RsSOS1 Responding to Salt Stress Might Be Involved in Regulating Salt Tolerance by Maintaining Na + Homeostasis in Radish ( Raphanus sativus L.)

: Radish is a kind of moderately salt-sensitive vegetable. Salt stress seriously decreases the yield and quality of radish. The plasma membrane Na + /H + antiporter protein Salt Overly Sensitive 1 (SOS1) plays a crucial role in protecting plant cells against salt stress, but the biological function of the RsSOS1 gene in radish remains to be elucidated. In this study, the RsSOS1 gene was isolated from radish genotype ‘NAU-TR17’, and contains an open reading frame of 3414 bp encoding 1137 amino acids. Phylogenetic analysis showed that RsSOS1 had a high homology with BnSOS1, and clustered together with Arabidopsis plasma membrane Na + /H + antiporter (AtNHX7). The result of subcellular localization indicated that the RsSOS1 was localized in the plasma membrane. Furthermore, RsSOS1 was strongly induced in roots of radish under 150 mmol/L NaCl treatment, and its expression level in salt-tolerant genotypes was signiﬁcantly higher than that in salt-sensitive ones. In addition, overexpression of RsSOS1 in Arabidopsis could signiﬁcantly improve the salt tolerance of transgenic plants. Meanwhile, the transformation of RsSOS1 (cid:52) 999 could rescue Na + efﬂux function of AXT3 yeast. In summary, the plasma membrane Na + /H + antiporter RsSOS1 plays a vital role in regulating salt-tolerance of radish by controlling Na + homeostasis. These results provided useful information for further functional characterization of RsSOS1 and facilitate clarifying the molecular mechanism underlying salt stress response in radish.


Introduction
Radish (Raphanus sativus L.), an essential root vegetable crop of the Brassicaceae family, playing an important role in vegetable production and supply. It has rich nutrition and high medicinal value and is very popular worldwide, especially in East Asia. Salt stress has become one of the major destructive abiotic stresses affecting crop growth, resulting in the decline of crop yield and quality [1]. It is predicted that by 2050, nearly half of the world's cultivated land will be salinized, posing a great threat to sustainable agricultural development and crop safety production [2]. Soil salinization and secondary salinization giving rise to salt stress could seriously affect the yield and quality of radish taproot. Therefore, the exploration and functional analysis of salt-tolerance-related genes in radish will provide a solid theoretical basis for further analyzing the mechanism underlying radish response to salt stress and developing salt-tolerant germplasm.
Plants display resistance to salt stress in several ways, including osmotic regulation, ion homeostasis regulation, hormone regulation, scavenging of reactive oxygen species (ROS) and signal transduction [3]. Reducing the content of Na + in the cytoplasm of plants is a vital way to maintain intracellular ion homeostasis under salt stress, mainly including reduction of Na + absorption, promotion of Na + efflux or Na + compartmentation [4]. Among them, exclusion and compartmentation of Na + are executed by Na + /H + antiporter proteins located on the plasma and vacuole membrane, respectively. The gene of the vacuolar membrane Na + /H + exchanger, RsNHX2, has been identified, which plays a key role in resistance to salt stress in radish [5]. The role of Salt Overly Sensitive 1 (SOS1), a plasma membrane Na + /H + antiporter, has been widely explored in salt tolerance. It eliminates Na + from cells under the action of plasma membrane H + -ATPase to reduce the accumulation of Na + in cells [6,7]. SOS1 homologues have been isolated in a variety of higher plant species, such as Sesuvium portulacastrum [8], Rice [9], Karelinia caspia [10], etc., while the isolation of the RsSOS1 gene and its functional response to salt stress in radish have not been reported.
In Arabidopsis, the SOS1 protein contains three conserved functional domains necessary to regulate its protein activity, namely Nhap, InhiBD and S2P under normal conditions, the inhibited region 'InhiBD' at the C-terminal of SOS1 combines with the adjacent functional active region 'Nhap' to inhibit its activity, and the Na + content in cells remains stable. When subjected to salt stress, the SOS signal transduction pathway is activated. Under this condition, the SOS2-SOS3 protein kinase complex phosphorylates the 1136th and 1138th serine sites of SOS1 S2P, which releases and activates the ion transport functional domain from the self-inhibitory state and plays the function of Na + /H + exchange transport under the action of H + ATPase [11]. However, the functional domain in RsSOS1 has not been explored.
Based on the previous transcriptome data, it was found that the RsSOS1 gene was significantly upregulated under salt stress [12], implying that RsSOS1 might play a significant role in the stress response to salt in radish. In this study, to explore molecular characteristics and function of the SOS1 gene in radish, the RsSOS1 gene was isolated and subcellular localization was analyzed. Additionally, the expression profiles of the RsSOS1 gene in different salt-tolerant radish genotypes under salt stress were investigated with real-time quantitative PCR. Furthermore, the biological function of RsSOS1 in salt stress response was investigated by overexpression in Arabidopsis and functional complement of yeast. The results of this study may facilitate elucidating the molecular mechanism of response to salt stress and improving the salt-tolerant traits in radish genetically.

