Genome-Wide Identification and Functional Characterization of the Cation Proton Antiporter (CPA) Family Related to Salt Stress Response in Radish (Raphanus sativus L.)

The CPA (cation proton antiporter) family plays an essential role during plant stress tolerance by regulating ionic and pH homeostasis of the cell. Radish fleshy roots are susceptible to abiotic stress during growth and development, especially salt stress. To date, CPA family genes have not yet been identified in radish and the biological functions remain unclear. In this study, 60 CPA candidate genes in radish were identified on the whole genome level, which were divided into three subfamilies including the Na+/H+ exchanger (NHX), K+ efflux antiporter (KEA), and cation/H+ exchanger (CHX) families. In total, 58 of the 60 RsCPA genes were localized to the nine chromosomes. RNA-seq. data showed that 60 RsCPA genes had various expression levels in the leaves, roots, cortex, cambium, and xylem at different development stages, as well as under different abiotic stresses. RT–qPCR analysis indicated that all nine RsNHXs genes showed up regulated trends after 250 mM NaCl exposure at 3, 6, 12, and 24h. The RsCPA31 (RsNHX1) gene, which might be the most important members of the RsNHX subfamily, exhibited obvious increased expression levels during 24h salt stress treatment. Heterologous over-and inhibited-expression of RsNHX1 in Arabidopsis showed that RsNHX1 had a positive function in salt tolerance. Furthermore, a turnip yellow mosaic virus (TYMV)-induced gene silence (VIGS) system was firstly used to functionally characterize the candidate gene in radish, which showed that plant with the silence of endogenous RsNHX1 was more susceptible to the salt stress. According to our results we provide insights into the complexity of the RsCPA gene family and a valuable resource to explore the potential functions of RsCPA genes in radish.


Introduction
Plants respond to salt stress by regulating the cell ion and pH balance through a variety of mechanisms, which are mainly dependent on ion transporters in cell membranes and organelle membranes [1,2]. Cation proton antiporters (CPAs) are mainly involved in the exchange and transport of monovalent cations in plants, which not only reverses the transport of Na + with H + but also exchanges and transports monovalent cations, such as K + and Li + [3][4][5]. The CPA family is divided and complete RsCPA members were identified among the radish genome, which were correspondingly named as RsCPA01-RsCPA60 (Table S1).
Through the physical and chemical properties analysis, the protein sizes of RsCPA ranged from 231 to 1172 amino acids (AAs) with molecular weight (MWs) from 25.97 to 126.11 kDa and the theoretical isoelectric point (pI) varied from 4.98 to 9.21. In addition, the instability coefficient reached from 27.56 to 46.90 and 42 members were <40.00, which were considered as stable proteins. The aliphatic index varied from 95.31 to 127.56, indicating that most RsCPA proteins contained a lot of aliphatic amino acids. The grand average of hydropathicity (GRAVY) ranged from 0.048 to 0.798, suggesting that all RsCPA proteins were hydrophobic proteins (Table S1).

Phylogenetic Analysis of RsCPA Members
To investigate the classification of the CPA subfamily of radish and the evolutionary relationship with other species, full-length CPA protein sequences of radish, Arabidopsis and Brassica rapa were extracted and aligned to construct a neighbor-joining (NJ) phylogenetic tree (Figure 1). A total of 166 CPA protein members in these three species (containing 60 radish, 42 Arabidopsis, and 64 Brassica rapa) were categorized into three subfamilies, namely NHX, KEA, and CHX. Among them, the NHX group had 28 members containing nine, eight, and 11 members of radish (15%), Arabidopsis (19.05%), and Brassica rapa (17.19%), respectively. The KEA group included 29 members with ten, six, and 13 members of radish (16.67%), Arabidopsis (14.29%), and Brassica rapa (20.31%), respectively. The CHX group was the most abundant subfamily and had 109 members with 41, 28, and 40 members in radish (68.33%), Arabidopsis (66.67%), and Brassica rapa (62.5%), respectively ( Table 1). The phylogenetic relationships indicated that the CPA proteins in radish had stronger homology with Brassica rapa than Arabidopsis.  Meanwhile, compared with several dicotyledon and monocotyledon crops, the number of CPA gene in radish was closer to Malus domestica and Pyrus bretschneideri, while it was significantly different from that in Prunus persica, Fragaria vesca, Prunus mume, and Vitis vinifera. Especially compared with monocotyledonous plants, such as Oryza sativa, Zea mays, and Sorghum bicolor, the difference of CPA gene numbers was very significant (Table 1).  Meanwhile, compared with several dicotyledon and monocotyledon crops, the number of CPA gene in radish was closer to Malus domestica and Pyrus bretschneideri, while it was significantly different from that in Prunus persica, Fragaria vesca, Prunus mume, and Vitis vinifera. Especially compared with monocotyledonous plants, such as Oryza sativa, Zea mays, and Sorghum bicolor, the difference of CPA gene numbers was very significant (Table 1).

