expression analysis of the NHX gene family under salt stress in wheat (Triticum aestivum L).

Salt stress affects plant growth and development, resulting in the loss of crop yield across the world, and sodium-proton antiporters (NHXs) are one of the genes known to promote salt tolerance in transgenic plants. In this study, we conducted a comprehensive genome-wide analysis and expression profile of NHX genes in wheat under salinity stress. We identified 30 TaNHX genes in wheat based on the Na+/H+ exchanger domain, with all genes containing an amiloride motif except one, a known for inhibiting Na+ ions in plants. Phylogenetic analysis classified these genes into three classes with subfamilies: 12 were localized in vacuoles, while 18 were in the endoplasmic reticulum and plasma membrane. Promoter analysis revealed stress-related cis-acting elements, indicating their potential role in abiotic stress tolerance. The non-synonymous (Ka)/synonymous (Ks) ratios highlighted that the majority of TaNHX genes experienced robust purifying selection throughout their evolutionary history. Transcriptomis data analysis and qRT-PCR demonstrated distinct expression patterns for TaNHX genes across various tissues when subjected to salt stress. Additionally, we predicted 20 different miRNA candidates targeting the identified TaNHX genes. Protein-protein interaction prediction revealed NHX6's involvement in the SOS1 pathway, while NHX1 gene exhibit proton antiporter activity. Molecular dynamics (MD) simulations were also conducted to examine the interactions of TaNHX1, TaNHX2, and TaNHX3. These results represent a significant advancement in our understanding of the molecular mechanisms governing Na+ transporters. This may also offer promising avenues for future studies aimed at unraveling the intricate details of their biological roles and applications.

A novel PGPR strain homologous to Beijerinckia fluminensis induces biochemical and molecular changes involved in Arabidopsis thaliana salt tolerance

The use of PGPR is widely accepted as a promising tool for a more sustainable agricultural production and improved plant abiotic stress resistance. This study tested the ability of PVr_9, a novel bacterial strain, homologous to Beijerinckia fluminensis, to increase salt stress tolerance in A. thaliana. In vitro plantlets inoculated with PVr_9 and treated with 150 mM NaCl showed a reduction in primary root growth inhibition compared to uninoculated ones, and a leaf area significantly less affected by salt. Furthermore, salt-stressed PVr_9-inoculated plants had low ROS and 8-oxo-dG, osmolytes, and ABA content along with a modulation in antioxidant enzymatic activities. A significant decrease in Na+ in the leaves and a corresponding increase in the roots were also observed in salt-stressed inoculated plants. SOS1, NHX1 genes involved in plant salt tolerance, were up-regulated in PVr_9-inoculated plants, while different MYB genes involved in salt stress signal response were down-regulated in both roots and shoots. Thus, PVr_9 was able to increase salt tolerance in A. thaliana, thereby suggesting a role in ion homeostasis by reducing salt stress rather than inhibiting total Na+ uptake. These results showed a possible molecular mechanism of crosstalk between PVr_9 and plant roots to enhance salt tolerance, and highlighted this bacterium as a promising PGPR for field applications on agronomical crops.

Identification and characterization of salt stress-responsive NHX gene family in chickpea

Chickpea (Cicer arietinum L.) is an important legume crop, primarily grown in the arid and semi-arid regions. It is a valuable protein source and rich in minerals, especially calcium and phosphorus. Salinity stress adversely affects chickpea germination, vegetative growth, and reproductive activities. The Na+/H+ exchanger (NHX), a gene family of membrane transporters, is well recognized for inducing salt tolerance in plants. The NHXs assist in maintaining ion and pH homeostasis in cells by exchanging K+ or Na+ for H+. This study was designed to identify and characterize the NHX genes for salt stress responsiveness in chickpea. Eight NHX genes were identified in chickpea. The phylogenetic analysis represented the evolutionary relationship of Cicer arietinum NHXs (CarNHXs) with Glycine max, Medicago truncatula, Lotus japonicus, Phaseolus vulgaris, Lens culinaris, Vigna unguiculata, and Oryza sativa. The in-silico gene expression analysis indicated high expression of CarNHX3 (Ca_19073) and CarNHX7 (Ca_02050) under salt stress. The qRT-PCR further validated that the expression of CarNHX3 and CarNHX7 increased when plants were exposed to salt stress. This study provides a way forward to plan functional research to exploit CarNHX genes to enhance salt-tolerance in chickpea.

