Identification of NHXs in Gossypium species and the positive role of GhNHX1 in salt tolerance

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.


Background
Soil salinization, caused by climate changes and human activities, is one of the most severe environmental challenges worldwide. The high concentration of salt in farmland soils was primarily from seawater and irrigation water containing toxic ions (Na + and Cl − ). Excess salts reduce the water uptake capacity of plants, block the absorption of nutrients, lead to excessive ROS production, disturb the cellular processes, and eventually inhibit plant growth [1]. The soil salinization poses a threat to the yield and quality of crops, which annually reduces arable farmland globally. On the other hand, as the world's population is growing rapidly, modern agriculture faces the challenge of producing more products with less available farmland. The development of saltresistant cultivars has been a major objective of crop improvement worldwide [2].
Plants have evolved elaborate mechanisms to adapt to salt stress, including ion efflux from cells and sequestration in the vacuole [3]. The ability to transport ions appears to be the key factor that determines plant adaptation in a high salt environment, and K + /Na + transporters play a fundamental role in this process [4]. The Na + /H + antiporters (NHXs) are membrane transporters that exchange Na + /K + for H + . NHX proteins exist in a broad range of eukaryotic organisms, including yeasts, plants, and animals. In Arabidopsis, so far eight NHX members have been identified [5]. Based on their sequence and localization, AtNHXs are categorized into three distinct functional groups: type I localized in the vacuole (AtNHX1/2/3/4), type II in endosome (AtNHX5/ 6), and type III in the plasma membrane (AtNHX7/8) [5]. Growing evidence highlights the important role of NHXs in plant growth and development under diverse hostile environments, especially in saline soil.
AtNHX7, known as AtSOS1 (salt overly sensitive 1), is essential for plant survival in saline soils. It maintains a low level of toxic Na + in the protoplast. The sos1 mutant showed > 20 times higher sensitivity of sanity than wild type [6]. The ortholog of AtSOS1 in other plant species showed similar functions, including OsSOS1 in rice and SlSOS1 in tomato [7,8]. SOS1, together with SOS2 and SOS3, plays an important role in plant response to salt stress, which is conserved in plants [9]. When plants are subjected to salt stress, the high concentration of Na + is perceived by cells that induce an instantaneous increase in endogenous Ca 2+ . SOS3 is a CBL (calcineurin B-Like) protein that activates SOS2 (CBL-interacting protein kinases) and forms SOS3-SOS2 complex in the presence of high Ca 2+ [10]. The active SOS3-SOS2 complex further activates the downstream SOS1, and SOS1 encodes a Na + /H + antiporter localized to the plasma membrane and is responsible for the extrusion of Na + [6,10].
Scientists have manipulated the expression of specific NHX isoforms to create new germplasm resources with elevated crop production in saline soils. AtNHX1 is a vacuolar-localized NHX protein that is important for osmotic regulation and plant development. Overexpression of AtNHX1 increases the salt tolerance of plant species [11][12][13]. For example, two NHXs (HtNHX1 and HtNHX2) were isolated from a salt-tolerant plant Helianthus tuberosus. Heterologous expression of these genes in rice improved salt tolerance. HtNHX2 improved grain yield as well as the harvest index of rice under both salt stress and nutrient deficient environment [13]. Type II NHXs are endosome-localized that provide salt tolerance by maintaining intracellular pH and ion balance [14]. A double mutant of the type II NHX proteins, nhx5/nhx6, significantly increased salt sensitivity of Arabidopsis [15].
Cotton (Gossypium spp.) is a globally cultivated crop with great economic value that provides fiber and oil.
Cotton has higher natural resistance to salt compared with many other crops (rice, wheat, and maize); however, the productivity of cotton is seriously threatened by increasing soil salinization [16,17]. Genetic engineering is an effective way to develop salt-resistant crops, and the study of the molecular mechanism of salt tolerance has provided candidate genes for genetic engineering in various species [18,19]. While the research on salt tolerance in cotton is still at the stage of gene cloning, phenotype analysis, and function prediction [17,[20][21][22]. Therefore, it is necessary to further understand the salt resistance mechanism in cotton through the study of potential genes.
Plant NHX family plays an important role in salt resistance; however, research involving NHXs is still rare in cotton. The rapid development of biotechnology accelerated the molecular breeding process in cotton that allows us to study cotton NHXs in the genome-wide level [23][24][25][26][27]. Recently, GhSOS1, homologous to AtSOS1, was isolated from a salt-tolerant genotype of G. hirsutum. Silencing of GhSOS1 significantly reduced cotton tolerance to salt, while the transgenic Arabidopsis with a heterogeneous expression of GhSOS1 showed enhanced tolerance to salt as well increased expression of stress-related genes [28,29]. In this study, we identified 47 putative NHXs from three different cotton varieties, including two diploid cotton varieties (A-genome species G. arboreum and D-genome species G. raimondii), and one tetraploid cotton variety (ADgenome species G. hirsutum). We further performed bioinformatics analysis, expression profiling, and virusinduced gene silencing (VIGS) to study the function of cotton NHXs. We proved that GhNHX1, which encodes vacuole-localized NHX protein, plays a positive role in cotton tolerance to salt. Therefore, GhNHX1 could be used as a candidate gene for the study of molecular mechanism in cotton response to salt-stress and breeding of salt-resistant cotton varieties.