Gene Structure and Motif Composition Analysis
All the 60 RsCPA members were divided into three subfamilies, including 9 RsNHXs, 10 RsKEAs, and 41 RsCHXs (Figure 2a). The distributions of RsCPA protein motifs were conducted by Multiple Em for Motif Elicitation (MEME) and 20 conserved motifs were generated (Figure 2b, Figure S1). Most RsCPA members in the same subfamily had similar motif compositions, suggesting that these proteins might have conservative functions. Among them, motif 1 and 12 were found in the CHX, KEA, and NHX subfamilies, indicating that were highly conserved in all RsCPA proteins. Additionally, some motifs were distributed in two subfamilies. For instance, motif 5, 11, and 17 were distributed in the CHX and NHX subfamilies, while motif 14 was distributed in the CHX and KEA subfamilies. Intriguingly, several motifs were only detected in specific RsCPA subfamilies. For example, the RsCHX subfamily independently contained diverse motifs, such as motif 3, 4, 6, 7, 8, 10, 13, 18, and 19. However, the KEA subfamily exclusively contained the motif 15 and 20, as well as the RsNHX subfamily that specifically contained motif 16 ( Figure 2b). similar, while the lengths of the exon and intron were different. Compared with the RsCHXs subfamily, the gene structures of RsNHXs and RsKEAs were more complex. Among them, the exon numbers of RsCHXs were generally one to five. However, the exon numbers of RsNHXs and RsKEAs ranged from 10 to 19 and 17 to 21, respectively. Additionally, all members in RsNHX subfamily as well as most RsKEA ones (except RsCPA01) had UTR (untranslated region), whereas several members of the RsCHX subfamily had no UTR region (Figure 2c).  Furthermore, exon-intron analysis was investigated to obtain the structure information of RsCPA genes. As shown in Figure 2c, the gene structures involved in the same subfamily were similar, while the lengths of the exon and intron were different. Compared with the RsCHXs subfamily, the gene structures of RsNHXs and RsKEAs were more complex. Among them, the exon numbers of RsCHXs were generally one to five. However, the exon numbers of RsNHXs and RsKEAs ranged from 10 to 19 and 17 to 21, respectively. Additionally, all members in RsNHX subfamily as well as most RsKEA ones (except RsCPA01) had UTR (untranslated region), whereas several members of the RsCHX subfamily had no UTR region (Figure 2c).

Promoter Elements and Transmembrane Region Analysis
The putative promoter sequence (2000 bp upstream region of transcription initiation site) of RsCPA genes was submitted to PlantCARE to search for cis-acting elements. A total of 103 cis-acting elements were identified. Except basic promoter elements, such as the CAAT box and TATA box, 18 other important cis-acting elements related to plant growth and development and various stresses were explored. In Figure 3, it was shown that the distribution pattern of cis-acting elements were diverse, indicating that the expression of RsCPA genes might be regulated by various factors. In the aspect of hormone regulation, Abscisic Acid (ABA), auxin, Gibberellin A 3 (GA 3 ), and Methyl Jasmonate (MeJA) responsiveness elements frequently existed. While in terms of stress, anaerobic induction, defense and stress, low temperature, and wound-responsive elements were resided. Moreover, the zein metabolism regulation element existed in 14 promoters of RsCPA genes, suggesting that RsCPA genes might participate in the process of metabolic regulation. Intriguingly, there were 12 promoters of the RsCPA gene involved in endosperm expression, among them, RsCPA27 and RsCPA38 participated in endosperm specific negative expression ( Figure 3 and Figure S2). Additionally, the transmembrane regions of RsCPA proteins showed that all RsCPA proteins, except RsCPA07, contained transmembrane regions that varied from three to 14 (Table S2).
there were 12 promoters of the RsCPA gene involved in endosperm expression, among them, RsCPA27 and RsCPA38 participated in endosperm specific negative expression (Figures 3 and S2). Additionally, the transmembrane regions of RsCPA proteins showed that all RsCPA proteins, except RsCPA07, contained transmembrane regions that varied from three to 14 (Table S2).
Futhermore, an interaction of CPA orthologs co-regulatory network was constructed based on the stress-inducible RsCPA orthologs in Arabidopsis ( Figure S3). It was found that the combination score of the ATCHX1 and KEA4 protein was highest at 0.868, suggesting that it was involved in a stronger relationship between some specific RsCPA proteins, such as RsCPA45, RsCPA47, RsCPA03, RsCPA01, and RsCPA02.  Futhermore, an interaction of CPA orthologs co-regulatory network was constructed based on the stress-inducible RsCPA orthologs in Arabidopsis ( Figure S3). It was found that the combination score of the ATCHX1 and KEA4 protein was highest at 0.868, suggesting that it was involved in a stronger relationship between some specific RsCPA proteins, such as RsCPA45, RsCPA47, RsCPA03, RsCPA01, and RsCPA02.

