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.


Introduction
High salinity stress is one of the main abiotic stress factors affecting plant growth and development, thereby posing a great threat to the sustainable development of agriculture and food security. It has been estimated that nearly 10% of the total land surface (950 Mha) and about 50% of all irrigated land (230 Mha) are salt-affected, therefore drastically reducing agricultural productivity and altering the geographical distribution of crops [1,2]. Salinity primarily causes ionic stress and osmotic stress in plants. The resulting salinityinduced water stress, oxidative stress, nutritional imbalances, ion toxicity, and disruption of metabolic processes restrict the normal growth of plants [3,4]. During the process of evolution, plants have developed multiple mechanisms involving genes and strategies at the physiological, molecular, and metabolic levels to improve salt resistance in various ways. To cope with salt stress, plants have evolved three distinct responses, including osmotic stress tolerance, sodium ion (Na + ) or chloride ion (Cl − ) exclusion, and the accumulation of Na + or Cl − in the tissues [5]. Therefore, it is suggested that some membrane proteins, including transporters or channels, play critical roles in maintaining ion homeostasis and osmotic adjustment, and regulating pH for plant survival under abiotic stress, such as high salinity/alkaline and drought. The Na + /H + exchangers (NHXs), belonging to the subfamily of monovalent cation-proton antiporters (CPA), are H + -coupled cotransporters that transfer Na + , potassium ion (K + ), or lithium ion (Li + ) across membranes in exchange for hydrogen ions (H + ), playing significant roles in cellular ion homeostasis, pH regulation, and plant salt tolerance [6].
Previous reports have shown that there are eight NHX members in the Arabidopsis genome. Based on the subcellular localization, the AtNHX protein family could be categorized into three subclasses: vacuolar (AtNHX1-AtNHX4), endosomal (AtNHX5 and AtNHX6), and plasma membrane (AtNHX7/SOS1 and AtNHX8) [7,8]. Among them, the distinctive plasma membrane AtNHX7/SOS1 (SOS1A) is the key factor of the well-known salt tolerance salt-overly-sensitive (SOS) pathway. AtNHX7 mainly mediates the efflux of Na + across the plasma membrane, thereby protecting cells from the deleterious effects of excessive Na + [9]. AtNHX7 also plays an important role in long-distance Na + transport, thus helping to regulate the Na + /K + ratio in the roots and shoots [10]. AtNHX8 (SOS1B) encodes a Li + transport that may be responsible for Li + extrusion, thereby maintaining Li + detoxification and ion homeostasis [11]. The other AtNHX members have been suggested as essential for Na + , K + , or Li + compartmentalization in the vacuole or other organelles, protecting cells from the deleterious effects of these ions and maintaining cytoplasmic ion homeostasis [12][13][14][15][16].
Numerous reports have confirmed the involvement of NHX proteins in salinity stress responses [8]. Based on the released genome sequences of various plant species, genomewide analyses of NHX families have recently gained remarkable traction in investigations of plant salt and alkaline tolerance mechanisms. Research on NHX genes in halophytes, xerophytes, or even extremophiles would improve the understanding of the adaptation of plants to environmental stress and provide a good foundation for formulating effective measures to modify the tolerance of crops to external stresses [17][18][19]. In recent years, the functional identification of NHX families from different plant species has broadly indicated that the NHX families are crucial for ion homeostasis, cellular pH regulation, plant development, vesicle trafficking, and salt-tolerance in plants. Mechanically speaking, the NHX transporters present in plant membrane systems maintain ionic homeostasis via the extrusion of Na + /K + ions out of the cells or the compartmentalization of Na + /K + ions into the vacuoles [14][15][16]20,21]. The NHX families have been systematically studied in rice [22], mulberry [23], poplar [24,25], alfalfa [26], sugar beet [27], grapevine [28], wheat [29], rapeseed [30], soybean [31], maize [32], bamboo [33], and cotton [34][35][36][37]. All of the above investigations established a substantial foundation for further functional studies of plant NHX genes and provided a systematic understanding of NHX families in regulating the salt tolerance of crops or plant species with specialized habitats.
To solve the growing crisis caused by soil salinity/alkaline, drought, and degradation, it is essential to improve the soil on saline-alkali land for agricultural purposes or for preserving the ecological environment. In addition, using effective gene resources to cultivate salt-, alkali-, and drought-resistant plant species is the most economical and productive measure to solve this problem. For some crops, genetic engineering strategies provide a viable alternative to conventional plant breeding and are now becoming more widely used throughout the world to produce salt-tolerant cultivars. Halophytes can thrive under high salinity by adopting special strategies to improve salt tolerance or enhance salt avoidance [38]. Canavalia rosea is a halophyte species and widely distributes in tropical and subtropical coastal regions or islands [39]. As we have sequenced the whole genome of C. rosea, a functional study of its gene/protein family could provide a reference for researching the conservation biology of plant species in specialized habitats.
In this study, we performed a genome-wide characterization of NHX genes in C. rosea, to explore the potential roles of these family members in the adaptation of C. rosea to tropical coastal regions or coral reefs. This study will provide significant insights into the function of plant NHXs as well as identify promising candidate genes for breeding salt-resistant crops. We further performed functional analysis of several CrNHXs via yeast functional complementation.

Plant Materials and Stress Treatments
Canavalia rosea plants growing in the South China Botanical Garden (SCBG, 23 • 18 76 N, 113 • 37 02 E) were used in this study. The different tissues from young seedlings (root and stem) or adult plants (leaf, flower bud, and young fruit) of C. rosea were gathered for further transcription analysis of gene expression. The seeds of C. rosea were gathered from the coastal regions of Hainan province, China and then cultivated under normal conditions in a growth chamber. The seeds were germinated and cultivated in soil/vermiculite mixture for one month to perform various abiotic stresses for the transcriptional analyses of CrNHX members. In brief, the seedlings were removed from the pots and carefully washed with distilled water to remove soil from the roots, after which they were transferred into different solutions. For high salinity, alkaline, drought stress, and heat treatments, seedlings were soaked in 600 mM NaCl, 150 mM NaHCO 3 (pH 8.2), 300 mM mannitol solutions, and 45 • C pre-warmed half-strength Hoagland's solution, respectively. The plant tissues were collected at different time points. For habitat-specific expression pattern analyses of CrNHX members, mature leaf samples were captured from C. rosea adult plants growing in SCBG (transplanted from Hainan province since 2012) and Yongxing (YX) Island (16 • 83 93 N, 112 • 34 00 E). All samples were immediately frozen in liquid nitrogen and stored at −80 • C for subsequent gene expression analysis. Three independent biological replicates were used.