Results
Evaluation of salt resistance in three Gossypium spp.
The seedlings of three Gossypium spp. (G. arboreum L. Shixiya1, G. raimondii, and G. hirsutum L. TM-1) were subjected to salt stress. Morphological changes such as severe dwarfing, leaf wilting, and chlorosis were observed in all three Gossypium spp. after irrigation with salt solution (300 mM NaCl) (Fig. 1a, b). The Na + concentration in the shoot and roots of stressed seedlings was measured. Both shoot and roots accumulated high Na + under salt stress (Fig. 1c). The results indicate that the seedlings of the three cotton species were all affected when grown in soils with high salt concentrations.

Identification of cotton NHXs
NHXs are evolutionarily and functionally conserved proteins. To identify and compare the NHX members of cotton, the NHX protein sequences of Arabidopsis, rice, tomato, soybean, and mulberry [30][31][32][33][34][35] were downloaded from NCBI and used to build HMM to search against cotton genome database [23,36,37]. All putative NHXs were further analyzed by InterPro and SMART to reconfirm their signature sequences and conserved domains. The predicted protein sequences with the Na + / H + exchanger domain were consider as cotton NHX. We identified 12, 12, and 23 NHXs from G. arboreum, G. raimondii, and G. hirsutum, respectively ( Table 1). The details of the identified NHX proteins are listed in Table S1, including accession number, sequence length, predicted isoelectric point (pI), and molecular weight (Mw). The NHXs of the three Gossypium spp. encoded proteins that varied from 211 to 1193 AA in size with predicted pI ranging from 5.05 to 9.14 and Mw ranging from 22.95 to 132.84 kD.