Chromosomal Localization and Gene Distribution Analysis
A total of 58 RsCPA genes (96.67%) were successfully mapped to the R1-R9 chromosomes of radish by TBtools, except RsCPA12 and RsCPA51 (Figure 4a). R1 and R4 harbored the most RsCPA genes (Ten, 17.67%), followed by R5 and R6 (Eight, 13.37%), while R3 and R8 contained the least RsCPA genes (Two, 3.33%). Genome duplication events have facilitated the expansion of plant gene families, including whole-genome duplication (WGD)/segmental duplication, dispersed duplication (DD), tandem duplication (TD), proximal duplication (PD), and transposed duplication (TRD) [34][35][36]. The duplication types driving expansion of the RsCPA gene family was explored by Multiple Collinearity Scan toolkit (MCScanX). Each RsCPA gene was mapped on the radish genome based on the position coordinates to deduce the evolutionary relationship ( Figure 4b). Totally, 24 pairs of CPA genes in radish had collinear relationships. Of these, 36 (60%) RsCPA genes were duplicated and retained in the WGD event, indicating that the WGD/segmental duplication type played an important role in expansion of the RsCPA gene family. 36]. The duplication types driving expansion of the RsCPA gene family was explored by Multiple Collinearity Scan toolkit (MCScanX). Each RsCPA gene was mapped on the radish genome based on the position coordinates to deduce the evolutionary relationship ( Figure 4b). Totally, 24 pairs of CPA genes in radish had collinear relationships. Of these, 36 (60%) RsCPA genes were duplicated and retained in the WGD event, indicating that the WGD/segmental duplication type played an important role in expansion of the RsCPA gene family.

Spatial and Temporal Expression Profiles of RsCPA Genes
According to the reads per kilobase per million (RPKM) values, the heatmap was generated to characterize the divergence in expression patterns of RsCPA genes among special tissues (cortical, cambium, xylem, root tip, and leaf) and different development stages (40, 60, and 90 d) ( Figure 6). In The non-synonymous/synonymous substitution ratio (Ka/Ks) for the 24 gene pairs was calculated to determine the selection pressure among duplicated RsCPA genes. Most of the RsCPA duplication genes (except RsCPA11-RsCPA23 and RsCPA21-RsCPA34) had a Ka/Ks < 1, indicating that they had experienced strong purifying selective pressure (Table S4).

Spatial and Temporal Expression Profiles of RsCPA Genes
According to the reads per kilobase per million (RPKM) values, the heatmap was generated to characterize the divergence in expression patterns of RsCPA genes among special tissues (cortical, cambium, xylem, root tip, and leaf) and different development stages (40,60, and 90 d) ( Figure 6). In general, the RPKM value varied from 0 to 103.06 and all RsCPA genes exhibited diverse expression patterns. Most of the RsNHX and RsKEA genes showed high expression levels among the five tissues, while the RsCHX genes had extremely low expression or were hardly expressed, as well as a few that showed tissue-specific expression (Figure 6a). It was found that RsCPA58 (RsCHX) was only highly expressed in leaves rather than other tissues, indicating that it might be a leaf-specific gene. Moreover, RsCPA35 was highly expressed in roots after 7 days, while it was down-regulated in roots at other development stages, suggesting it might be a spatiotemporal-specific gene. Furthermore, 68.