Isolation of RsSOS1 Gene in Radish
Total RNA was extracted from the root tissue of radish advanced inbred line 'NAU-TR17' using the RNA Simple Total RNA Kit (Tiangen biotech Ltd. Co., Beijing, China). The cDNA was synthesized using PrimeScript RT reagent Kit (TaKaRa, Dalian, China). The RsSOS1 gene was isolated with the cDNA template. The primer RsSOS1-F/R was designed using homologous recombination primer-design software CEDesign (Vazyme Biotechnology Co., Ltd., Nanjing, China) according to the CDS sequence of RsSOS1 (XM_018601043.1) in NCBI (Table S1). The target fragment was combined with pCAM-BIA1300 vector (named 35S::RsSOS1-GFP) by the homologous recombination method, and then transferred into E.coli DH5α (TransGen Biotechnology Co., Ltd., Beijing, China) with the heat shock method [13]. The positive clones identified by PCR were sequenced (Springen Biotechnology Co., Ltd., Nanjing, China). The gene-specific primers for isolating RsSOS1 genomic DNA were designed according to the gDNA sequence of RsSOS1 in genomic database (Table S1) [14]. The PCR products were recovered and purified, and after the T-A cloning, the positive clones were then sequenced to obtain the full-length sequence of RsSOS1 gDNA.

Bioinformatic Analysis of RsSOS1
The CDS sequence of the RsSOS1 gene sequencing was translated into protein.
The transmembrane domain (http://www.cbs.dtu.dk/services/TMHMM/, accessed on 1 April 2021), physical and chemical properties (https://web.expasy.org/protparam/, accessed on 1 April 2021) and signal peptides (http://www.cbs.dtu.dk/services/SignalP-5.0/, accessed on 1 April 2021) of RsSOS1 were analyzed. To compare the genetic relationship of SOS1 from different species and identify the membrane localization of RsSOS1, a phylogenetic tree of 12 SOS1 proteins from different species coupled with eight different membrane located Arabidopsis Na + /H + antiporter proteins was mapped with Mega 6.0 software (Mega Limited, Auckland, New Zealand). It has been reported that AtNHX1-AtNHX6 were located on cell endomembrane, while AtNHX7 and AtNHX8 were located on plasma membrane [15,16]. An unrooted phylogenetic tree was generated subsequently with the neighbor-joining (NJ) method with: p-distance (Model/Method), pairwise deletion (Gaps/Missing Data treatment) and 1000 bootstrap replicates (Test of phylogeny) [17,18].

Identification of Salt Tolerance of Radish Genotypes
Using the radish advanced inbred lines, 'NAU-TR17' and 'NAU-TR12' as materials, plants with the same growth status were selected and hydroponic stress treatment was conducted with 250 mmol/L NaCl contained nutrient solution [20], and nutrient solution without NaCl was used as control. After treatment for 10 d, the relative values of growth indexes including relative plant height, root length, shoot fresh weight, root fresh weight and total biomass were calculated after measurement. The calculation was as follows: Relative value = measured value in the treatment group/measured value in the control group × 100%; Biomass (fresh weight/g) = shoot fresh weight + root fresh weight.