Phylogenetic tree of NHXs in cotton
To investigate the evolutionary relationships between NHXs of G. arboreum, G. raimondii, G. hirsutum and other plant species such as rice, mulberry, and Arabidopsis (Table S2), we built an unrooted phylogenetic tree using ClustalW and MEGA 6.0 with neighbor-joining  (N-J) method. Similar to the previous studies in other plant species [30][31][32][33][34], the 47 NHX candidates of Gossypium were divided into three distinct classes based on the sequence conservation (Table 1, Fig. 2). The majority of NHXs (75%) of Gossypium spp. were categorized into class I (9 NHXs of G. arboreum, 9 of G. raimondii, and 17 of G. hirsutum). Class II had 2 GaNHXs, 2 GrNHXs, and 4 GhNHXs. According to previous research, class III is the smallest group, and only 1 GaNHX, 1 GrNHX, and 2 GhNHXs were categorized into this class in our study.
All NHXs of G. arboreum had orthologs in G. raimondii. For instance, Ga10585 and Gr10021256 of class I are high homologous. They shared 99.26% identity in the protein sequence with only a few single nucleotide polymorphisms observed ( Figure S1) and had similar predicted pI and Mw values (Table S1). The phylogenetic tree and sequence alignment showed common gene duplication events associated with chromosome doubling in G. hirsutum compared to G. arboreum and G.
raimondii, which were also found in the previous reports of gene family studies in G. hirsutum [38]. These gene duplication events contributed to the expansion of NHX family in G. hirsutum, which has twice as many NHXs in diploid cotton ( Table 1). The only exception is Gr10038122 of class I. The NHX corresponding to Gr10038122 in the D sub-genome of G. hirsutum could be lost during the evolution process after G. hirsutum formation.
We further analyzed the genomic distribution of cotton NHXs. The genomic sequences of NHXs were used to query with BLAST to assess their chromosomal locations. We observed that the distribution pattern of NHXs in G. hirsutum is symmetrical and most members split evenly between A and D sub-genomes, while NHX genes of diploid cotton are distributed uniformly on the genome ( Figure S2). The Circos cycle demonstrated the syntenic relationship between cotton and Arabidopsis ( Figure S3), which revealed that most of the NHXs in Arabidopsis are homologous with the syntenic genes Fig. 2 Phylogenetic relationships of diploid and tetraploid cotton NHXs. The phylogenetic tree depicts 8 NHXs from Arabidopsis, 7 from mulberry (Morus atropurpurea), 7 from rice (Oryza sativa), and 47 predicted NHXs from cotton. All NHXs were classified into three groups (class I-III). The unrooted tree was generated with MEGA 6.0 using the N-J method with a p-distance model (1000 replicates). Red triangle, green circle, and blue rhombus represent the NHX genes from G. hirsutum, G. arboreum, and G. raimondii, respectively. Numbers on the tree branches represent bootstrap values pairs in cotton. For example, AtNHX7 located on At2 chromosome of Arabidopsis is highly homologous to GhD02G1385 and GhA03G0996 in D02 and A03 of G. hirsutum.

Structural divergence of cotton NHX genes
The structural analysis of cotton NHX gene family revealed that all the identified NHXs possess several introns (Fig. 3). Generally, the class I NHX genes from G. arboreum and G. raimondii are conserved and contain 13 introns. Contrarily, one-third of the class I GhNHXs evolved different structure compared to the orthologous in G. arboreum or G. raimondii (eg., Ga36304/GhA09G0594/Gr10009549/GhD09G0593). Ga36304 and Gr10009549 with conserved protein sequence and gene structure contain 14 exons, and the longest intron is 1 kb and 0.85 kb, respectively. On the other hand, GhA09G0594 and GhD09G0593 evolved several extremely long introns. The longest intron of GhA09G0594 is 3.8 kb and GhD09G0593 is 5.2 kb. As a consequence, the genome sequences of these two GhNHX genes expanded to 18 kb and 16 kb, respectively, and the genome sequences of Ga36304 and Gr10009549 are both 7 kb. Besides, the predicted protein sequences of these four orthologs were different ( Figure S4). Similar evolutionary events were observed in class III. Gh02G1385 has a 17.2 kb intron at the 3′ end and a total genome sequence of 31 kb. These findings revealed the complexity of the gene structure of cotton NHXs that may be related to the biological roles of NHX family.