Understanding the salt overly sensitive pathway in Prunus: Identification and characterization of NHX, CIPK, and CBL genes.

Salinity is a major abiotic stress factor that can significantly impact crop growth, and productivity. In response to salt stress, the plant Salt Overly Sensitive (SOS) signaling pathway regulates the homeostasis of intracellular sodium ion concentration. The SOS1, SOS2, and SOS3 genes play critical roles in the SOS pathway, which belongs to the members of Na+/H+ exchanger (NHX), CBL-interacting protein kinase (CIPK), and calcineurin B-like (CBL) gene families, respectively. In this study, we performed genome-wide identifications and phylogenetic analyses of NHX, CIPK, and CBL genes in six Rosaceae species: Prunus persica, Prunus dulcis, Prunus mume, Prunus armeniaca, Pyrus ussuriensis × Pyrus communis, and Rosa chinensis. NHX, CIPK, and CBL genes of Arabidopsis thaliana were used as controls for phylogenetic analyses. Our analysis revealed the lineage-specific and adaptive evolutions of Rosaceae genes. Our observations indicated the existence of two primary classes of CIPK genes: those that are intron-rich and those that are intron-less. Intron-rich CIPKs in Rosaceae and Arabidopsis can be traced back to algae CIPKs and CIPKs found in early plants, suggesting that intron-less CIPKs evolved from their intron-rich counterparts. This study identified one gene for each member of the SOS signaling pathway in P. persica: PpSOS1, PpSOS2, and PpSOS3. Gene expression analyses indicated that all three genes of P. persica were expressed in roots and leaves. Yeast two-hybrid-based protein-protein interaction analyses revealed a direct interaction between PpSOS3 and PpSOS2; and between PpSOS2 and PpSOS1C-terminus region. Our findings indicate that the SOS signaling pathway is highly conserved in P. persica.

SiPLATZ12 transcription factor negatively regulates salt tolerance in foxtail millet (Setaria italica) by suppressing the expression of SiNHX, SiCBL and SiSOS genes

Salt tolerance seriously limits crop production. Foxtail millet (Setaria italica) is an important crop with high salt and drought tolerance in arid and semi-arid regions. However, the genetic basis underlying the salt tolerance remains poorly described in it. Here, we identify 16 PLATZ genes that mainly expressed in roots and panicles and induced by abiotic stresses in foxtail millet. SiPLATZ12 overexpression results in more Na+ but lower K+ accumulation and lower vacuolar pH than the control. SiPLATZ12 overexpression represses the transcription of most NHX, SOS, and CBL genes. Further investigation reveals that SiPLATZ12 directly binds the A/T rich sequences in the SiNHX2, SiCBL4, and SiCBL7 promoters. Both SiNHX2 and SiCBL4 can improve salt tolerance of ‘Ci846′ and SiPLATZ12 overexpression millet seedlings when overexpressed in them, respectively. Moreover, natural variations in the SiPLATZ12 promoter have significant effects on the different expression of SiPLATZ12 and salt tolerance between millet (Hap1 genotype) and the wild ancestors (Hap18 genotype). The expression levels of SiNHX1, SiNHX2, SiNHX3, and SiNHX7 are highly correlated with the salt tolerance of Hap1 and Hap18 millet seedlings. Therefore, our results uncover a possible domesticated site that can be used to improve salt tolerance in millet.

Foliar Spraying of NaHS Alleviates Cucumber Salt Stress by Maintaining N+/K+ Balance and Activating Salt Tolerance Signaling Pathways.