The RsCPA Genes Expression Levels under Abotic Stresses
Based on our previous RNA-Seq. data in radish taproots, the differential expression levels of RsCPA genes under various abiotic stresses were investigated, including heavy metal (HM, such as Cadmium (Cd), Chromium (Cr), and lead (Pb), temperature, and salt exposure. As a result, a total of 35, 38, 35, 33, and 29 RsCPA genes were differentially expressed during Cd, Pb, Cr, heat, and salt stress (Fold change >1, p-value < 0.05), respectively ( Figure S4). For instance, RsCPA13 was upregulated in response to Cd, Pb, Cr, and salt stresses, while it was down-regulated under high temperatures. RsCPA08 was up-regulated in response to Cd and Pb, whereas it was down-regulated under Cr and salt stress. Additionally, RsCPA35 was up-regulated in response to Cd, Pb, high temperature, and salt stresses, but it was down-regulated under Cr stress.

The RsCPA Genes Expression Levels under Abotic Stresses
Based on our previous RNA-Seq. data in radish taproots, the differential expression levels of RsCPA genes under various abiotic stresses were investigated, including heavy metal (HM, such as Cadmium (Cd), Chromium (Cr), and lead (Pb), temperature, and salt exposure. As a result, a total of 35, 38, 35, 33, and 29 RsCPA genes were differentially expressed during Cd, Pb, Cr, heat, and salt stress (Fold change >1, p-value < 0.05), respectively ( Figure S4). For instance, RsCPA13 was up-regulated in response to Cd, Pb, Cr, and salt stresses, while it was down-regulated under high temperatures. RsCPA08 was up-regulated in response to Cd and Pb, whereas it was down-regulated under Cr and salt stress. Additionally, RsCPA35 was up-regulated in response to Cd, Pb, high temperature, and salt stresses, but it was down-regulated under Cr stress.
Furthermore, RT-qPCR was conducted to explore the expression levels of RsNHXs subfamily genes under the stress of salt exposure. On the whole, all of the RsNHX genes were significantly up-regulated under the 250 mM salt stress treatment. The expression level of RsCPA13, RsCPA29, RsCPA31, and RsCPA35 were highly increased during the 24 h salt exposure. Moreover, several genes were significantly up-regulated after 12 h salt exposure, such as RsCPA21 and RsCPA34. Notably, the expression level of RsNHXs recovered to normal levels after 96 h salt exposure, implying that RsNHXs might play a crucial role in the process of salt stress response ( Figure S5).

Ectopic Expression of the RsNHX1 Gene in Arabidopsis Can Influence Salt Tolerance
To confirm the biologic function of RsNHX1 gene in the salt stress response of plant, over-expression (OE-RsNHX1) and amiRNA-induced inhibit-expression (amiR-RsNHX1) constructs were introduced into wild-type Arabidopsis (WT). Four and six independent transgenic lines were respectively generated for OE-RsNHX1 (#2, 4, 5 and 6) and amiR-RsNHX1 vectors (#1, 2, 3, 4, 5, and 6) ( Figure S6). Following, each of the six OE-RsNHX1 and amiR-RsNHX1 transgenic lines were compared with nine WT lines under the 200 mM NaCl stress. It was found that the OE-RsNHX1 transgenic lines were slightly yellowed and grew better than the control lines, while most of the amiR-RsNHX1 transgenic lines turned markedly yellow and their growth was inferior to control lines (Figure 7a). For salt stress assays at the seedling stage, the germination ratio of two transgenic seedlings and WT seedlings were counted under 0, and 100 mM NaCl treatment ( Figure 7b). As shown in Figure 7b,c, the germination rate of OE-RsNHX1 seedlings treated with 50mM NaCl was higher than the WT, while amiR-RsNHX1 seedlings treated with 100 mM NaCl was significantly inferior to the WT. Furthermore, the root length of transgenic seedlings and the WT were measured under 0 and 100 mM NaCl treatment (Figure 7d). The root of OE-RsNHX1 seedlings exhibited continuing elongation, whereas the amiR-RsNHX1 seedlings were significantly inhibited under 100 mM NaCl exposure (Figure 7e). These results indicated that RsNHX1 might play a positive role in the salt tolerance of radish.
shown in Figure 7b,c, the germination rate of OE-RsNHX1 seedlings treated with 50mM NaCl was higher than the WT, while amiR-RsNHX1 seedlings treated with 100 mM NaCl was significantly inferior to the WT. Furthermore, the root length of transgenic seedlings and the WT were measured under 0 and 100 mM NaCl treatment (Figure 7d). The root of OE-RsNHX1 seedlings exhibited continuing elongation, whereas the amiR-RsNHX1 seedlings were significantly inhibited under 100 mM NaCl exposure (Figure 7e). These results indicated that RsNHX1 might play a positive role in the salt tolerance of radish.

