Genome-Wide Identification of the Gossypium hirsutum NHX Genes Reveals that the Endosomal-Type GhNHX4A is Critical for the Salt Tolerance of Cotton.
ABSTRACT: Soil salinization, which is primarily due to excessive Na+ levels, is a major abiotic stress adversely affecting plant growth and development. The Na+/H+ antiporter (NHX) is a transmembrane protein mediating the transport of Na+ or K+ and H+ across the membrane to modulate the ionic balance of plants in response to salt stress. Research regarding NHXs has mainly focused on the vacuolar-type NHX family members. However, the biological functions of the endosomal-type NHXs remain relatively uncharacterized. In this study, 22 NHX family members were identified in Gossypium hirsutum. A phylogenetic analysis divided the GhNHX genes into two categories, with 18 and 4 in the vacuolar and endosomal groups, respectively. The chromosomal distribution of the NHX genes revealed the significant impact of genome-wide duplication during the polyploidization process on the number of GhNHX genes. Analyses of gene structures and conserved motifs indicated that GhNHX genes in the same phylogenetic cluster are conserved. Additionally, the salt-induced expression patterns confirmed that the expression levels of most of the GhNHX genes are affected by salinity. Specifically, in the endosomal group, GhNHX4A expression was substantially up-regulated by salt stress. A yeast functional complementation test proved that GhNHX4A can partially restore the salt tolerance of the salt-sensitive yeast mutant AXT3. Silencing GhNHX4A expression decreased the resistance of cotton to salt stress because of an increase in the accumulation of Na+ in stems and a decrease in the accumulation of K+ in roots. The results of this study may provide the basis for an in-depth characterization of the regulatory functions of NHX genes related to cotton salt tolerance, especially the endosomal-type GhNHX4A. Furthermore, the presented data may be useful for selecting appropriate candidate genes for the breeding of new salt-tolerant cotton varieties.
Project description:BACKGROUND:Plant Na+/H+ antiporters (NHXs) are membrane-localized proteins that maintain cellular Na+/K+ and pH homeostasis. Considerable evidence highlighted the critical roles of NHX family in plant development and salt response; however, NHXs in cotton are rarely studied. RESULTS:The comprehensive and systematic comparative study of NHXs in three Gossypium species was performed. We identified 12, 12, and 23 putative NHX proteins from G. arboreum, G. raimondii, and G. hirsutum, respectively. Phylogenetic study revealed that repeated polyploidization of Gossypium spp. contributed to the expansion of NHX family. Gene structure analysis showed that cotton NHXs contain many introns, which will lead to alternative splicing and help plants to adapt to high salt concentrations in soil. The expression changes of NHXs indicate the possible differences in the roles of distinct NHXs in salt response. GhNHX1 was proved to be located in the vacuolar system and intensively induced by salt stress in cotton. Silencing of GhNHX1 resulted in enhanced sensitivity of cotton seedlings to high salt concentrations, which suggests that GhNHX1 positively regulates cotton tolerance to salt stress. CONCLUSION:We characterized the gene structure, phylogenetic relationship, chromosomal location, and expression pattern of NHX genes from G. arboreum, G. raimondii, and G. hirsutum. Our findings indicated that the cotton NHX genes are regulated meticulously and differently at the transcription level with possible alternative splicing. The tolerance of plants to salt stress may rely on the expression level of a particular NHX, rather than the number of NHXs in the genome. This study could provide significant insights into the function of plant NHXs, as well as propose promising candidate genes for breeding salt-resistant cotton cultivars.