Identification, Ka/Ks Calculation, and Evolutionary Analyses of the CrNHX Family in C. rosea
Whole-genome sequencing was performed with C. rosea plants growing in SCBG, Guangzhou city, China. Then, the genome sequence was submitted to GenBank (Accession No.: JACXSB000000000), which will be released on 16 September 2024. The assembled genome data of C. rosea were annotated with different programs, including Inter-Pro [40] and Pfam [41] for gene identification, and DIAMOND [42] and InterProScan [40] were used to acquire the information of all proteins with conserved domains and motifs (e < 1 × 10 −5 ). After that, a Pfam ID (Na_H_Exchanger, PF00999) search was performed in the C. rosea protein annotation database, and putative sequences of CrNHX proteins were further identified with the AtNHXs sequence alignment to eliminate the cation/H + exchanger (CHX) and K + efflux antiporter (KEA). Then, the corresponding putative CrNHX protein sequences were submitted to the NCBI Conserved Domain Database (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi, accessed on 1 October 2021) and PfamScan (https://www.ebi.ac.uk/Tools/pfa/pfamscan/, accessed on 1 October 2021) to confirm the presence of the Na_H_Exchanger domain. Finally, the candidate CrNHXs were named based on their sequence homology after AtNHXs or other plant species NHXs and the C. rosea genome annotation.
The CrNHX genomic DNA and cDNA sequences were extracted from the C. rosea genome database. Gene segmental duplication events of the CrNHX family were analyzed using MCScanX software (http://chibba.pgml.uga.edu/mcscan2/, accessed on 1 October 2021). The number of synonymous substitutions per synonymous site (Ka), the number of non-synonymous substitutions per non-synonymous site (Ks), and the probability (p-value) of Fisher's exact test of neutrality were calculated using the Nei-Gojobori model with 1000 bootstrap replicates [43]. The diagrams of exon/intron organization, protein structure, chromosomal location, and gene duplication event were drawn using TBtools software [44]. The exon-intron structures within the coding sequences and the genomic sequences of each CrNHX were predicted with the Gene Structure Display Server (GSDS, http://gsds.cbi.pku. edu.cn, accessed on 1 October 2021). The conserved motifs of CrNHXs were detected using Multiple Em for Motif Elicitation (MEME) software (http://meme-suite.org/tools/meme, accessed on 1 October 2021), with the maximum number of motifs set as 10.

Cis-Regulatory Element Analysis of CrNHX Promoters
The promoter regions (2000 bp upstream from the translation start site) of all CrNHXs were retrieved from the genome database of C. rosea. The cis-regulatory elements present in these regions were predicted with PlantCARE (http://bioinformatics.psb.ugent.be/webtools/ plantcare/html/, accessed on 1 October 2021). These cis-regulatory elements were summarized with Microsoft Excel 2010 software (Microsoft Corp., Albuquerque, NM, USA), and several selected CrNHX promoters were visualized using TBtools.

RNA-Seq of Different C. rosea Tissues under Different Stress Treatments
The C. rosea RNA-seq datasets were constructed using Illumina HiSeq X sequencing technology, by commissioning a commercial biotechnology company (Oebiotech, Shanghai, China). First, seven different tissues from C. rosea plants (root, stem, young leaf, flower bud, and young silique samples collected from C. rosea plants growing in SCBG; mature leaf samples from C. rosea growing in SCBG and on YX Island) were used for isolating the total RNA. Then, the high-quality RNAs were constructed into libraries with enriched mRNA by Oligo (dT) magnetic bead. The libraries were randomly sequenced, and the data were examined using FastQC (http://www.bioinformatics.babraham.ac.uk/ projects/fastqc/, accessed on 1 October 2021) based on the primary 5.65-7.16 Gb clean reads each sample and were mapped to the C. rosea reference genome using Tophat v.2.0.10 (http://tophat.cbcb.umd.edu/, accessed on 1 October 2021). Second, the C. rosea seedling tissues under different abiotic stress challenges mentioned above (including 600 mM NaCl, 150 mM NaHCO 3 (pH 8.2), 300 mM mannitol solutions, and 45 • C pre-warmed halfstrength Hoagland's solution) were also sequenced at the transcriptome level, with the same above RNA isolation, library construction, cDNA sequencing, and gene read alignment procedures. These four types of stress treatments were used for simulating the high salinity, alkaline, drought, and heat environmental conditions on tropical coral reefs. All of the EST information was mapped to the C. rosea reference genome. Gene expression levels were calculated as fragments per kilobase (kb) of transcript per million mapped reads (FPKM) according to the length of the gene and the read counts mapped to the gene: FPKM = total exon fragments/(mapped reads (millions) × exon length (kb)) Expression levels (log2 values of FPKM) of CrNHXs were visualized as clustered heatmaps using TBtools.

Expression Patterns Analysis by Quantitative Reverse Transcription (qRT)-PCR
The total RNA was extracted from different C. rosea tissues using the Quick RNA isolation Kit (Huayueyong, Beijing, China) according to the manufacturer's specifications. The RNA yield was determined using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA), and the integrity was evaluated using agarose gel electrophoresis stained with ethidium bromide. The cDNA was synthesized from total RNA using TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China) with Oligo (dT) 15 primers according to the manufacturer's instructions. The concentration of cDNA was also measured using NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA).
The qRT-PCR was performed using a LightCycler ® 480 II Real-Time PCR Instrument (Roche, Basel, Switzerland) with a 10 µL PCR reaction mixture that included 1 µL of cDNA (100 ng µL −1 ), 5 µL of 2 × PerfectStart™ Green qPCR SuperMix (TransGen Biotech, Beijing, China), 0.2 µL of forward primer, 0.2 µL of reverse primer, and 3.6 µL of nucleasefree water. Reactions were incubated in a 384-well optical plate (Roche, Basel, Switzerland) at 94 • C for 30 s, followed by 45 cycles of 94 • C for 5 s, 60 • C for 30 s. Each sample was run in triplicate for analysis. At the end of the PCR cycles, melting curve analysis was performed to validate the specific generation of the expected PCR product. The primer sequences were designed in the laboratory and synthesized (listed in Table S2). The expression levels of the mRNAs were normalized to the reference gene CrEF-α and were calculated using the 2 −∆∆Ct method. Three technical replicates were performed per sample. GraphPad Prism v8 (San Diego, CA, USA) was used to calculate the mean ± standard deviation (SD; n ≥ 3) and perform the statistical analyses.