Expression Profiling of RsSOS1
Radish genotypes 'NAU-TR17' and 'NAU-TR12' were used as materials. Salt stress treatment with 150 mmol/L NaCl [13] was performed at seedling stage, and the non-NaCl treated material was used as control. The roots and leaves were collected at 0 h, 3 h, 6 h, 12 h, 24 h, 48 h and 72 h after treatment and three biological replicates were performed. Total RNA was extracted using the RNA Simple Total RNA Kit (Tiangen biotech Ltd. Co., Beijing, China). The cDNA was synthesized using PrimeScript RT reagent Kit (TaKaRa, Dalian, China). Primer-Blast program (https://www.ncbi.nlm.nih.gov/, accessed on 1 April 2021) was used to design real-time quantitative PCR (RT-qPCR) primers (RsSOS1-qRT-F/R, RsActinq-RT-F/R) (Table S1), and RsActin gene was used as an internal reference gene [21]. RT-qPCR was performed according to the instructions of SYBR Premix Ex Taq kit (TaKaRa, Dalian, China) and the reaction procedures were 95 • C for 10 min, 95 • C for 10 s, 56 • C for 10 s, 72 • C for 20 s and 50 • C for 30 s, 40 cycles were set from step 2 to step 4. The expression level was estimated using the 2 − CT method [22].

Heterologous Expression of RsSOS1 in Arabidopsis and Yeast
Engineered Agrobacterium carrying 35S::RsSOS1-GFP were activated and used to infect the inflorescence of Arabidopsis [23]. The positive transgenic plants were screened continuously on 1/2 Murashige Skoog medium containing 36 mg/L hygromycin to obtain the homozygous T 3 generation plants. Using DNA of wildtype (WT) and transgenic A. thaliana as templates, the transgenic positive plants were identified by amplification with pCAMBIA1300 vector universal primer. The expression of RsSOS1 was analyzed by RT-PCR. The wildtype and transgenic Arabidopsis plants were sown on 1/2 MS medium containing 0, 75 and 150 mmol/L NaCl, respectively, and the germination rate, root length and fresh weight (Fw) were counted and calculated.
To analyze the sodium transport function of RsSOS1 protein, AXT3 yeasts (endogenous vacuolar membrane Na + transporter NHX1, plasma membrane Na + transporter ENA1-4 and NHA1-deficient: nhx1::TRP1, ean1::HIS3::ena4, nha1::LEU2) were used for functional complement analysis (kindly provided by Professor Yu L., College of Resources and Environmental Sciences, Nanjing Agricultural University). The alignment was carried out between amino acid sequences of RsSOS1 and AtSOS1 to the conjectured self-activity inhibited region of RsSOS1. It was speculated that the self-activity inhibited region of RsSOS1 was located behind the 999th amino acid according to the known self-activity inhibited region of AtSOS1. The cloning primers of RsSOS1 and RsSOS1 999 were designed by the homologous recombination method, respectively (RsSOS1-JM-F/R, RsSOS1 999-JM-F/R) ( Table S1). The sequence of RsSOS1 999, which has been removed from the C-terminal inhibited region of RsSOS1, contains first 2997 bp nucleotide sequence of RsSOS1 plus the stop codon. The clone product of RsSOS1 and RsSOS1 999 were combined with yeast expression vector pYES2 (http://www.youbio.cn, accessed on 23 October 2021), respectively.
The pYES2 plasmid, recombinant plasmid pYES2-RsSOS1 and pYES2-RsSOS1 999 were transformed into AXT3 yeast by the lithium acetate method, respectively. These three kinds of transformed yeast were incubated overnight in SD-Ura liquid medium (Coolaber Technology Co., Ltd., Beijing, China), and then resuspended with sterile water after centrifugation, respectively. In total, 6 µL yeast liquid was added to the SC-Ura+Gal solid medium with concentration of 0, 35, 75, 150, 300 mmol/L NaCl after diluting these three kinds of yeast liquid according to the proportion of 1, 10 −1 , 10 −2 , 10 −3 , 10 −4 , respectively. Then, the growth of yeast was observed [24]. The yeast were collected and baked at 80 • C for 72 h after cultivating these three kinds of yeast for 48 h in SC-Ura+Gal liquid medium containing 0 and 75 mmol/L NaCl, respectively. Then, 0.1 g samples were dissolved to determine the contents of potassium and sodium ions by plasma emission spectrometer (iCAP7000 plus, Thermo Fisher, Waltham, MA, USA) [25].

Statistical Analysis
The experimental data were analyzed using SPPS (SPSS Inc., Chicago, IL, USA). Significance tests for differences between control and stress treatments were assessed at a p ≤ 0.05 level of significance. All experiments were performed and analyzed separately based on three biological replicates.