Functional Analysis of RsNHX1 in Radish Confirms that It Can Positively Regulates Salt Tolerance
We further identify the function of RsNHX1 gene responding to salt stress in radish by using turnip yellow mosaic virus (TYMV)-induced gene silencing (VIGS) technique to silence RsNHX1 expression. Phytoene desaturase (PDS) was employed as a reporter gene to test whether the TYMV-

Functional Analysis of RsNHX1 in Radish Confirms that It Can Positively Regulates Salt Tolerance
We further identify the function of RsNHX1 gene responding to salt stress in radish by using turnip yellow mosaic virus (TYMV)-induced gene silencing (VIGS) technique to silence RsNHX1 expression. Phytoene desaturase (PDS) was employed as a reporter gene to test whether the TYMV-derived vector can silence the endogenous gene of radish. Plants treated with the pTY-S virus vector designed to silence RsNHX1 expression (pTY-RsNHX1), with the wide type (WT), empty pTY-S silencing vector as well as the pTY-RsPDS gene served as mock, empty vector, and positive controls, respectively. Three weeks after particle gun bombardment in radish seedlings, typical phenotype of chlorophyll photobleaching and TYMV spots were separately observed on the leaves of pTY-RsPDS, pTY-RsNHX1, and pTY-S plants, indicating that TYMV-VIGS system was effective in radish (Figure 8a). 522bp PCR products were amplified in four pTY-RsPDS plants and five pTY-RsNHX1 plants, indicating that they were successfully silenced in radish plants (Figure 8b). Furthermore, RT-qPCR revealed that the expression of positive pTY-RsNHX1 was significantly decreased compared to the WT and positive pTY-S plants (Figure 8c). Subsequently, the positive pTY-RsNHX1 plants showed more severe yellowing and wilting than positive pTY-S plants after 7 days of 250mM NaCl exposure (Figure 8d), showing that RsNHX1 might be a salt sensitive gene.

Genome-Wide Identification and Phylogenetic Analysis of CPA Genes in Radish
The CPA gene encoded a conserved Na + /H + domain and played an important role in diverse biology processes, including salt stress, cell expansion, pH and ion balance regulation, osmotic regulation, vesicle transport, protein processing, and floral organ development [15][16][17][18][19]. With the completion of genome sequencing in many plant species, CPA family members have been reported in various plant species, including model plant Arabidopsis (42) and Oryza sativa (30), as well as several horticulture plants, such as Vitis vinifera (29) and Pyrus bretschneideri (53) [11][12][13][14]. Herein, 60 RsCPA members were identified from the radish genome, which showed more members than other reported species indicating that the CPA gene family in radish may be duplicated and expanded.
Phylogenetic analysis indicated that a total of 166 CPAs among the three species containing radish, Brassica rapa, and Arabidopsis were divided into three subfamilies: CHX (109, 65.66%), KEA (29,17.47%), and NHX (28, 16.87%), which was largely consistent with previous studies in Arabidopsis, The total RNA was extracted from the diseased leaves of the suspected radish. Gel electrophoresis showed that the 488 bp PCR products were amplified in two pTY-S plants, the 522bp PCR products were amplified in four pTY-RsPDS plants and five pTY-RsNHX1 plants, indicating that they were successfully silenced in radish plants (Figure 8b). Furthermore, RT-qPCR revealed that the expression of positive pTY-RsNHX1 was significantly decreased compared to the WT and positive pTY-S plants (Figure 8c). Subsequently, the positive pTY-RsNHX1 plants showed more severe yellowing and wilting than positive pTY-S plants after 7 days of 250mM NaCl exposure (Figure 8d), showing that RsNHX1 might be a salt sensitive gene.

Genome-Wide Identification and Phylogenetic Analysis of CPA Genes in Radish
The CPA gene encoded a conserved Na + /H + domain and played an important role in diverse biology processes, including salt stress, cell expansion, pH and ion balance regulation, osmotic regulation, vesicle transport, protein processing, and floral organ development [15][16][17][18][19]. With the completion of genome sequencing in many plant species, CPA family members have been reported in various plant species, including model plant Arabidopsis (42) and Oryza sativa (30), as well as several horticulture plants, such as Vitis vinifera (29) and Pyrus bretschneideri (53) [11][12][13][14]. Herein, 60 RsCPA members were identified from the radish genome, which showed more members than other reported species indicating that the CPA gene family in radish may be duplicated and expanded.
Phylogenetic analysis indicated that a total of 166 CPAs among the three species containing radish, Brassica rapa, and Arabidopsis were divided into three subfamilies: CHX (109, 65.66%), KEA (29,17.47%), and NHX (28, 16.87%), which was largely consistent with previous studies in Arabidopsis, Vitis vinifera, and Pyrus bretschneideri [11][12][13][14]. Through the phylogenetic relationships analysis, it was showed that RsCPAs exhibited closer relations to BraCPAs than AtCPAs, demonstrating that the CPA proteins in radish had stronger homology with the Brassica rapa rather than Arabidopsis (Figure 1). Furthermore, gene structure and motif analysis indicated that the RsCPAs family harbored similar exon-intron structure and shared motif composition with other species, such as Vitis vinifera and Pyrus bretschneideri [13,14] (Figure 2).