Expression pattern of NHXs in cotton under salt stress
The expression pattern of NHX genes in G. hirsutum under salt stress was studied using previously published transcriptome data [23]. RPKM (reads per kilobase per million mapped reads) values in the leaves of G. hirsutum under NaCl treatment at indicated time points were used to create the salt-induced heat map of GhNHXs. As shown in Fig. 4, The 2 genes from class III, homologous to SOS1, were upregulated within 1 h of salt treatment. Four genes from class II showed no significant difference. NHX members of class I showed significantly different expression patterns. Seven genes were significantly induced and 3 genes were significantly suppressed under salt stress, while 6 NHXs were not expressed in all samples. These findings indicated differences in the potential roles of NHX classes under salt response. We cloned the gene pair GhA11G2132/GhD11G2440 from TM-1 transcripts for further study.
qPCR was performed to reconfirm the expression of GhA11G2132/GhD11G2440 in different tissues and in respond to salt treatment. Because of the high similarities of GhA11G2132/GhD11G2440 gene pair and high requirements for qPCR primers, it is very difficult to study the expression changes of the two genes individually with qPCR-based methods. The gene-specific primers were designed to amplify the GhA11G2132 and GhD11G2440 together. As shown in Fig. 5, GhA11G2132/GhD11G2440 gene pair was expressed in root, stem, and leaf, and the expression level of GhA11G2132/GhD11G2440 in leaf and root was two times than that in stem. GhA11G2132/ GhD11G2440 was significantly induced by 300 mM NaCl, which was consistent with the results of heat map analysis.

Analysis of GhNHX1A/D protein sequence
The sequences of GhA11G2132/GhD11G2440 were studied. Phylogenetic tree (Fig. 2) and sequence alignment (Fig. 6) revealed that GhA11G2132/GhD11G2440 pair was homologous to NHX1 and NHX2 from Arabidopsis. GhA11G2132 from sub-genome A was denoted as GhNHX1A, and the GhD11G2440 from sub-genome D was denoted as GhNHX1D. Prediction methods highlighted 12 transmembrane domains for GhNHX1A and GhNHX1D, and they showed high similarity (96.17%) with an extra five amino acid segment at 444~448 AA in GhNHX1D. The putative amiloride-binding site (LFFIYLLPPI) that inhibits eukaryotic NHX function was found in transmembrane domain III.
The biological roles of plant NHXs were closely related to their subcellular localization. To study the possible roles of the GhNHX1A/D in upland cotton, we fused GFP to the N-terminal of GhNHX1A/D. The vacuolar marker protein δ-TIP [39] fused with C-terminal RFP was cotransferred with GFP-NHX to the Arabidopsis protoplast. As shown in Fig. 7, GFP expressed throughout the protoplast, while red fluorescence expressed only on the vacuolar membrane in the protoplast transformed with GFP and δ-TIP-RFP vectors. The coexpression of GFP-GhNHX1A/D and δ-TIP-RFP in the protoplast, represented by overlapped red and green fluorescent signals, indicated that majority of GhNHX1A/D localized to the vacuolar system where the δ-TIP protein was located.

GhNHX1A/D regulate salt tolerance in cotton
The potential roles of GhNHX1A/D in cotton response to salt stress were studied using the VIGS method. A 376 bp fragment from 3′ end of GhNHX1A was cloned and inserted into the VIGS vector to silence both GhNHX1A/ D. After 3 weeks, the silencing efficiency was detected in cotton leaves using qPCR. GhNHX1 expression in GhNHX1-silenced (TRV:NHX1) plants reduced to 10% compared with the empty vector transferred control (TRV:00) plants (Fig. 8a). The phenotypic appearance of TRV:00 and TRV:NHX1 was studied and no significant difference was observed under normal conditions. Cotton seedlings were subjected to water or 300 mM NaCl treatment for 6 days. High salt concentrations restricted cotton growth and development; salt treatment resulted in shorter plants with wilting and yellowing symptoms on leaves. Compared to TRV:00, the TRV: NHX1 showed reduced tolerance to salt stress with more wilting of leaves and less biomass of both roots and leaves (Fig. 8b, c). The Na + content was measured in the roots and shoots of TRV:00 and TRV:NHX1 plants after water or salt treatment (Fig. 8d). After 6 days of salt treatment, the Na + concentration in TRV:NHX1 was increased compared with TRV:00. These findings indicated a positive role of GhNHX1 in cotton response to salt stress.