Isolation and Phylogenetic Analysis of RsSOS1 Gene
The cDNA coding sequence of the RsSOS1 gene with a length of 3414 bp was isolated with the cDNA from 'NAU-TR17', and the corresponding gDNA sequence of RsSOS1 is 6113 bp in length and contains 23 exons ( Figure S1). These sequences were deposited in GenBank with the Acc. No. MZ484951 and No. MZ484950, respectively. Sequence analysis suggested that RsSOS1 encoded 1137 amino acids with a relative molecular weight of 125.61 kDa and an isoelectric point of 6.61. The N-terminal region contains 12 transmembrane domains, with a long hydrophilic tail in the C-terminal part ( Figure S2). Moreover, the signal peptide does not exist in this protein.
Phylogenetic analysis showed that RsSOS1 has a high homology with BnSOS1 ( Figure 1), and is closely related to the plasma membrane Na + /H + antiporter of Arabidopsis (AtNHX7, AtNHX8) in evolutionary relationship, while is far less related to the vacuolar Na + /H + antiporters of Arabidopsis (AtNHX1-6), suggesting that RsSOS1 is a plasma membranelocated protein.

Subcellular Localization Analysis of RsSOS1
The subcellular localization of RsSOS1 protein in tobacco leaves was observed under laser confocal microscope ( Figure 2). The results indicated that the green fluorescence signal of 35S::RsSOS1-GFP was concentrated on the cell membrane, indicating that the RsSOS1 of radish plays corresponding functions in the cell membrane.

Identification of Salt Tolerance of Genotype 'NAU-TR17' and 'NAU-TR12'
The growth and development of genotype 'NAU-TR17' were normal after salt treatment (250 Mm NaCl), with no obvious yellowing leaves and normal of fibrous roots compared with the control plants. However, the genotype 'NAU-TR12' suffered serious damage after salt treatment, with retarded fibrous root growth and yellowing wilted leaves ( Figure 3A).

Subcellular Localization Analysis of RsSOS1
The subcellular localization of RsSOS1 protein in tobacco leaves was observed under laser confocal microscope ( Figure 2). The results indicated that the green fluorescence signal of 35S::RsSOS1-GFP was concentrated on the cell membrane, indicating that the RsSOS1 of radish plays corresponding functions in the cell membrane.

Subcellular Localization Analysis of RsSOS1
The subcellular localization of RsSOS1 protein in tobacco leaves was observed under laser confocal microscope ( Figure 2). The results indicated that the green fluorescence signal of 35S::RsSOS1-GFP was concentrated on the cell membrane, indicating that the RsSOS1 of radish plays corresponding functions in the cell membrane.

Identification of Salt Tolerance of Genotype 'NAU-TR17' and 'NAU-TR12'
The growth and development of genotype 'NAU-TR17' were normal after salt treatment (250 Mm NaCl), with no obvious yellowing leaves and normal of fibrous roots compared with the control plants. However, the genotype 'NAU-TR12' suffered serious damage after salt treatment, with retarded fibrous root growth and yellowing wilted leaves ( Figure 3A).

Identification of Salt Tolerance of Genotype 'NAU-TR17' and 'NAU-TR12'
The growth and development of genotype 'NAU-TR17' were normal after salt treatment (250 Mm NaCl), with no obvious yellowing leaves and normal of fibrous roots compared with the control plants. However, the genotype 'NAU-TR12' suffered serious damage after salt treatment, with retarded fibrous root growth and yellowing wilted leaves ( Figure 3A). The relative plant height, the relative underground fresh weight and the relative biomass of 'NAU-TR17' remained at a high level under 250 mmol/L NaCl condition. However, the relative value of 'NAU-TR12' decreased seriously, and the plant growth was seriously inhibited ( Figure 3B). It could be concluded that genotype 'NAU-TR17' has strong salt tolerance, while genotype 'NAU-TR12' is sensitive to salt stress.