Functional Identification with a Yeast Expression System
The full-length cDNA sequences of the CrNHX genes were obtained from the genome database of C. rosea (listed in Supplementary materials, Table S1). Then, the open reading frames (ORFs) of the CrNHX genes were PCR amplified from different cDNA samples of C. rosea with gene-specific primer pairs (listed in Table S2). The PCR steps were set as usual: 94 • C for 3 min, followed by 30 cycles of 94 • C for 30 s, 53-60 • C for 30 s (depending on the gene-specific primer sequences), and 72 • C for 90 s. After several different PCR procedures, the PCR fragments were purified and cloned into the BamHI and EcoRI sites of pYES2 using In-Fusion ® techniques (In-Fusion HD ® Cloning System, Clontech, Mountain view, CA, USA) to yield recombinant CrNHXs-pYES2, and sequenced. The yeast wild-type (WT) strain W303 (MATα ura3-1 leu2-3112 his3-11,15 trp1-1 ade2-1 can1-100) and triple mutant strain AXT3 (∆ena1::HIS3::ena4, ∆nha1::LEU2, ∆nhx1::KanMX4) were obtained from Zhou et al. [45]. The plasmids were introduced into yeast using the LiAc/PEG method [46]. Yeast growth and metal sensitivity tests were performed as described previously with minor modifications [47]. Briefly, transformed yeasts were grown on solid SDG-Ura medium (Synthetic Dropout medium plus 2% Galactose, Uracil deficiency) plates for two days, and single colonies of the yeast transformants were picked out and inoculated in liquid SDG-Ura medium overnight or longer at 30 • C, diluted with fresh pre-warmed SDG medium (volume ratio 1:10), and then incubated with vigorous shaking for about two days at 30 • C to reach an optical density of just 1 at OD600 (optical density at 600 nm). Then, the cells were serially diluted in 10-fold steps, and 2-µL aliquots of each were finally spotted onto APG medium (arginine phosphate medium, supplied with 2% galactose) plates [37] with or without different concentrations of NaCl, KCl, or hygromycin B stressors. The plates were incubated at 30 • C for two to five days and photographed.

Overview of the C. rosea CrNHX Genes
A total of eight CrNHX genes were identified from the C. rosea genome through sequence similarity searches and the conserved Na_H_Exchanger domain identification (Table S1). The chromosomal map of the CrNHX family showed that only five chromosomes of C. rosea held the CrNHX genes, and most genes were on chromosome 1 (three genes), followed by chromosome 4 with two genes. Chromosomes 2, 6, 7, 8, 9, and 10 carried no CrNHX genes ( Figure 1A). The identified orthologs were named CrNHXs based on their sequence similarity with Arabidopsis AtNHXs and the gene nomenclature system, and the basic features of the CrNHX cDNAs and proteins were also analyzed to obtain the primary information of this family ( Table 1). Members of the CrNHX gene family were subdivided into three subfamilies: vacuole type (Vac), endomembrane type (Endo), and plasma membrane (PM) type, according to their homologies with AtNHXs ( Figure 1B). The gene exon-intron organization for each CrNHX was examined to further analyze the evolution of this gene family. As shown in Figure 1B, all the CrNHX genes contained multi-introns structures; the Vac-type CrNHXs possessed 13-14 introns, the Endo-type CrNHXs (CrNHX5 and CrNHX6) held 18 and 20 introns, and the PM-type CrNHX7 had the most introns (23). Basically, the intron number, exon length, and intron phase were relatively conserved among the members of the same subfamily. The number of exons and introns of CrNHXs was similar to that of NHXs from other plant species [23,24,30]. (CrNHX4-1), indicated that the CrNHXs were generally hydrophobic, which was consistent with the biochemical functions of NHXs as transmembrane Na + /H + exchangers (Table 1). Transmembrane helices (TMHs) and topology prediction were conducted with TMHMM, which further indicated that the CrNHXs maintained special secondary structures by these transmembrane helices and formed pores for cation transport ( Figure S1). The two programs used (Plant-mPLoc and WoLF_PSORT) indicated similar results, with most CrNHXs located in the vacuoles, and only CrNHX7 showed typical features of cellular membrane localization ( Table 1). The number of synonymous substitutions per synonymous site (Ks) and number of nonsynonymous substitutions per nonsynonymous site (Ka) values were calculated to explore the selective pressures on the duplication of CrNHXs based on all nucleotide sequences. The results revealed that one gene pair, CrNHX5/CrNHX6, possessed Ka/Ks ratios greater than 0.1 but lower than 1, indicating that this gene pair underwent some purifying selection and demonstrated segmental duplications, which might suggest their evolutionary and functional divergence ( Table 2).    Table 1 shows the genomic locus, molecular features, structural features, and subcellular prediction of the CrNHXs. In general, the length of the CrNHX cDNA coding region sequences ranged from 1578 bp (CrNHX3) to 3570 bp (CrNHX7), with 525-1189 amino acid residues. Their genomic size was around 4-7 kb, except for CrNHX4 and CrNHX6, which were 11 and 15 kb, respectively ( Figure 1B). The isoelectric point (pI) value of the CrNHXs varied between 5.22 and 9.24, with the Vac-type CrNHXs being alkalescent, the Endo-type CrNHXs being sub-acid, and the PM-type CrNHX7 being nearly neutral. The grand average of hydropathicity (GRAVY) results, ranging from 0.101 (CrNHX7) to 0.666 (CrNHX4-1), indicated that the CrNHXs were generally hydrophobic, which was consistent with the biochemical functions of NHXs as transmembrane Na + /H + exchangers (Table 1). Transmembrane helices (TMHs) and topology prediction were conducted with TMHMM, which further indicated that the CrNHXs maintained special secondary structures by these transmembrane helices and formed pores for cation transport ( Figure S1). The two programs used (Plant-mPLoc and WoLF_PSORT) indicated similar results, with most CrNHXs located in the vacuoles, and only CrNHX7 showed typical features of cellular membrane localization ( Table 1).
The number of synonymous substitutions per synonymous site (Ks) and number of nonsynonymous substitutions per nonsynonymous site (Ka) values were calculated to explore the selective pressures on the duplication of CrNHXs based on all nucleotide sequences. The results revealed that one gene pair, CrNHX5/CrNHX6, possessed Ka/Ks ratios greater than 0.1 but lower than 1, indicating that this gene pair underwent some purifying selection and demonstrated segmental duplications, which might suggest their evolutionary and functional divergence ( Table 2).

Multiple Sequence Alignment and Phylogenetic Analysis of the CrNHX Family
We further aligned the CrNHX protein sequences according to a previous report [23] and labeled the twelve transmembrane (TM) regions in all of the CrNHX proteins ( Figure S2). Basically, the CrNHX proteins showed high similarities in their TM regions, which were also assigned as the conserved Na_H_Exchanger domains. This feature implies the high conservation of NHX proteins as univalent cation channels. To study the evolutionary relationships between the CrNHX proteins and other known NHXs from Arabidopsissoybean, alfalfa, rice, mulberry, and grapevine-an unrooted neighbor-joining phylogenetic tree was created based on multiple alignments of the predicted sequences of the NHX proteins from these above plants. The phylogenetic tree revealed the formation of three different clusters, which were classified as Vac (vacuolar), Endo (endosomal), and PM (plasma membrane) subfamilies, as illustrated in Figure 2. The clustering results clearly showed that within the same subfamily, the NHX proteins were highly conservative, and different members showed some degree of homology, which is consistent with species evolution, especially in the three leguminous plants.