Project description:Soil salinity is a main abiotic stress in agriculture worldwide. The Na+/H+ antiporters (NHXs) play pivotal roles in intracellular Na+ excretion and vacuolar Na+ compartmentalization, which are important for plant salt stress resistance (SSR). However, few systematic analyses of NHXs has been reported in allotetraploid rapeseed so far. Here, a total of 18 full-length NHX homologs, representing seven subgroups (NHX1-NHX8 without NHX5), were identified in the rapeseed genome (AnAnCnCn). Number variations of BnaNHXs might indicate their significantly differential roles in the regulation of rapeseed SSR. BnaNHXs were phylogenetically divided into three evolutionary clades, and the members in the same subgroups had similar physiochemical characteristics, gene/protein structures, and conserved Na+ transport motifs. Darwin´s evolutionary pressure analysis suggested that BnaNHXs suffered from strong purifying selection. The cis-element analysis revealed the differential transcriptional regulation of NHXs between the model Arabidopsis and B. napus. Differential expression of BnaNHXs under salt stress, different nitrogen forms (ammonium and nitrate), and low phosphate indicated their potential involvement in the regulation of rapeseed SSR. Global landscapes of BnaNHXs will give an integrated understanding of their family evolution and molecular features, which will provide elite gene resources for the genetic improvement of plant SSR through regulating the NHX-mediated Na+ transport.
Project description:The Arabidopsis vacuolar Na+/H+ transporters (NHXs) are important regulators of intracellular pH, Na+ and K+ homeostasis and necessary for normal plant growth, development, and stress acclimation. Arabidopsis contains four vacuolar NHX isoforms known as AtNHX1 to AtNHX4. The quadruple knockout nhx1nhx2nhx3nhx4, lacking any vacuolar NHX-type antiporter activity, displayed auxin-related phenotypes including loss of apical dominance, reduced root growth, impaired gravitropism and less sensitivity to exogenous IAA and NAA, but not to 2,4-D. In nhx1nhx2nhx3nhx4, the abundance of the auxin efflux carrier PIN2, but not PIN1, was drastically reduced at the plasma membrane and was concomitant with an increase in PIN2 labeled intracellular vesicles. Intracellular trafficking to the vacuole was also delayed in the mutant. Measurements of free IAA content and imaging of the auxin sensor DII-Venus, suggest that auxin accumulates in root tips of nhx1nhx2nhx3nhx4. Collectively, our results indicate that vacuolar NHX dependent cation/H+ antiport activity is needed for proper auxin homeostasis, likely by affecting intracellular trafficking and distribution of the PIN2 efflux carrier.
Project description:The endosomal-type Na+, K+/H+ antiporters (NHXs) play important roles in K+, vesicle pH homeostasis, and protein trafficking in plant. However, the structure governing ion transport mechanism and the key residues related to the structure-function of the endosomal-type NHXs remain unclear. Here, the structure-function relationship of the only endosomal-type NHX from mulberry, MnNHX6, was investigated by homology modeling, mutagenesis, and localization analyses in yeast. The ectopic expression of MnNHX6 in arabidopsis and Nhx1 mutant yeast can enhance their salt tolerance. MnNHX6's three-dimensional structure, established by homology modeling, was supported by empirical, phylogenetic, and experimental data. Structure analysis showed that MnNHX6 contains unusual 13 transmembrane helices, but the structural core formed by TM5-TM12 assembly is conserved. Localization analysis showed that MnNHX6 has the same endosomal localization as yeast Nhx1/VPS44, and Arg402 is important for protein stability of MnNHX6. Mutagenesis analysis demonstrated MnNHX6 contains a conserved cation binding mechanism and a similar charge-compensated pattern as NHE1, but shares a different role in ion selectivity than the vacuolar-type NHXs. These results improve our understanding of the role played by the structure-function related key residues of the plant endosomal-type NHXs, and provide a basis for the ion transport mechanism study of endosomal-type NHXs.