Hydrogen sulfide (H2S) is involved in the regulation of plant salt stress as a potential signaling molecule. This work investigated the effect of H2S on cucumber growth, photosynthesis, antioxidation, ion balance, and other salt tolerance pathways. The plant height, stem diameter, leaf area and photosynthesis of cucumber seedlings were significantly inhibited by 50 mmol·L-1 NaCl. Moreover, NaCl treatment induced superoxide anion (O2·-) and Na+ accumulation and affected the absorption of other mineral ions. On the contrary, exogenous spraying of 200 μmol·L-1 sodium hydrosulfide (NaHS) maintained the growth of cucumber seedlings, increased photosynthesis, enhanced the ascorbate-glutathione cycle (AsA-GSH), and promoted the absorption of mineral ions under salt stress. Meanwhile, NaHS upregulated SOS1, SOS2, SOS3, NHX1, and AKT1 genes to maintain Na+/K+ balance and increased the relative expression of MAPK3, MAPK4, MAPK6, and MAPK9 genes to enhance salt tolerance. These positive effects of H2S could be reversed by 150 mmol·L-1 propargylglycine (PAG, a specific inhibitor of H2S biosynthesis). These results indicated that H2S could mitigate salt damage in cucumber, mainly by improving photosynthesis, enhancing the AsA-GSH cycle, reducing the Na+/K+ ratio, and inducing the SOS pathway and MAPK pathway.

Understanding physiological and molecular adaptations of three diverse halophytic grasses under saline and sodic stresses

Physiological and biochemical analyses of three halophytes were conducted to explore their tolerance and phytoremediation potential. The halophytes Urochondra setulosa, Sporobolus marginatus and Leptochloa fusca were categorised based on gas exchange attributes, antioxidant system, biomass production under soil salinity (ECe 30-50 dS/m) and sodicity (pH 9.5-10). L. fusca and S. marginatus produced slightly higher biomass under the sodic condition of pH 9.5, while in U. setulosa, it increased under salinity stress. Under sodic conditions, L. fusca showed less reduction in potassium content and maintained a higher K+/Na+ ratio in their leaf tissues. A maximum decrease in net photosynthesis was observed in L. fusca (28.55%) at ECe ~ 50 dS m-1 while minimum in S. marginatus (13.73%) at pH ~ 10.0. Comparatively, U. setulosa showed higher stomatal conductance and transpiration rate than L. fusca and S. marginatus. At the highest pH and salinity, the antioxidant activities of enzymes APX, SOD, GR and POX increased in all three halophytes. Quantitative expression of MnSOD, NHX1 and FuSOS1 genes in all three halophytes increased with salt stresses. Based on these indicators, these halophytes were categorised as salt-tolerant or alkaline-stress-tolerant.

WITHDRAWN: HKT1;5 transporter gene expression and NHX-type Na+/H+ exchanger activity regulate adaptation of Echinacea species to salt stress

Sodium (Na+) toxicity, as one of the main physiological threats to plant growth, may be used for maintaining cell turgor and solute concentrations. Salt-sensitive Echinacea purpurea and Echinacea angustifolia were compared regarding the mechanisms of Na+ uptake, exclusion, energetics, and expression of SOS1, NHX, and HKT1 transporter genes. The species differed in strategies to control Na+ concentration. Echinacea purpurea increased the xylem Na+ concentration upon exposure to 60 mM NaCl by increasing the activity of a high-affinity K+ transporter HKT1;5 and a Na+/H+ exchanger NHX. Echinacea angustifolia decreased the Na+ concentration in leaf sap at the beginning of salinity stress but could not maintain it for long. Higher net Na+ uptake was found in E. angustifolia than E. purpurea, whereas stronger activity of root-cell plasma membrane Na+/H+ (SOS1) exchanger was found in E. purpurea than E. angustifolia. It appeared that passive root entry of Na+ was limited in E. purpurea under salinity stress due to the SOS1-mediated Na+ exclusion. In conclusion, E. purpurea and E. angustifolia possess mechanisms to utilize rather than exclude Na+ as a means of adapting to the salinity stress.

Genome-wide identification of NHX (Na+/H+ antiporter) gene family in Cucurbita L. and functional analysis of CmoNHX1 under salt stress.