Discussion
It has been reported that more than 20% of the farmland globally, including 50% of the irrigated land, is threatened by soil salinization. Most crops are sensitive to high salt concentrations that indicate the urgent need to develop new varieties with enhanced productivity in saline soils [19,40]. Cotton is an economically important crop.
Improving the yield and quality of cotton and enhancing the salt tolerance of cotton cultivars are priorities in cotton breeding [17,41]. NHX genes play important roles in salt stress. Therefore, studying the function of cotton NHXs could provide a theoretical basis of salt tolerance mechanism and help identify the candidate genes for breeding salt-tolerant cotton.
NHX family is relatively small and conserved in plant species. In diploids, the number of NHX genes is around seven (Table 1) with the exception of soybean that contains 11 NHX genes. Soybean is a diploid crop derived from a palaeo tetraploid. This typical evolutionary process with duplications, deletions, mutations, and DNA rearrangement events resulted in 75% of soybean genes with two or more copies [42]. The diploid cotton also experienced complex polyploidy during its evolution. The repeated polyploidization of Gossypium resulted in a large and unexpected complex genome [36,37,43]. Wang et al. revealed that the ancestors of cotton experienced more multiplication events (five times) followed by extensive whole genome reshuffle and large-scale chromosome loss [44]. We identified 12 NHXs from both diploid cotton cultivars. This suggested that the number of NHX genes in a plant species is correlated with the size and complexity of its genome. Chromosome polyploidization also contributes to the expansion of NHX family. The heterotetraploid cotton was produced by the hybridization of A and D genomes approximately 1~2 Myr ago [23,24,43]. The chromosome doubling resulted in double the number of NHX genes in G. hirsutum compared to G. arboreumii and G. raimondii. The number of NHX genes in a plant species is not directly related to its tolerance to salt stress. There are 7 NHXs in both rice and mulberry; however, they exhibit different tolerance levels in saline soils. Rice is the model plant of Poaceae family that is sensitive to salt stress, while mulberry is a perennial tree that adapts to adverse environments. Compared with diploid cotton, the tetraploid cotton has twice the number of NHX genes; however, the tetraploid cotton showed no absolute increase in salt tolerance.
NHXs of different plant species are divided into three classes based on their sequence similarity. Previous researches highlighted the distinct functional roles and subcellular localization of plant NHXs of three classes [31,34], which also suggest the sequence conservation of NHX orthologs among species and the close correlation between the function of NHXs and their localization. Class I NHXs localized to vacuole membrane, class II NHXs localized to the endosome, and class III NHXs localized to the plasma membrane. 75% of cotton NHXs are divided into class I, while only 50% of Arabidopsis members belong to class I NHXs [34]. These results revealed that the expansion of NHX family in diploid cotton was due to the large number of NHX genes in class I. The expression of NHXs in G. hirsutum under salt stress was studied and a distinct expression pattern was observed among three classes (Fig. 4). The 2 genes from class III, orthologous to SOS1, were induced by salt. SOS1 is pivotal for plant survival in saline soils [6], which suggests the possible role of class III GhNHXs in salt response. The expression of 4 NHXs of class II was not closely related to salt treatment. According to the previous study, the endosome-localized NHXs are involved in vesicle pH homeostasis and endomembrane trafficking [5,14,45]. The GhNHXs of class I exhibit diverse expression pattern under salt stress. These results were corroborated by the phylogenetic study, which indicates that NHXs from the three classes are regulated meticulously and differently at the transcription level.
NHX genes in cotton contain large number of long introns (Fig. 3), which is also found in other plant species [30][31][32][33][34]. The number of introns is related to the organism's ability to adapt to adverse environmental conditions [46,47]. The introns are molecular basis of alternative splicing. More number of introns in a gene produces more functional transcripts by alternative splicing. Alternative splicing has been considered as a powerful source of functional innovation in evolutionary adaptation. Evidence has shown alternative splicing of at least half of the human genes [48]. In fact, the studies on plant NHXs have proved high frequency of alternative splicing in post-transcriptional regulation [30]. This study provides information on the multilevel regulation of NHXs.
NHXs are closely related to environmental adaptation. The present study on NHXs has promising prospects in crop breeding for salinity. In this study, the salt-induced Fig. 8 Silencing of GhNHX1 compromises cotton tolerance to salt stress. a qPCR analysis of GhNHX1 silencing efficiency in root and leaf of TRV:00 and TRV:NHX1. UB7 was used as internal control. The standard deviations were calculated from the results of four replicates (n ≥ 5). b Representative plants of TRV:00 and TRV:NHX1 after 300 mM NaCl treatment for 6 days. c Quantitative measurement of biomass after water (mock) and salt treatment (n ≥ 14). d Na + content of cotton seedlings after water (mock) and salt treatment (n ≥ 14). The standard deviations were calculated from the results of three independent experiments (p < 0.05) gene pair GhNHX1A and GhNHX1D were silenced to study their function. Both genes were upregulated after salt treatment, and their proteins were located to the vacuolar system. Silencing of GhNHX1A and GhNHX1D together resulted in reduced tolerance of cotton seedlings to high salt concentrations, suggesting that GhNHX1 plays positive role in cotton tolerance to salt stress. These findings provide candidate genes that enable us to produce new germplasm for research and breeding of cotton cultivars resistant to salt stress.