Expression Profiling of RsSOS1 under Salt Stress
The expression level of the RsSOS1 gene in the salt-tolerant genotype 'NAU-TR17' root was upregulated after 6-72 h of salt treatment (Figure 4). In the leaves of 'NAU-TR17', there was no significant difference in expression level within 12 h after salt treatment, and the expression level was downregulated after 12 h. In the salt-sensitive genotype 'NAU-TR12' root, the expression level of RsSOS1 was significantly upregulated only at 12 h to 24 h after salt treatment, while the expression difference was not significant during other treatment. In 'NAU-TR12' leaves, the expression level of RsSOS1 was upregulated at 48 h after salt treatment, while the expression difference was not significant during other treatments. The significance of differential expression level of RsSOS1 gene in roots of salt sensitive genotype 'NAU TR12' was far less than that in salt tolerant genotype 'NAU TR17' under salt stress, implying that the RsSOS1 gene contributed greatly to the salt tolerance of radish. The relative plant height, the relative underground fresh weight and the relative biomass of 'NAU-TR17' remained at a high level under 250 mmol/L NaCl condition. However, the relative value of 'NAU-TR12' decreased seriously, and the plant growth was seriously inhibited ( Figure 3B). It could be concluded that genotype 'NAU-TR17' has strong salt tolerance, while genotype 'NAU-TR12' is sensitive to salt stress.

Expression Profiling of RsSOS1 under Salt Stress
The expression level of the RsSOS1 gene in the salt-tolerant genotype 'NAU-TR17' root was upregulated after 6-72 h of salt treatment (Figure 4). In the leaves of 'NAU-TR17', there was no significant difference in expression level within 12 h after salt treatment, and the expression level was downregulated after 12 h. In the salt-sensitive genotype 'NAU-TR12' root, the expression level of RsSOS1 was significantly upregulated only at 12 h to 24 h after salt treatment, while the expression difference was not significant during other treatment. In 'NAU-TR12' leaves, the expression level of RsSOS1 was upregulated at 48 h after salt treatment, while the expression difference was not significant during other treatments. The significance of differential expression level of RsSOS1 gene in roots of salt sensitive genotype 'NAU TR12' was far less than that in salt tolerant genotype 'NAU TR17' under salt stress, implying that the RsSOS1 gene contributed greatly to the salt tolerance of radish.

Overexpression of RsSOS1 in Arabidopsis to Improve Salt Stress Tolerance
PCR amplification results showed that the RsSOS1 was successfully transferred into Arabidopsis ( Figure 5A). RT-PCR results indicated that RsSOS1 could be expressed in transgenic plants ( Figure 5B). The germination rates of three transgenic Arabidopsis lines with different overexpression levels (OE1-OE3) and wildtype (WT) were compared under 0, 75 and 150 mmol/L NaCl treatment. There was no significant difference in germination rate between WT and OE1-OE3 in the control medium, while the germination rate was totally decreased under NaCl stress at 75 mmol/L and 150 mmol/L ( Figure 5C). The germination rates of WT and OE1-OE3 under NaCl treatment at 75 mmol/L were 82.82% and 90.71-94.29%, respectively. Under NaCl treatment at 150 mmol/L, the germination rates of OE1-OE3 were 82.14-84.52% but higher than that of control (71.42%) ( Figure 5D). It could be concluded that overexpression of RsSOS1 gene could increase the seed germination rates under salt stress.
When compared with transgenic plants, the WT plants showed more wilted and yellow leaves under NaCl condition at 150 mmol/L ( Figure 5E). The growth of WT and transgenic plants was inhibited under NaCl treatment at 75 mmol/L, but the fresh weight of OE-RsSOS1 transgenic plants was higher than that of WT, and root length was longer than that of WT ( Figure 5F, G), indicating that the overexpression of RsSOS1 gene could improve the salt tolerance of transgenic plants.

Overexpression of RsSOS1 in Arabidopsis to Improve Salt Stress Tolerance
PCR amplification results showed that the RsSOS1 was successfully transferred into Arabidopsis ( Figure 5A). RT-PCR results indicated that RsSOS1 could be expressed in transgenic plants ( Figure 5B). The germination rates of three transgenic Arabidopsis lines with different overexpression levels (OE1-OE3) and wildtype (WT) were compared under 0, 75 and 150 mmol/L NaCl treatment. There was no significant difference in germination rate between WT and OE1-OE3 in the control medium, while the germination rate was totally decreased under NaCl stress at 75 mmol/L and 150 mmol/L ( Figure 5C). The germination rates of WT and OE1-OE3 under NaCl treatment at 75 mmol/L were 82.82% and 90.71-94.29%, respectively. Under NaCl treatment at 150 mmol/L, the germination rates of OE1-OE3 were 82.14-84.52% but higher than that of control (71.42%) ( Figure 5D). It could be concluded that overexpression of RsSOS1 gene could increase the seed germination rates under salt stress.
When compared with transgenic plants, the WT plants showed more wilted and yellow leaves under NaCl condition at 150 mmol/L ( Figure 5E). The growth of WT and transgenic plants was inhibited under NaCl treatment at 75 mmol/L, but the fresh weight of OE-RsSOS1 transgenic plants was higher than that of WT, and root length was longer than that of WT ( Figure 5F,G), indicating that the overexpression of RsSOS1 gene could improve the salt tolerance of transgenic plants.