Conserved Motifs and Transmembrane Domains of CrNHX Members
To learn more about the diversity of motif compositions among different CrNHXs, the conserved motifs were predicted using MEME, which further reflected the evolutionary conservation of some specific functional amino acids in the CrNHXs. We identified a total of 10 putative motifs based on the protein sequences of all CrNHXs. The predicted motifs of CrNHXs ranged from 6 to 50 amino acids in length. Predictably, the members in the same subfamily had a common motif composition. Motifs 1 and 7 were widely found in all CrNHXs, and motifs 2, 5, and 10 were widely present in the Vac-type and Endo-type CrNHX subfamilies ( Figure 3A). A Pfam database search showed that all CrNHXs contained the Na_H_Exchanger domain (PF00999) (Figure 3B), and the TMHMM analysis indicated that the transmembrane regions presenting in the Na_H_Exchanger domain were all conserved ( Figure 3C), which almost indicated a one-to-one relationship with the predicted motifs predicted by MEME. In general, the C-termini of all CrNHXs were less conservative, which were predicted as being hydrophilic and playing crucial parts in channel activity regulation and protein trafficking by protein-protein interaction.

Conserved Motifs and Transmembrane Domains of CrNHX Members
To learn more about the diversity of motif compositions among different CrNHXs, the conserved motifs were predicted using MEME, which further reflected the evolutionary conservation of some specific functional amino acids in the CrNHXs. We identified a total of 10 putative motifs based on the protein sequences of all CrNHXs. The predicted motifs of CrNHXs ranged from 6 to 50 amino acids in length. Predictably, the members in the same subfamily had a common motif composition. Motifs 1 and 7 were widely found in all CrNHXs, and motifs 2, 5, and 10 were widely present in the Vac-type and Endotype CrNHX subfamilies ( Figure 3A). A Pfam database search showed that all CrNHXs contained the Na_H_Exchanger domain (PF00999) (Figure 3B), and the TMHMM analysis indicated that the transmembrane regions presenting in the Na_H_Exchanger domain were all conserved ( Figure 3C), which almost indicated a one-to-one relationship with the predicted motifs predicted by MEME. In general, the C-termini of all CrNHXs were less conservative, which were predicted as being hydrophilic and playing crucial parts in channel activity regulation and protein trafficking by protein-protein interaction.

Abiotic Stress-Related Cis-regulatory Elements in CrNHX Promoters
To gain further insights into the putative biological function of CrNHX genes in C. rosea, cis-regulatory elements located in the promoter (ATG upstream 2000 bp) of each CrNHX gene were investigated. We particularly focused on the hormone-responsive cisregulatory elements (gibberellin-responsive element, MeJA (methyl jasmonate)-responsiveness element, auxin-responsive element, salicylic acid responsiveness, ethylene-responsive ERE, and ABA-responsive element ABRE) and abiotic stresses cis-regulatory elements (light-responsive element, MYC, and MYB), and other cellular functioning related cis-regulatory elements (ERE, MBS, and TC-rich repeats) were specially identified. As shown in Figure 4, the light-responsive elements accounted for a larger proportion of all elements, which indicated that the CrNHX expression responses regulated by light were under complex programmed control. Among all the hormone-related cis-regulatory elements, ABREs were found in seven of the eight CrNHX promoters (except CrNHX3-1),

Abiotic Stress-Related Cis-Regulatory Elements in CrNHX Promoters
To gain further insights into the putative biological function of CrNHX genes in C. rosea, cis-regulatory elements located in the promoter (ATG upstream 2000 bp) of each CrNHX gene were investigated. We particularly focused on the hormone-responsive cis-regulatory elements (gibberellin-responsive element, MeJA (methyl jasmonate)-responsiveness element, auxin-responsive element, salicylic acid responsiveness, ethylene-responsive ERE, and ABA-responsive element ABRE) and abiotic stresses cis-regulatory elements (lightresponsive element, MYC, and MYB), and other cellular functioning related cis-regulatory elements (ERE, MBS, and TC-rich repeats) were specially identified. As shown in Figure 4, the light-responsive elements accounted for a larger proportion of all elements, which indicated that the CrNHX expression responses regulated by light were under complex programmed control. Among all the hormone-related cis-regulatory elements, ABREs were found in seven of the eight CrNHX promoters (except CrNHX3-1), and ERE was also present in all but the CrNHX3-1 promoter ( Figure 4A). The salicylic acid responsiveness, MeJA-responsive elements, and ethylene responsive ERE were also specifically absent in the CrNHX3-1 promoter. The most frequent abiotic stress-related elements were MYB and ABRE ( Figure 4B), which further indicated that the CrNHXs were responsible for various stresses, including drought, high salinity, or other temperature abnormalities. The promoter sequence information of all CrNHXs is listed in Table S1, and the categories of those cis-regulatory elements found in CrNHX promoter DNA regions are listed in Table S3. The cis-regulatory element analysis data suggested that CrNHX genes might respond to multiple hormones and abiotic stresses, thus playing a role in high salinity/alkaline stress adaptation in C. rosea.
Genes 2022, 12, x FOR PEER REVIEW 11 of 25 and ERE was also present in all but the CrNHX3-1 promoter ( Figure 4A). The salicylic acid responsiveness, MeJA-responsive elements, and ethylene responsive ERE were also specifically absent in the CrNHX3-1 promoter. The most frequent abiotic stress-related elements were MYB and ABRE ( Figure 4B), which further indicated that the CrNHXs were responsible for various stresses, including drought, high salinity, or other temperature abnormalities. The promoter sequence information of all CrNHXs is listed in Table S1, and the categories of those cis-regulatory elements found in CrNHX promoter DNA regions are listed in Table S3. The cis-regulatory element analysis data suggested that CrNHX genes might respond to multiple hormones and abiotic stresses, thus playing a role in high salinity/alkaline stress adaptation in C. rosea.

Expression Profiles of CrNHXs in different Tissues or under different Habitat Environmental Conditions
To investigate whether the predicted CrNHX genes were actually transcribed, we examined their transcription levels in different tissues using RNA-seq to further confirm that these CrNHXs were not pseudogenes. Five tissues, including the root, stem, young leaf, flowering bud, and young fruit collected from adult C. rosea plants growing in SCBG, were selected for this study. As shown in Figure 5, three genes (CrNHX3-1, CrNHX5, and CrNHX7) were highly expressed in all detected five tissues, while CrNHX4, CrNHX4-1, and CrNHX6 showed moderate expression levels in most of the tissues. Both CrNHX1 and CrNHX3 were expressed at extremely low levels in most of the detected tissues (Figure