Conclusions
To identify salt-resistant genes and study the evolution of NHXs in cotton, bioinformatics analysis, and molecular characterization were performed. We identified 47 NHXs from three Gossypium spp.. The cotton NHXs contained many introns, and the complexity of gene structure may be related to the adaption of cotton to salt stress. The expression pattern of NHXs indicates the possible differences in the roles of distinct NHX classes under salt stress. In addition, functional analysis of GhNHX1 using VIGS proved that GhNHX1 is a positive factor in cotton response to salt stress. These findings could contribute to the study of cotton response to salt stress and provide candidate genes for breeding saltresistant cotton cultivars.

Plant materials and salt treatment
The seeds of Arabidopsis Columbia (Col-0) were suspended in 0.1% (w/v) agarose solution and sown in pots filled with growth medium consisting of perlite, vermiculite, and peat. The pots were placed in a growth chamber (Conviron CMP6010, Canada) at 22/20°C, 16 h light/8 h dark (day/night) for germination and growth.
Seeds of three different cotton varieties, including G. hirsutum L. TM-1, G. arboreum L. Shixiya1, and G. raimondii, were collected from the germplasm bank of the state key laboratory of cotton biology of China. The seeds were soaked in sterile water for 4 h and allowed to germinate on wet filter paper at 25°C for 1-2 days. The germinating seeds were planted in growth medium in a growth chamber at 25/22°C and 16 h light/8 h dark (day/night). After 4 weeks, the well-grown plants were treated with water or salt. For salt treatment, each cotton seedling was watered with 50 mL of 300 mM NaCl solution. Cotton seedlings treated with deionized water were used as mock. For phenotypic observation, photographs were taken after 12 days of treatment. Salt ion concentration was detected after photographing.

Identification of cotton NHXs
Sequences of the NHX proteins of various plant species were downloaded from NCBI (http://www.ncbi.nlm.nih. gov/). These proteins were used as reference proteins to build a Hidden Markov Model (HMM). HMM was used as a query to search against protein database of G. hirsutum, G. arboreum, and G. raimondii. The nucleotide and protein sequences of G. hirsutum, G. arboreum, and G. raimondii were obtained from CottonGen database (https://www.cottongen.org/) [49]. The conserved domains in each candidate sequence were scanned by motif scan using SMART (http://smart.embl-heidelberg.de/ smart/) and InterPro (http://www.ebi.ac.uk/interpro/). The molecular weight (Mw) and isoelectric point (pI) of candidate proteins were predicted using Compute pI/ Mw tool (http://web.expasy.org/compute_pi/). The unrooted phylogenetic tree was built with the aligned NHX protein sequences using the neighbor-joining method in MEGA6 with 1000 bootstrap replicates with a p-distance model and partial deletion.