Functional Complement Analysis of RsSOS1 Protein in AXT3 Yeast
On the medium with the supplement of 0 and 35 mmol/L NaCl, there was no significant difference among the growth of pYES2, RsSOS1 and RsSOS1△999 transformed yeast ( Figure 6). Under the conditions of 75, 150 or 300 mmol/L NaCl treatment, there was no significant difference between the growth of pYES2 and RsSOS1 transformed yeast, and the growth became weaker with the increase of salt concentration. When the salt concentration reached 300 mmol/L, these two yeast transformers could hardly grow. With the increase of salt concentration, the growth of RsSOS1△999 transformed yeast was significantly better than that of pYES2 and RsSOS1 transformed yeast, and it could grow well when the concentration of NaCl was up to 300 mmol/L. These findings indicate that transfection of RsSOS1△999 could restore the growth of the dysfunctional yeast mutant AXT3 under high salt conditions.

Functional Complement Analysis of RsSOS1 Protein in AXT3 Yeast
On the medium with the supplement of 0 and 35 mmol/L NaCl, there was no significant difference among the growth of pYES2, RsSOS1 and RsSOS1 999 transformed yeast ( Figure 6). Under the conditions of 75, 150 or 300 mmol/L NaCl treatment, there was no significant difference between the growth of pYES2 and RsSOS1 transformed yeast, and the growth became weaker with the increase of salt concentration. When the salt concentration reached 300 mmol/L, these two yeast transformers could hardly grow. With the increase of salt concentration, the growth of RsSOS1 999 transformed yeast was significantly better than that of pYES2 and RsSOS1 transformed yeast, and it could grow well when the concentration of NaCl was up to 300 mmol/L. These findings indicate that transfection of RsSOS1 999 could restore the growth of the dysfunctional yeast mutant AXT3 under high salt conditions.

Determination of Ion Content in Transgenic Yeast
There was no significant difference in the content of Na + or K + among RsSOS1, RsSOS1△999 and pYES2 transformed yeast under normal conditions. However, when the content of Na + in yeast increased, K + content decreased under 75 mmol/L NaCl treatment (Figure 7). Under 75 mmol/L NaCl treatment, there was no significant difference in K + or Na + contents between transformers pYES2 and RsSOS1, while RsSOS1△999 strains had higher K + content, lower Na + content and higher K + /Na + ratio, indicating that the first 999 amino acids of RsSOS1 contain Na + efflux functional structures, which played a critical role in maintaining intracellular ion homeostasis. Error bars indicate SD, letters represent significant differences at p < 0.05 level based on Duncan's test.

Discussion
The Na + /H + antiporter encoded by the SOS1 gene enables plants to adapt to the saline environment [26]. The ORF length of SOS1 genes in most plant is 3410 bp-3500 bp, encoding 1129-1169 amino acids, with a relative molecular weight of about 127 kDa and 10-12 transmembrane domains [27]. The results of RsSOS1 sequence analysis in this study are consistent with those of previous studies. Subcellular localization indicated that RsSOS1 was localized on the cell membrane. Phylogenetic tree analysis suggested that RsSOS1 was closely related to plasma membrane Na + /H + antiporters in evolutionary relationship, but far less related to inner membrane Na + /H + antiporters, suggesting that RsSOS1 was a plasma membrane Na + /H + antiporter, and played a corresponding role of maintaining Na + /K + ion homeostasis in cell membrane. Previous studies have indicated

Determination of Ion Content in Transgenic Yeast
There was no significant difference in the content of Na + or K + among RsSOS1, RsSOS1 999 and pYES2 transformed yeast under normal conditions. However, when the content of Na + in yeast increased, K + content decreased under 75 mmol/L NaCl treatment (Figure 7). Under 75 mmol/L NaCl treatment, there was no significant difference in K + or Na + contents between transformers pYES2 and RsSOS1, while RsSOS1 999 strains had higher K + content, lower Na + content and higher K + /Na + ratio, indicating that the first 999 amino acids of RsSOS1 contain Na + efflux functional structures, which played a critical role in maintaining intracellular ion homeostasis.