Expression Profiles of CrNHXs in Different Tissues or under Different Habitat Environmental Conditions
To investigate whether the predicted CrNHX genes were actually transcribed, we examined their transcription levels in different tissues using RNA-seq to further confirm that these CrNHXs were not pseudogenes. Five tissues, including the root, stem, young leaf, flowering bud, and young fruit collected from adult C. rosea plants growing in SCBG, were selected for this study. As shown in Figure 5, three genes (CrNHX3-1, CrNHX5, and CrNHX7) were highly expressed in all detected five tissues, while CrNHX4, CrNHX4-1, and CrNHX6 showed moderate expression levels in most of the tissues. Both CrNHX1 and CrNHX3 were expressed at extremely low levels in most of the detected tissues ( Figure 5A). The FPKM values are listed in Supplementary Material Table S4. We also performed qRT-PCR to measure the relative expression level of the CrNHX genes in different organ tissues. In general, the results from the qRT-PCR and RNA-seq were consistent, with a few exceptions ( Figure 5B and Figure S3A). For instance, CrNHX5 showed the greatest expression levels in all tested tissues in the RNA-seq results ( Figure S3A), while in our qRT-PCR confirmation assay, CrNHX5 presented relatively lower expression levels than most CrNHXs ( Figure 5B). Our results indicated that the CrNHX genes diverged in expression pattern in different organs, while they were all truly expressed and acted as functional genes.
HXs ( Figure 5B). Our results indicated that the CrNHX genes diverged in expressio tern in different organs, while they were all truly expressed and acted as functional The expression profiles of the C. rosea NHX genes were also analyzed in m leaves collected from adult plants growing in different habitats (SCBG and YX Isla explore the possible roles that CrNHXs play in the response of C. rosea plants to spec habitats with high salinity/alkaline features. As we can see from Figure 6, the expr of most CrNHXs was obviously lower in the YX sample than in the SCBG samp only CrNHX1 and CrNHX4 were expressed slightly higher in the YX sample than SCBG sample ( Figure S3B). These results suggest that, under the extreme circums on the tropical coral reef, the expression of CrNHXs in C. rosea leaves was most suppressed to some degree, implying the decreased transporting of salt ions in the part of this halophyte to alleviate the toxic effects of high salinity/alkaline. This resu implied that the biological functions of the CrNHX family are closely related to the tation to coral reef habitats. The expression profiles of the C. rosea NHX genes were also analyzed in mature leaves collected from adult plants growing in different habitats (SCBG and YX Island) to explore the possible roles that CrNHXs play in the response of C. rosea plants to specialized habitats with high salinity/alkaline features. As we can see from Figure 6, the expression of most CrNHXs was obviously lower in the YX sample than in the SCBG sample, and only CrNHX1 and CrNHX4 were expressed slightly higher in the YX sample than in the SCBG sample ( Figure S3B). These results suggest that, under the extreme circumstances on the tropical coral reef, the expression of CrNHXs in C. rosea leaves was most likely suppressed to some degree, implying the decreased transporting of salt ions in the aerial part of this halophyte to alleviate the toxic effects of high salinity/alkaline. This result also implied that the biological functions of the CrNHX family are closely related to the adaptation to coral reef habitats.

Expression Profiles of CrNHXs under different Abiotic Stress Treat
To further verify that CrNHXs are involved in the specific resp to different stresses, we analyzed the transcriptional expression salinity/alkaline, high osmotic stress, and heat treatment, to mimic in the specialized habitat of the tropical coral reef as closely as pos ures 7 and S4, the expression of eight CrNHXs showed different p downregulated) under these stress challenges in the seedling roots the expression of CrNHXs in the C. rosea seedlings showed more changes under high salinity stress than under alkaline or high o might be closely related to their biochemical functions being ion ch 2 h of salt stress, CrNHX5 and CrNHX7 were obviously induced while the expression of CrNHX4 was downregulated (by −42.95% only induced CrNHX7 expression (by 35.21%) and decreased the 1 (by −48.98%), CrNHX4 (by −90.09%), and CrNHX4-1 (by −57.71%

Expression Profiles of CrNHXs under Different Abiotic Stress Treatments
To further verify that CrNHXs are involved in the specific responses of C. rosea plants to different stresses, we analyzed the transcriptional expression of CrNHXs under high salinity/alkaline, high osmotic stress, and heat treatment, to mimic each single stress factor in the specialized habitat of the tropical coral reef as closely as possible. As shown in Figures 7 and S4, the expression of eight CrNHXs showed different patterns (upregulated or downregulated) under these stress challenges in the seedling roots and leaves. In general, the expression of CrNHXs in the C. rosea seedlings showed more obvious transcriptional changes under high salinity stress than under alkaline or high osmosis stresses, which might be closely related to their biochemical functions being ion channels. In the root, after 2 h of salt stress, CrNHX5 and CrNHX7 were obviously induced (by 33.71 and 136.83%), while the expression of CrNHX4 was downregulated (by −42.95%); high alkaline stress only induced CrNHX7 expression (by 35.21%) and decreased the expression of CrNHX3-1 (by −48.98%), CrNHX4 (by −90.09%), and CrNHX4-1 (by −57.71%) in the root at the 2 h time point, while high osmotic stress also greatly decreased the expression of CrNHX3-1 (by −39.85%) and CrNHX4 (by −60.95%) at 2 h in root ( Figure 7A). In contrast, high salt stress for 2 h caused the decreased expression of CrNHX5 (by −39.24%) and CrNHX7 (by -32.48%) in the leaf samples, as well as the high alkaline challenge (by −43.02% and −40.13%, respectively); the high osmotic stress also obviously decreased the expression of CrNHX5 (by −43.30%) in C. rosea leaf at the 2 h time point ( Figure 7B). In the root, after 48 h of salt change, only CrNHX5 showed obviously upregulated expression patterns (by 62.59%), while the high alkaline and osmotic stresses caused irregular transcriptional changes ( Figure 7C). Comparatively, in the leaf after a 48 h stress challenge, the expression of four CrNHXs was affected, and CrNHX5 showed an obviously upregulated expression pattern (by 97.41, 69.65, and 79.74%, respectively); CrNHX1 showed an obviously downregulated expression pattern responding to these three challenges (by -99.33%, −81.24%, and −89.25%, respectively). The expression of CrNHX3-1 was decreased by high salt stress (by −72.03%), while the expression of CrNHX7 was slightly induced by high alkaline and osmotic stresses (by 54.26 and 16.23%) ( Figure 7D). The FPKM values and the percentage changes of up-or downexpression patterns are listed in Supplementary Material Table S4.