Evolutionary Characterization of the RsCPA Family
The expansion of the gene family was mainly caused by gene duplication [34,35]. In the process of plant evolution, duplicated genes could obtain new functions or segment existing functions to improve the adaptability of plants [34]. For instance, expansion of the RsHSF gene family was primarily driven by WGD or segmental duplication, which might be largely related with gene duplication [36]. It was previously reported that WGD and PDs event were mainly involved in the expansion of the CPA family in Pyrus bretschneideri [14]. In the present study, the predicted gene duplication was also found in radish among the CPA genes, indicating that the WGD event played an important role in the expansion of the CPA gene family (Figure 4b). In addition, Ka/Ks could be used to identify whether selective pressure existed on the RsCPA gene family, including positive, negative, and neutral selection. Herein, except RsCPA11-RsCPA23 and RsCPA21-RsCPA34, all of the RsCPA duplication genes displayed a Ka/Ks < 1, suggesting that they had experienced strong purifying selective pressure. A similar Ka/Ks was also reported in the Pyrus bretschneideri and cotton, further confirming that the evolutionary pattern of CPA genes was very conservative (Table S4) [14,37].
Combined with evolutionary classification and synteny analysis, a large number of the RsCPAs were identified as orthologous genes in Arabidopsis. For example, eleven pairs seemed to be single radish-to-Arabidopsis pairs, presuming that these genes might exist in the genome of the last common ancestor of the two species. There also existed more complex relationships, such as single radish and multiple Arabidopsis genes, one Arabidopsis and multiple radish genes, and two Arabidopsis and multiple radish genes, etc. This phenomenon was similar to the evolutionary relationship of bZIP genes in radish and Arabidopsis [38]. The close relationship of orthologous genes may exhibit similar functions in different species. For instance, AtNHX1 gene was reported high salt sensitivity [24], accordingly in our study its orthologues comparising RsCPA31, RsCPA32, and RsCPA34 also exhibited high induction under 250 Mm NaCl salt treatments ( Figure S5). The collinearity-orthologues analysis of radish and Arabidopsis could provide a valuable reference for further exploring the functions of these highly homologous genes in radish.

Roles of RsCPA Genes in Response to Different Abiotic Stresses
Increasing evidences indicated that CPA genes played vital roles in a variety of abiotic stresses, including HM, temperature, and salt exposure [39][40][41][42]. For instance, AtCHX17 mutant accumulated less K + than wide type under the salt stress and K + deficiency environment, indicating that AtCHX17 was involved in the absorption and transport of K + [28]. Among the CPA genes in other plants, it was NHX1 that could decrease the salt-tolerance ability of kallar grass at the concentration of 100 and 150 µM cadmium concentrations [43]. Moreover, NHX1 regulated Cd 2+ and H + flow during short-term Cd 2+ shock and confirmed that it could enhance tolerance during Cd 2+ stress [44]. Recent studies have shown that NHX2 homologues had a high expression under salinity stress at higher time intervals in G. barbadense and G. hirsutum [45]. The expressions of NHX were up-regulated in root tissues of wheat under salinity stress [46]. In this study, the transcriptome data of the radish taproot showed that nearly one-half of RsCPA genes displayed diverse expression profiles under HM, heat, and salt stress, indicating that they might play important roles in the plant response to abiotic stress. For example, RsCPA09 was significantly up-regulated under HM and heat stress, while exhibited down-regulated under salt stress, indicating that the expression of RsCPA09 might be a repress factor during salt stress. While RsCPA13 and RsCPA31 were all up-regulated under the HM and salt stress, indicating these two genes might play positive roles in response the various abiotic stresses of radish. A recent study in genome-wide identification of the Gossypium hirsutum NHX genes showed that most of the GhNHX genes were affected by salinity through salt-induced expression patterns analysis [47]. Here, according to the RT-qPCR analysis of RsNHX subfamily genes, we found all of them were significantly up-regulated under the 250 mM salt treatment, which indicated that the RsNHX genes may be the critical characters for the salt response of radish.