Project description:The Na+/H+ antiporters (NHXs) are secondary ion transporters to exchange H+ and transfer the Na+ or K+ across membrane, they play crucial roles during plant development and stress responses. To gain insight into the functional divergence of NHX genes in poplar, eight PtNHX were identified from Populus trichocarpa genome. PtNHXs containing 10 transmembrane helices (TMH) and a hydrophilic C-terminal domain, the TMH compose a hollow cylinder to provide the channel for Na+ and H+ transport. The expression patterns and cis-acting elements showed that all the PtNHXs were response to single or multiple stresses including drought, heat, cold, salinity, MV, and ABA. Both the co-expression network and protein-protein interaction network of PtNHXs implying their functional divergence. Interestingly, although PtNHX7 and PtNHX8 were generated by whole genome duplication event, they showed significant differences in expression pattern, protein structure, co-expressed genes, and interacted proteins. Only PtNHX7 interact with CBL and CIPK, indicating PtNHX7 is the primary NHX involved in CBL-CIPK pathway during salt stress responses. Natural variation analysis based on 549?P. trichocarpa individuals indicated the frequency of SNPs in PtNHX7 was significantly higher than other PtNHXs. Our findings provide new insights into the functional divergence of NHX genes in poplar.
Project description:The sodium/hydrogen antiporter (NHX) gene family with the Na+/H+ exchange protein domain is a transporter of sodium and hydrogen ions and plays an important role in the response of plants to salt stress. Studying the response of cotton to salt stress through comprehensive identification and analysis of NHX genes in several species and their roles in salt tolerance mechanisms is of great significance. In this study, 23, 24, 12, and 12 NHX genes were identified from Gossypium hirsutum (Gh), G. barbadense, G. arboreum and G. raimondii, respectively. Phylogenetic analysis showed that these genes were mainly divided into three clades with significant subcellular localization, namely, endosome (Endo-class), plasma membrane (PM-class) and vacuole (Vac-class). By analyzing the structure of NHX genes and proteins, each branch of the NHX gene family was found to be structurally conserved, and collinearity analysis showed that NHX genes were mainly expressed through whole genome and segmental duplication. The non-synonymous (Ka)/synonymous (Ks) values showed that the NHX gene family experienced strong purifying selection during long-term evolution. Cis-acting element analysis showed that the NHX gene family may be related to the regulation of abscisic acid (ABA) and methyl jasmonate (MeJA) hormones. Additionally, transcriptomic data analysis and qRT-PCR showed that GhNHXs exhibited different expression patterns in each tissue and under different salinities. These results provide an important reference for us to further understand and analyze the molecular regulation mechanism of cotton NHX genes.
Project description:Cotton is an important economic crop affected by different abiotic stresses at different developmental stages. Salinity limits the growth and productivity of crops worldwide. Na+/H+ antiporters play a key role during the plant development and in its tolerance to salt stress. The aim of the present study was a genome-wide characterization and expression pattern analysis under the salinity stress of the sodium-proton antiporter (NHX) of Gossypium barbadense in comparison with Gossypium hirsutum. In G. barbadense, 25 NHX genes were identified on the basis of the Na+_H+ exchanger domain. All except one of the G. barbadense NHX transporters have an Amiloride motif that is a known inhibitor of Na+ ions in plants. A phylogenetic analysis inferred three classes of GbNHX genes-viz., Vac (GbNHX1, 2 and 4), Endo (GbNHX6), and PM (GbNHX7). A high number of the stress-related cis-acting elements observed in promoters show their role in tolerance against abiotic stresses. The Ka/Ks values show that the majority of GbNHX genes are subjected to strong purifying selection under the course of evolution. To study the functional divergence of G. barbadense NHX transporters, the real-time gene expression was analyzed under salt stress in the root, stem, and leaf tissues. In G. barbadense, the expression was higher in the stem, while in G. hirsutum the leaf and root showed a high expression. Moreover, our results revealed that NHX2 homologues in both species have a high expression under salinity stress at higher time intervals, followed by NHX7. The protein-protein prediction study revealed that GbNHX7 is involved in the CBL-CIPK protein interaction pathway. Our study also provided valuable information explaining the molecular mechanism of Na+ transport for the further functional study of Gossypium NHX genes.