Soil salinization, which is the accumulation of salt in soil, can have a negative impact on crop growth and development by creating an osmotic stress that can reduce water uptake and cause ion toxicity. The NHX gene family plays an important role in plant response to salt stress by encoding for Na+/H+ antiporters that help regulate the transport of sodium ions across cellular membranes. In this study, we identified 26 NHX genes in three cultivars of Cucurbita L., including 9 Cucurbita moschata NHXs (CmoNHX1-CmoNHX9), 9 Cucurbita maxima NHXs (CmaNHX1-CmaNHX9) and 8 Cucurbita pepo NHXs (CpNHX1-CpNHX8). The evolutionary tree splits the 21 NHX genes into three subfamilies: the endosome (Endo) subfamily, the plasma membrane (PM) subfamily, and the vacuole (Vac) subfamily. All the NHX genes were irregularly distributed throughout the 21 chromosomes. 26 NHXs were examined for conserved motifs and intron-exon organization. These findings suggested that the genes in the same subfamily may have similar functions while genes in other subfamilies may have functional diversity. The circular phylogenetic tree and collinearity analysis of multi-species revealed that Cucurbita L. had a substantially greater homology relationship than Populus trichocarpa and Arabidopsis thaliana in terms of NHX gene homology. We initially examined the cis-acting elements of the 26 NHXs in order to investigate how they responded to salt stress. We discovered that the CmoNHX1, CmaNHX1, CpNHX1, CmoNHX5, CmaNHX5, and CpNHX5 all had numerous ABRE and G-box cis-acting elements that were important to salt stress. Previous transcriptome data showed that in the mesophyll and veins of leaves, many CmoNHXs and CmaNHXs, such as CmoNHX1, responded significantly to salt stress. In addition, we heterologously expressed in A. thaliana plants in order to further confirm the response of CmoNHX1 to salt stress. The findings demonstrated that during salt stress, A. thaliana that had CmoNHX1 heterologously expression was found to have decreased salt tolerance. This study offers important details that will aid in further elucidating the molecular mechanism of NHX under salt stress.

Identification and expression analysis of NHX gene family in Chinese cabbage

Na+/H+ antiporter (NHX) gene subfamily plays an important role in plant response to salt stress. In this study, we identified the NHX gene family members of Chinese cabbage and analyzed the expression patterns of BrNHXs gene in response to abiotic stresses such as high temperature, low temperature, drought and salt stress. The results showed that there were 9 members of the NHX gene family in Chinese cabbage, which were distributed on 6 chromosomes respectively. The number of amino acids was 513-1 154 aa, the relative molecular weight was 56 804.22-127 856.66 kDa, the isoelectric point was 5.35-7.68. Members of BrNHX gene family mainly existed in vacuoles, the gene structure is complete, and the number of exons is 11-22. The secondary structures of the proteins encoded by the NHX gene family in Chinese cabbage had alpha helix, beta turn and random coil, and the alpha helix occurred more frequently. Quantitative real-time PCR (qRT-PCR) analysis showed that the gene family members had different responses to high temperature, low temperature, drought and salt stress, and their expression levels differed significantly in different time periods. BrNHX02 and BrNHX09 had the most significant responses to these four stresses, and their expression levels were significantly up-regulated at 72 h after treatments, which could be used as candidate genes to further verify their functions.

Arbuscular mycorrhizal fungi affect the expression of PxNHX gene family, improve photosynthesis and promote Populus simonii×P. nigra growth under saline-alkali stress.

Saline-alkali stress seriously endangers the normal growth of Populus simonii×P. nigra. Arbuscular mycorrhizal (AM) fungi can enhance the saline-alkali tolerance of plants by establishing a symbiotic relationship with them. In this study, a pot experiment was conducted to simulate a saline-alkali environment where Populus simonii×P. nigra were inoculated with Funneliformis mosseae to explore their effects on the saline-alkali tolerance of Populus simonii×P. nigra. Our results show that a total of 8 NHX gene family members are identified in Populus simonii×P. nigra. F. mosseae regulate the distribution of Na+ by inducing the expression of PxNHXs. The pH value of poplar rhizosphere soil is reduced, result in the promote absorption of Na+ by poplar, that ultimately improved the soil environment. Under saline-alkali stress, F. mosseae improve the chlorophyll fluorescence and photosynthetic parameters of poplar, promote the absorption of water, K+ and Ca2+, thus increase the plant height and fresh weight of aboveground parts, and promote the growth of poplar. Our results provide a theoretical basis for further exploring the application of AM fungi to improve the saline-alkali tolerance of plants.

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.