Potential Functions of RsNHXs Genes in Salt Stress
Emerging evidence indicates the Plant NHX proteins play critical roles for salt tolerance through biological function verification. For instance, over-expression of the soybean gene GmNHX1 in Arabidopsis thaliana could enhance salt tolerance through maintaining higher Na + efflux rate and K + /Na + ratio, while silencing it may cause soybean plants became more susceptible to salt stress [48]. Similar, over-expression of wheat TaNHX2 gene in transgenic sunflower improved salinity stress tolerance and growth performance, which showed better growth performance and accumulated higher Na + ,K + contents in leaves and roots under 200 mM NaCl salt stress [49]. Addtionally, a yeast functional complementation test proved that GhNHX4A can partially restore the salt tolerance of the salt-sensitive yeast mutant AXT3, while silencing it decreased the resistance of cotton [47]. In the present study, ectopic over-expression and inhibited-expression of the RsNHX1 gene in Arabidopsis significantly affected salt tolerance. Soil culture experiments showed that the growth of Arabidopsis OE-RsNHX1 responded more positively and amiR-RsNHX1 responded more negatively than the non-transgenic control plants. Furthermore, a TYMV-based VIGS system was used to functionally characterize the RsNHX1 gene, which was the first time to be employed for silencing the endogenous gene of radish. The expression levels of RsNHX1 gene were successfully silenced in pTY-RsNHX1 lines and the seedlings showed more salt damage than controls, which clarified that RsNHX1 may be a potential regulator in response to salt stress of radish.
According to our results, 60 CPA candidate genes of radish were firstly identified on the whole genome level. These genes could be clustered into three subfamilies, including nine RsNHXs, ten RsKEAs, and 41 RsCHXs. 58 genes were mapped to the nine chromosomes based on radish genome sequences. All the 60 RsCPA genes had various expression levels in the leaves, roots, cortex, cambium, and xylem at different development stages, as well as under various abiotic stresses. RT-qPCR analysis indicated that all nine RsNHXs genes showed upregulated trends after 250 mM NaCl exposure. The RsCPA31 (RsNHX1) gene, which might be the most important members of the RsNHX subfamily, exhibited obvious increased expression levels during 24h salt stress treatment. Heterologous over-expression and inhibited expression of RsNHX1 in Arabidopsis showed that RsNHX1 had a positive function in salt tolerance. Meanwhile, TYMV-based gene silence system was firstly used to functionally characterize the candidate gene in radish, and the silence of endogenous RsNHX1 in radish was more susceptible to the salt stress. The results would be useful to understand the complexity of the RsCPA gene family and could provide a valuable resource to explore the potential functions of RsCPA genes in radish.

Synteny Analysis and Chromosomal Localization
Synteny analysis was performed by the method described in the Plant Duplicate Gene Database (PlantDGD, http://pdgd.njau.edu.cn:8080/) [67]. The collinear block was identified by RsCPA duplication events in the MCScanX [68]. The data were integrated and plotted by using Circos [69]. Based on the annotation information from RGD and duplications of the RsCPA genes, the corresponding location distributions of RsCPA genes in chromosomes were displayed by TBtools [62].

Expression Analysis of RsCPAs Based on the RNA-Seq. Data
Illumina RNA-Seq. data were downloaded from the NODAI Radish Genome Database and used for the transcriptional profiling of RsCPA genes in five tissues (cortical, cambium, xylem, root tip, and leaf) and six stages (7,14,20,40,60, and 90 days after sowing) [70]. The RPKM method was used to analyze the expression level for each RsCPA gene and the heatmap was displayed by TBtools [62]. Furthermore, the expression patterns of RsCPA genes during abiotic stress, including heavy metal (HM, such as Cd, Cr and Pb), high temperature, and salt stress were extracted and analyzed from the transcriptome data of radish taproots [71][72][73][74][75].