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Heat stress can cause cellular metabolic disorders and even the death of plants. High temperatures are often the norm on tropical coral reefs and affect the growth of C. rosea. Our results showed that the expression levels of most CrNHXs were lower after 2 h heat stress (HS) than in the controls (Figure 8), which indicated that this gene family might respond to heat stress by decreasing its transcription, thereby coping with energy imbalances and metabolic disorders. In general, our RNA-seq results demonstrated that this gene family might be involved in multiple stresses and may play different roles in these stresses.  Heat stress can cause cellular metabolic disorders and even the death of plants. High temperatures are often the norm on tropical coral reefs and affect the growth of C. rosea. Our results showed that the expression levels of most CrNHXs were lower after 2 h heat stress (HS) than in the controls (Figure 8), which indicated that this gene family might respond to heat stress by decreasing its transcription, thereby coping with energy imbalances and metabolic disorders. In general, our RNA-seq results demonstrated that this gene family might be involved in multiple stresses and may play different roles in these stresses.
(B) after 2 h abiotic stress challenge; (C,D) the expression differences of eight CrNHXs in the root (C) and leaf (D) after 48 h abiotic stress challenge. Expression levels for each CrNHX were shown with the log2 values of FPKM. Yellow denotes high expression levels, and dark-blue denotes low expression levels.  We further confirmed the expression of the CrNHXs under high salinity, alkaline, and high osmosis challenges by qRT-PCR in C. rosea seedlings (Figure 9). The RNA was isolated from the leaf and root tissues at 2 h or 48 h after the same stress treatment that the RNA-seq assays were initiated. Genes with changes in transcription levels greater than 1.5-fold were considered to be significantly regulated. Of the eight CrNHX genes, only seven genes were detected in all the tested tissues, including CrNHX1, CrNHX3-1, CrNHX4, CrNHX4-1, CrNHX5, CrNHX6, and CrNHX7. The results of the qRT-PCR analysis showed that seven CrNHXs responded to different stresses to varying degrees, and similarly, the expression of CrNHXs under salt stress was more greatly regulated in both the root and leaf samples, and the alkali stress also resulted in variable expression patterns of several CrNHX members, such as CrNHX1 and CrNHX4. In brief, after 2 h challenges, the expression of

Functional Characterization of CrNHXs in Yeast
For the functional characterization of different CrNHX members, we first attempted to clone all CrNHX members by RT-PCR. We ultimately succeeded in cloning only five CrNHX cDNAs, including CrNHX1, CrNHX3, CrNHX5, CrNHX6, and CrNHX7. The coding sequences of these five CrNHXs were separately inserted into the yeast expression vector pYES2 under the control of the GAL1 promoter. Each vector (recombinant vectors and control pYES2) was introduced into the yeast triple mutant strain AXT3, which lacks the functions of four plasma membrane Na + -ATPases (ScENA1-4), the plasma membrane Na + , K + /H + antiporter ScNHA1, and the vacuolar Na + , K + /H + exchanger ScNHX1. This mutant strain is highly sensitive to Na + stress and is relatively sensitive to K + and hygromycin B stress when grown on AP medium plate [48]. The corresponding wild-type strain W303 was set as a positive control.
The different levels of salt tolerance generated by specific CrNHX members (CrNHX1, CrNHX3, CrNHX5, CrNHX6, and CrNHX7) are demonstrated in Figure 10. The results showed that the transformed AXT3 lines expressing these genes grew much better than the AXT3 mutant transformed with the empty vector pYES2 under 6, 8, 10, and 12 mM NaCl stress ( Figure 10A), with the wild-type strain control W303 always growing well. Under 4 mM NaCl treatment, the growth of AXT3 with a series of expressed CrNHXs or AXT3/W303 with the control vector pYES2 all exhibited regular growth on APG plates. It seemed that CrNHX1 and CrNHX3 led to greater increases in the tolerance to NaCl than the other CrNHXs (at 10 and 12 mM NaCl). However, although all of the five CrNHX members increased the tolerance of AXT3 to KCl, only CrNHX3 led to the greatest increase

Functional Characterization of CrNHXs in Yeast
For the functional characterization of different CrNHX members, we first attempted to clone all CrNHX members by RT-PCR. We ultimately succeeded in cloning only five CrNHX cDNAs, including CrNHX1, CrNHX3, CrNHX5, CrNHX6, and CrNHX7. The coding sequences of these five CrNHXs were separately inserted into the yeast expression vector pYES2 under the control of the GAL1 promoter. Each vector (recombinant vectors and control pYES2) was introduced into the yeast triple mutant strain AXT3, which lacks the functions of four plasma membrane Na + -ATPases (ScENA1-4), the plasma membrane Na + , K + /H + antiporter ScNHA1, and the vacuolar Na + , K + /H + exchanger ScNHX1. This mutant strain is highly sensitive to Na + stress and is relatively sensitive to K + and hygromycin B stress when grown on AP medium plate [48]. The corresponding wild-type strain W303 was set as a positive control.
The different levels of salt tolerance generated by specific CrNHX members (CrNHX1, CrNHX3, CrNHX5, CrNHX6, and CrNHX7) are demonstrated in Figure 10. The results showed that the transformed AXT3 lines expressing these genes grew much better than the AXT3 mutant transformed with the empty vector pYES2 under 6, 8, 10, and 12 mM NaCl stress ( Figure 10A), with the wild-type strain control W303 always growing well. Under 4 mM NaCl treatment, the growth of AXT3 with a series of expressed CrNHXs or AXT3/W303 with the control vector pYES2 all exhibited regular growth on APG plates. It seemed that CrNHX1 and CrNHX3 led to greater increases in the tolerance to NaCl than the other CrNHXs (at 10 and 12 mM NaCl). However, although all of the five CrNHX members increased the tolerance of AXT3 to KCl, only CrNHX3 led to the greatest increase in tolerance to KCl (at 500, 550, and 600 mM KCl) ( Figure 10B). Similar results were also observed in the hygromycin B tolerance test, and only CrNHX3 resulted in much stronger tolerance than the other CrNHXs (at 6, 8, and 12 mg L −1 hygromycin B) ( Figure 10C). These results confirmed the functions of CrNHX1, CrNHX3, CrNHX5, CrNHX6, and CrNHX7 in salt tolerance and also proved the biochemical functions of CrNHX members in transmembrane transport in a single-cell system. Our findings also demonstrate that the CrNHX members showed different functional specificities for salt tolerance in yeast cells, though their biological functions in plants need to be further explored. in tolerance to KCl (at 500, 550, and 600 mM KCl) ( Figure 10B). Similar results were also observed in the hygromycin B tolerance test, and only CrNHX3 resulted in much stronger tolerance than the other CrNHXs (at 6, 8, and 12 mg L −1 hygromycin B) ( Figure 10C). These results confirmed the functions of CrNHX1, CrNHX3, CrNHX5, CrNHX6, and CrNHX7 in salt tolerance and also proved the biochemical functions of CrNHX members in transmembrane transport in a single-cell system. Our findings also demonstrate that the CrNHX members showed different functional specificities for salt tolerance in yeast cells, though their biological functions in plants need to be further explored.