Gene structure analysis
The coding sequence and corresponding genomic DNA sequence were downloaded and submitted to Gene Structure Display Server 2.0 (http://gsds.cbi.pku.edu.cn/), a webbased bioinformatics tool, to visualize gene features concerning intron-exon organization of cotton NHX genes.

Genomic distribution of NHX genes
The chromosomal location and syntenic diagram were generated as previously reported [38]. Cotton NHX genes were mapped onto corresponding chromosomes by MapInspect program (http://www.plantbreeding.wur. nl/UK/software_mapinspect.html) to draw the chromosomal locations of each NHX genes. The syntenic analysis showing the genomic distribution of NHXs and the synteny relationships between cotton NHXs and Arabidopsis NHXs was performed with Circos software [50].

RNA isolation
Total RNA was isolated from G. hirsutum using the EASYspin Plus Plant RNA Kit (AidLab, Beijing, China) according to the manufacturer's instructions. Approximately 1 μg of total RNA was used for the first-strand cDNA synthesis using M-MLV Reverse Transcript System (Promega, USA). The cDNA samples were diluted 50 times and stored at − 20°C for further experiments.

Heat map and qPCR analysis
We obtained the released transcriptome data of G. hirsutum L. TM-1 from NCBI and translated the raw data into RPKM values, which represented the expression levels of NHXs. Heat map was generated using Genesis 1.8.1 program [51]. Four replicates of qPCR analysis were performed on an ABI 7500Fast Real-Time PCR System (Applied Biosystems, USA) with SYBR Green Master Mix (Vazyme Biotech, China) as described in our previous publication [52]. The relative fold-changes of target genes were calculated using the 2 -ΔCt method, with cotton ubiquitin 7 gene (UB7), amplified as an internal control to normalize the cDNA amplification in each reaction.

Gene clone and vector construction
The target NHX genes were amplified by PCR and ligated to the expression vector pK7FWG2 with GFP fused to the N-terminus. The marker protein δ-TIP was fused to red fluorescence protein (RFP) [39]. The gene silencing vectors used for VIGS were constructed as described previously and was transferred into Agrobacterium tumefaciens [53]. The primers used in this study were designed with Primer Premier 5 software and were listed in Table S3.

Transient transformation of Arabidopsis protoplast
Young leaves of Arabidopsis seedlings were collected and used for protoplast isolation as described previously [54,55]. For transient transformation, 15 μg of plasmid was gently mixed with 100 μL of the protoplast (10 5 /mL) and incubated at room temperature for 30 min. Green and red fluorescence were observed using a confocal laser scanning microscope (BioRad Radiance 2100, USA).

VIGS in cotton
Transformed A. tumefaciens cells containing pTRV1, pTRV2, and pTRV-NHX1 were collected and resuspended in suspension buffer. pTRV1 was mixed with pTRV2 and pTRV-NHX1 in equal amounts, and the concentration of the bacterial suspension was adjusted to 0.6-0.8 (OD 600 ) and incubated at room temperature for 3 h. The A. tumefaciens suspension was infiltrated into the cotyledons of 1-week-old cotton seedlings by syringe infiltration. The infiltrated seedlings were kept in dark for 12 h and moved into growth incubator [56]. After 3 weeks, the cotton seedlings were subjected to gene silencing efficiency analysis and salt treatment. To study the silencing efficiency of NHX1, the roots and leaves of TRV:00 and TRV:NHX1 were harvested for RNA extraction and qPCR analysis. To study the salt tolerance of TRV:00 and TRV:NHX1, the TRV:00 and TRV:NHX1 seedlings were subject to water or 300 mM NaCl treatment for 6 days, the quantitative measurement of plant biomass and Na + content were conducted after the water or NaCl treated TRV:00 and TRV:NHX1 seedlings being photographed.

Measurement of Na + concentration
The roots and leaves of cotton seedlings were harvested, dried, and ground into powder. Approximately, 0.05 g of the tissue powder was dissolved in 5 mL of concentrated nitric acid, and the nitric acid solution was diluted to 5% with water. The supernatant was used to detect the ion concentration using an atomic absorption spectrophotometer [57].