Project description:Plant NHX antiporters are responsible for monovalent cation/H+ exchange across cellular membranes and play therefore a critical role for cellular pH regulation, Na+ and K+ homeostasis, and salt tolerance. Six members of grapevine NHX family (VvNHX1-6) have been structurally characterized. Phylogenetic analysis revealed their organization in two groups: VvNHX1-5 belonging to group I (vacuolar) and VvNHX6 belonging to group II (endosomal). Conserved domain analysis of these VvNHXs indicates the presence of different kinds of domains. Out of these, two domains function as monovalent cation-proton antiporters and one as the aspartate-alanine exchange; the remaining are not yet with defined function. Overall, VvNHXs proteins are typically made of 11-13 putative transmembrane regions at their N-terminus which contain the consensus amiloride-binding domain in the 3rd TM domain and a cation-binding site in between the 5th and 6th TM domain, followed by a hydrophilic C-terminus that is the target of several and diverse regulatory posttranslational modifications. Using a combination of primary structure analysis, secondary structure alignments, and the tertiary structural models, the VvNHXs revealed mainly 18 ? helices although without ? sheets. Homology modeling of the 3D structure showed that VvNHX antiporters are similar to the bacterial sodium proton antiporters MjNhaP1 (Methanocaldococcus jannaschii) and PaNhaP (Pyrococcus abyssi).
Project description:Salinity is one of the major environment factors that limits the growth of plants and the productivity of crops worldwide. It has been shown that Na+ transporters play a central role in salt tolerance and development of plants. The objective of this study was to identify Na+/H+ antiporter (NHX) genes and investigate their expression patterns in sugar beet (Beta vulgaris L.) subjected to various concentrations of NaCl. A total of five putative NHX genes were identified and distributed on four chromosomes in sugar beet. Phylogenetic analysis revealed that these BvNHX genes are grouped into three major classes, viz Vac- (BvNHX1, -2 and -3), Endo- (BvNHX4), and PM-class NHX (BvNHX5/BvSOS1), and within each class the exon/intron structures are conserved. The amiloride-binding site is found in TM3 at N-terminus of Vac-class NHX proteins. Protein-protein interaction (PPI) prediction suggested that only BvNHX5 putatively interacts with calcineurin B-like proteins (CBL) and CBL-interacting protein kinases (CIPK), implying it might be the primary NHX involved in CBL-CIPK pathway under saline condition. It was also found that BvNHX5 contains one abscisic acid (ABA)-responsive element (ABRE), suggesting that BvNHX5 might be involved in ABA signal responsiveness. Additionally, the qRT-PCR analysis showed that all the BvNHX genes in both roots and leaves are significantly up-regulated by salt, and the transcription levels under high salinity are significantly higher than those under either low or moderate salinity. Taken together, this work gives a detailed overview of the BvNHX genes and their expression patterns under salt stress. Our findings also provide useful information for elucidating the molecular mechanisms of Na+ homeostasis and further functional identification of the BvNHX genes in sugar beet.
Project description:Sodium/proton exchangers (NHX) are key players in plant responses to salinity and have a central role in establishing ion homeostasis. NHXs can be localized in tonoplast or plasma membranes, where they exchange sodium ions for protons, resulting in the removal of ions from the cytosol into vacuole or extracellular spaces. In the present study, the expression pattern of the gene encoding Na+/H+ antiporter in the vacuolarmembrane (NHX1 gene) in Leptochloa fusca (Kallar grass) was measured by a semi- quantitative RT-PCR method under different treatments of NaCl and CdCl2. Results indicated that NaCl positively affected expression levels of LfNHX1, and that the amount of LfNHX1 mRNA increased in conjunction with the rise of salinity pressure, This finding suggests that vacuolar Na+/H+ antiporter might play an important role in the salt tolerance ability of kallar grass. The results also showed that cadmium exposure significantly modulated the mRNA expression of the LfNHX1 gene, suggesting that cadmium exposure disturbed Na+ homeostasis across the tonoplast and decreased the salt tolerance ability of kallar grass.