Discussion
Climate change is causing rising sea levels and increased drought, thereby elevating salinization in many regions of the world [49]. Salinity is a key environmental factor and one of the most important abiotic stresses affecting physiological pathways and inhibiting development and growth in plants. Globally, over 11% of the irrigated land used for agriculture is affected by high salinity [50], which is also responsible for water deficit, nutritional imbalance, and toxicity challenges. To tolerate salt stress, plants have developed a

Discussion
Climate change is causing rising sea levels and increased drought, thereby elevating salinization in many regions of the world [49]. Salinity is a key environmental factor and one of the most important abiotic stresses affecting physiological pathways and inhibiting development and growth in plants. Globally, over 11% of the irrigated land used for agriculture is affected by high salinity [50], which is also responsible for water deficit, nutritional imbalance, and toxicity challenges. To tolerate salt stress, plants have developed a series of physiological, biochemical, and ecological strategies. A halophyte is an extremophile that can survive and even flourish under highly saline conditions. Halophytes are likely to possess some morphological, physiological, or molecular mechanisms associated with development and growth regulation that have evolved as adaptive responses to salt tolerance [38]. In general, halophytes can adjust osmotically to the surrounding high salinity by accumulating ions and sequestering them into the vacuoles or out of the cytoplasm to maintain cellular water or solute homeostasis and to prevent adverse effects on metabolism [51]. It is well documented that NHX proteins are involved in salinity tolerance by mediating Na + transport, K + homeostasis, pH regulation, and cellular vesicle trafficking, thereby regulating the ion balance in plants under salt stress [52].
It has been confirmed that plant NHXs play vital roles in various cellular processes in response to high salinity stress conditions. These stress response mechanisms mainly involve H + -coupled cation transportation and cellular compartments regulated by NHXs and their interacting proteins, which produce channels across the membrane. Thus, the different classes of NHXs work to maintain cellular salinity tolerance and K + homeostasis [52]. In this study, we performed a genome-wide analysis of CrNHXs in the C. rosea genome and obtained genomic and transcriptional information concerning the possible roles of this family in adaptation to specialized habitats on tropical coral reefs and islands. High salinity/alkaline and drought have been demonstrated to be detrimental factors that severely affect the growth and distribution of vegetation on coral reefs and islands. It is thus of great importance to study how plants respond to high salinity/alkaline and related hyperosmotic or drought stresses with regard to growth, development, and even natural ecological adaptability. In this study, eight CrNHX genes were comprehensively investigated for the first time based on the C. rosea genome data, and the expression profiles of these CrNHXs were analyzed to explore their functions in the abiotic stress response in different C. rosea tissues.
The NHX gene families are conserved in all eukaryotes, mainly mediating the transmembrane transport of Na + /K + and maintaining cellular Na + /K + homeostasis. To date, the composition of the NHX family has been fully described in model or cash crop species as well as in some specialized habitat plants. Compared with the number of NHXs in other species, the number in C. rosea is similar to that in Arabidopsis and other leguminous plants. The plant NHX family consists of three subgroups, including Vac-NHX, Endo-NHX (intracellular proteins), and PM-NHX (plasma membrane-bound proteins) (Figures 1 and 2). Some members identified from other plant species have been well characterized and are proved to be associated with salt tolerance in plants, mainly including NHX7/SOS1 genes in Arabidopsis [10], soybean [53], rice [54], cotton [55], and wheat [56]. Vac-NHX or Endo-NHX can form convenient channels for vacuolar or endomembrane system Na + /K + sequestration, while PM-NHX is mainly responsible for the Na + /K + efflux out of the cell in exchange for H + influx into the cell [52,57]. Besides several Arabidopsis Vac-NHX or Endo-NHX members [12][13][14][15][16], some other Vac-NHX or Endo-NHX genes have been confirmed to be involved in the compartmentalization of Na + /K + into the vacuoles or intracellular compartmentalization and are critical of salt tolerance in other plant species [48,58,59]. In recent years, the functional identification of NHX genes from plant species growing in specialized habitats has been explored, including in eremophytes and halophytes [60][61][62][63]. Understanding the specific mechanisms by which NHX family members are involved in salt tolerance and stress regulation will be of great significance for elucidating the environmental adaptation mechanisms of plants in specialized habitats.
Analysis of gene structures and conserved motifs showed that CrNHXs exhibited high conservation during evolution. Interestingly, in the plant monovalent cation/proton antiporters (CPAs) superfamily, the NHX families often show multi-intron characteristics compared with other ion channel gene families, for example, the cation/H + exchanger (CHX) families [33,64], and in other species, the introns of NHX families seem to be fairly conservative [23,24,65]. It is reported that multiple numbers of exons in genes might be positively correlated with active chromatin structures, which could represent a type of possible gene regulation pattern for epigenetic or expression complexity [24]. Corresponding to the gene structures, the conserved motifs are more related to the biochemical functions of proteins. The Pfam search identified the conserved Na_H_Exchanger domain in each CrNHX protein, and in addition, the motif identification and transmembrane region prediction indicated that these conserved motifs in CrNHXs are highly consistent (Figure 3), concentrating mainly at the transmembrane domain that could form hollow ion channels in each CrNHX protein ( Figure S1). Except for CrNHX3-1, the other four Vac-CrNHXs also have the typical amiloride-binding site of the NHX gene (FFI/LY/FLLPPI), but this motif is not so conserved in two Endo-CrNHX, CrNHX5, and CrNHX6. The five Vac-CrNHXs also have the putative calmodulin (CaM) binding site in their C-termini ( Figure S2). It is suggested that the possession of amiloride-binding sites in plant NHX proteins is closely related to their role in the tolerance of high salinity conditions [36].
The evolutionary process of gene families usually undergoes tandem duplication, large-scale segmental duplication, and whole-genome duplication (WGD) to maintain the size of each family. Our previous genome sequencing for C. rosea indicates that this species is a typical diploid (data not published). Here, we checked the tandem and segmental duplications of CrNHX family. Although these duplications have been suggested to play important roles in the formation of gene families, in this study, only one gene pair, CrNHX5/CrNHX6, showed the segmental duplication pattern, further suggesting the conservative feature of NHX families (Table 2). In other two leguminous plants, alfalfa [26] and soybean [31], the numbers of NHX families are six and nine; however, they seem to be incomplete. Compared to other species, for example, the NHX families hold seven members in mulberry [23], eight members in poplar [24], six member in grapevine [28], 11-12 members in diploid cotton species Gossypium raimondii and G. arboreum, 23-24 members in the tetraploid cotton G. hirsutum and G. barbadense [36], and 10 members in gramineous bamboo plant Phyllostachys edulis [33]; the CrNHX family seems to be relatively small and stable, and the gene duplication did not obviously expand this family in this specialized species.