Plant Material, Salt Stress Treatment, and RT-qPCR Expression Analysis
The radish variety 'NAU-XBC' was used, which is an advanced inbred line with white flesh and white skin in taproot, and sensitive to salt stress. The seeds were germinated at 25 • C in the dark for 3 days and then cultivated in the growth chamber with a day/night temperature of 25/18 • C (16/8 h), 60% humidity, and 12,000 lx light. The three-week-old radish seedlings were grown in a plastic container with half-strength Hoagland nutrient solution, as previously described [76]. One week later, seedlings were treated with 250 mM NaCl and the NaCl-free solution was used as a control. Leaves were collected in triplicate at 0, 3, 6, 12, 24, 48, and 96 h after NaCl treatment and immediately frozen in liquid nitrogen and stored at −80 • C. Total RNA was isolated using the TRIzol reagent RNAsimple total RNA kit (Tiangen, Beijing, China) and reverse transcribed into cDNA using the PrimeScript™ RT reagent kit (Takara, Dalian, China) according to the instructions. RT-qPCR analysis was carried out on a LightCycler ® 480 System (Roche, Mannheim, Germany). The 2 −∆∆CT formula was used to calculate the relative expression level [77]. RsActin was regarded as the internal reference gene. The primers used for RT-qPCR are listed in Table S5.
Arabidopsis thaliana (ecotype: Col-0) plants were used for heterologous over-expression and inhibited expression RsNHX1. In addition, the advanced inbred line 'NAU-YH' seedlings were used to perform the VIGS experiment [78].

Genetic Transformation and Generation of RsNHX1 Transgenic Lines in Arabidopsis
To generate over-expression lines, the ORF sequence of RsNHX1 was cloned into the pCAMBIA2301 vector, using with BamHI/KpnI restriction sites, and driven by a 35S promoter [79]. For amiRNA construction, the natural miR319a sequence as the backbone was exchanged with the amiRRsNHX1 via the overlapping PCR method [80]. The 418 bp specific sequence was inserted with the XbaI and SacI digestion sites and then transferred into the pCAMBIA2301 vector [76]. These fusion plasmids were introduced into Arabidopsis using A. tumefaciens-mediated transformation with the floral dip method [81,82]. Transgenic lines were screened by 1 2 Murashige and Skoog ( 1 2 MS) solid medium with 100 mg·L −1 kanamycin. Following, the 14-day-old wide type and transgenic line seedlings were planted in sterilized soil one week later and treated with 200 mM NaCl solution every other day [83]. Additionally, the seeds were sown on 1 2 MS solid media containing 0 and 100 mM NaCl to calculate the germination ratio as well as the root length after two weeks [24].

VIGS-Mediated Silencing of RsNHX1 in Radish
The VIGS experiment was conducted to functionally characterize RsCPA, as previously described [78]. The 40 bp specific sequence and its reverse complementary fragment of RsNHX1 and RsPDS were separately synthesized and phosphorylated, and inserted into the pTY-S vector by digestion with SnaBI and transformed to obtain positive clones. The primers used for vector construction to identify RsNHX1-silenced plants are listed in Table S6. The pTY-S empty vector and self-hybridized palindromic oligonucleotide of RsPDS-silencing vector were regarded as negative and positive controls, respectively. Particle bombardment was performed on the two-four fully expanded leaves from 'NAU-YH' plants using the PDS-1000/He bio-gun (Bio-Rad, Hercules, CA, USA) to trigger RsNHX1 silencing [84]. Five plants were bombarded in each experiment. Three weeks later, the inoculated plant leaf phenotype was observed and sampled to analyze the downstream gene level and silencing efficiency. RT-qPCR was used to further confirm RsNHX1-silencing. The primers used for RT-qPCR are listed in Table S5. Afterwards, the two positive plants were treated with 250 mM NaCl solution to observe the phenotypes.
Supplementary Materials: Supplementary Materials can be found at http://www.mdpi.com/1422-0067/21/21/ 8262/s1. Figure S1. LOGO of 20 amino acid motifs in RsCPA proteins. Figure S2. Number of cis-acting elements on promoters of RsCPA genes. Figure S3. Functional interaction networks of 60 RsCPA proteins. Figure S4. RNA-Seq of RsCPA genes under different treatments in the radish taproot. Figure S5. The expression levels of RsNHX genes at different times under 250 mM NaCl treatment. Figure S6. PCR analysis of over-expression and inhibited-expression in T 3 transgenic Arabidopsis plants. Table S1. Basic data for the CPA proteins in radish. Table S2. Number of transmembrane regions in RsCPA proteins. Table S3. Synteny blocks of CPA genes between radish and Arabidopsis genomes. Table S4. Ka/Ks analysis of 24 RsCPA duplicated gene pairs. Table S5. Specific primers of RsCPA genes for qRT-PCR. Table S6. Specific primers of RsNHX1 for vector construction.

Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.