In response to environmental changes, plants must trigger their gene regulatory network, primarily mediated by a series of transcription factors (TFs), and then these TFs can bind to specific cis-regulatory elements located at the promoter regions of genes and determine gene expression patterns. In this study, we mainly focused on some hormoneresponsive and abiotic stress-related elements that have been confirmed in other plant species, and we propose that these elements hold some function similarities in C. rosea. These elements include light response elements, gibberellin-responsive elements, MeJAresponsive elements, auxin response elements, salicylic acid responsiveness elements, ABA response elements (ABRE), ethylene response elements (ERE), MYC transcription factor binding elements (MYC), MYB transcription factor binding elements (MYB and MBS), and TC-rich repeats. The ABRE, MYC-, MYB-, and TC-rich repeat elements are believed to be involved in plant responses to dehydration, low temperature, salt stress, and other abiotic stresses. CrNHX1, CrNHX4-1, CrNHX6, and CrNHX7 harbored more stress-related cis-regulatory elements (dismissing the light response elements) (Figure 4), which might be the reason for these genes presenting significant transcriptional changes to abiotic stresses. ABA is a critical signaling hormone that adjusts the physiological or metabolic processes of plants in vivo in response to various environmental abiotic stresses [66], and ABREs are kind of the critical regulatory elements found in the promoters of stress-responsive genes. They are the direct target of ABRE binding factors (AREBs/ABFs). ABFs belong to the basicdomain leucine zipper (bZIP) transcription family and induce the transcriptional activation of the stress-inducible genes [67]. Seven of the CrNHX promoters (except CrNHX3-1) contained one or more ABREs, indicating that these CrNHXs might be involved in the ABA signal pathway. Although fewer cis-regulatory elements (including ABREs) were found in the promoter of CrNHX3-1, combined with our RNA-seq results, it was also speculated that this gene might be expressed consistently to maintain its transcription at a certain high level (Figures 5 and 6) and that other regulatory mechanisms might exist in the stress responsiveness of CrNHX3-1 (Figures 7 and 8).
At the genetic level, except for the gene structure (including promoter sequences), transcriptional regulation is the most direct method that directly reflects the biological functions of genes, which have been confirmed as being involved in various biological processes, such as hormonal responses, abiotic stress responses, and development in plants. Canavalia rosea is a typical salt and drought-tolerant sea-drifting plant that is significant for ecological reconstruction on tropical reefs and islands. On YX Island, this species demonstrates stronger adaptability and better growth potential than most other plants and is used as a pioneer and dominant species for environmental greening. The habitat-specific RNA-seq data further indicated that most of the CrNHXs had lower expression levels in the leaf samples of C. rosea plants growing in coastal C. rosea (YX) than in inland C. rosea (SCBG; Figure 6), which suggests that the high salinity/alkaline and environmental extremes probably severely limited the expression levels and subsequent Na + /K + transport activities in the aerial parts of C. rosea. The differential expression of CrNHXs might be associated with Na + and K + /H + absorption and transport in different habitats, and the lower expression level of CrNHXs in coastal C. rosea plants might be an adaptive mechanism to deal with intracellular and extracellular high salinity/alkaline stress, while the transcriptional characteristics of CrNHXs in YX-native C. rosea plant roots are unclear and need further clarification. This may be because this protein family fulfills an important function in Na + /K + and H + transportation across cellular membranes, which is closely related to the salinity/alkaline tolerance of C. rosea, thereby regulating its environmental adaptation to tropical coastal regions.
Given the extreme salt/alkaline, drought, and heat environment on YX Island, we mimicked the main stress factors to detect the expression patterns of CrNHXs, including high salinity, alkaline, high osmosis, and heat stress. In general, the transcript changes in the root of the CrNHXs showed more variability than that in the leaves under different abiotic stress challenges (Figures 7, 8 and S4). Our results also implied that some CrNHXs in the same class had distinct expression changes. For instance, the Vac-typeCrNHX1 and CrNHX4 were rapidly induced by salinity, alkaline, and high osmosis in the leaf at 2 h time point, while their expression changes in the leaf were completely at odds at 48 h time point (Figure 9). The Endo-type CrNHX5 and CrNHX6 were more induced in the leaf than in the root and even demonstrated opposite expression patterns to some degree (Figures 8 and 9). This can be attributed to the fact that in the roots the high salinity/alkaline was stronger and more direct, which might result in the immediate promotion of cellular Na + /K + distribution in the root, while in the leaf, the transcriptional response was slow until 48 h, and CrNHX5 and CrNHX6 demonstrated induced expression after those abiotic stress challenges (Figures 7 and 8).
Although plenty of previous reports have confirmed that overexpressing NHXs in plants could result in the elevated salt tolerance of the whole plant, probably by maintaining the balance of Na + /K + and controlling saline ions and pH homeostasis in cytoplasm [33,34,37,48,[53][54][55][56], the expression of CrNHXs in C. rosea was not always induced by the high salinity/alkaline challenges. It probably boiled down to the regulatory complexity of maintaining the saline ions homeostasis in different tissues, due to C. rosea being a multicellular organism. In the stressful habitat, the CrNHXs members in different parts of the plant might be subject to different regulatory mechanisms and then play differentiating roles in regulating the translocation of the saline ions. Given that, we cloned the CrNHX genes and performed the further functional confirmation in the unicellular yeast system.
Over-expression in yeast systems is widely used to study the function of genes under biotic stresses. The uptake and translocation of Na + and K + /H + play essential roles in plant halotolerance, signal transduction, growth, and development, especially for the tropical coral reef-growing plant species. Among them, Na + toxicity is a principal cause of the deleterious effects associated with salinity stress, whereas H + is essential for balancing cellular acid-alkali levels. In this study, CrNHX1, CrNHX3, CrNHX5, CrNHX6, and CrNHX7 were cloned and expressed in the salt-sensitive yeast mutant strain AXT3. These five CrNHXs could partially restore the Na + /K + tolerance of AXT3 ( Figure 10A,B), while the recovery from salt resistance showed some member-specific variance. In contrast to MnNHXs in mulberry [48], two Vac-type CrNHXs, CrNHX1 and CrNHX3, showed greatly enhanced tolerance to salt-stress (Na + /K + tolerance) compared to the two Endo-type CrNHXs (CrNHX5 and CrNHX6) and PM-type CrNHX7, and the hygromycin B tolerance seemed to be similar ( Figure 10C). Hygromycin B is a large, toxic alkali cation that accumulates intracellularly in response to the electrochemical proton gradient. According to previous reports, hygromycin B sensitivity is a common phenotype of the yeast nhx1 mutant [12], and some identified plant NHX proteins have been confirmed being transporters for hygromycin B, as well as the Na + , K + , or Li + transporters [48]. This further confirmed that CrNHXs transport Na + /K + and hygromycin B in vivo, while their exact functions or mechanisms in salinity tolerance need to be further confirmed in transgenic plants.

Conclusions
In brief, we carried out a detailed analysis of the complete NHX gene family in specialized habitat halophyte species, C. rosea. A total of eight CrNHXs were identified based on their composition, nomenclature, chromosomal locations, and phylogenetic relationships with NHXs from other plant species. Further detailed analysis of expression profiles for CrNHXs performed using RNA-seq data, as well as using the heterologous expression assays with the yeast system to investigate the transporting activities of several CrNHXs, proved that this family is involved in salt/alkaline stress responses and plays vital roles in ecological adaptability for natural extreme adversity on tropical coral islands and reefs. The results in this study offer a foundation for future work aimed at both elucidating the molecular mechanisms underlying NHXs functioning in salt resistance and high salinity/alkaline environmental adaptation mechanisms from the starting point concerning plant NHX genes. However, this is only the tip of the iceberg in terms of studying this species from a molecular perspective.