Determination of Ion Content in Transgenic Yeast
There was no significant difference in the content of Na + or K + among RsSOS1, RsSOS1△999 and pYES2 transformed yeast under normal conditions. However, when the content of Na + in yeast increased, K + content decreased under 75 mmol/L NaCl treatment (Figure 7). Under 75 mmol/L NaCl treatment, there was no significant difference in K + or Na + contents between transformers pYES2 and RsSOS1, while RsSOS1△999 strains had higher K + content, lower Na + content and higher K + /Na + ratio, indicating that the first 999 amino acids of RsSOS1 contain Na + efflux functional structures, which played a critical role in maintaining intracellular ion homeostasis. Error bars indicate SD, letters represent significant differences at p < 0.05 level based on Duncan's test.

Discussion
The Na + /H + antiporter encoded by the SOS1 gene enables plants to adapt to the saline environment [26]. The ORF length of SOS1 genes in most plant is 3410 bp-3500 bp, encoding 1129-1169 amino acids, with a relative molecular weight of about 127 kDa and 10-12 transmembrane domains [27]. The results of RsSOS1 sequence analysis in this study are consistent with those of previous studies. Subcellular localization indicated that RsSOS1 was localized on the cell membrane. Phylogenetic tree analysis suggested that RsSOS1 was closely related to plasma membrane Na + /H + antiporters in evolutionary relationship, but far less related to inner membrane Na + /H + antiporters, suggesting that RsSOS1 was a plasma membrane Na + /H + antiporter, and played a corresponding role of maintaining Na + /K + ion homeostasis in cell membrane. Previous studies have indicated Figure 7. K + /Na + ion content in transgenic yeast: (A) Na + content; (B) Na + content; (C). K + /Na + ratio. Error bars indicate SD, letters represent significant differences at p < 0.05 level based on Duncan's test.

Discussion
The Na + /H + antiporter encoded by the SOS1 gene enables plants to adapt to the saline environment [26]. The ORF length of SOS1 genes in most plant is 3410-3500 bp, encoding 1129-1169 amino acids, with a relative molecular weight of about 127 kDa and 10-12 transmembrane domains [27]. The results of RsSOS1 sequence analysis in this study are consistent with those of previous studies. Subcellular localization indicated that RsSOS1 was localized on the cell membrane. Phylogenetic tree analysis suggested that RsSOS1 was closely related to plasma membrane Na + /H + antiporters in evolutionary relationship, but far less related to inner membrane Na + /H + antiporters, suggesting that RsSOS1 was a plasma membrane Na + /H + antiporter, and played a corresponding role of maintaining Na + /K + ion homeostasis in cell membrane. Previous studies have indicated that OsSOS1 was highly induced in the root after 15 h of salt stress treatment in rice [28]. In tuber mustard, both the expression levels of BjSOS1-1 and BjSOS1-2 were significantly induced in the root by salt treatment at 12 h [29], and the expression induction of PabSOS1 was also found in Populus [30]. It was also found that SOS1 was mainly induced in the root under salt stress in Medicago sativa [31] and Cucurbita maxima [32]. In this study, the RsSOS1 gene was highly induced in root after 6 h of salt stress treatment in the salt-tolerant genotype 'NAU-TR17' taproot, which is in accordance with the reported results [28][29][30][31][32]. However, the SOS1 gene of Populus and Mesembryanthemum is mainly upregulated in leaves [33,34]. Those results indicated that the functions of SOS1 gene are relatively conserved and have various organ expression specificity among different species. In addition, the expression level of the RsSOS1 gene in roots of the salt-sensitive genotype 'NAU-TR12' was far less than that in the salt-tolerant genotype 'NAU-TR17' under salt stress. It is speculated that the higher RsSOS1 gene expression level of 'NAU-TR17' radish (salt tolerant) makes its root cells have a stronger ability to excrete sodium ions, so as to reduce the toxic effect of sodium ion accumulation in cells, which suggests that the expression level of the RsSOS1 gene may be mainly responsible for the difference in salt tolerance among radish genotypes.
Since the first identification of the Na + /H + antiporter gene SOS1 in A. thaliana [35], the function of the SOS1 gene in several plants has been investigated. Overexpression of the TrSOS1 gene in Tamarix can supplement salt sensitivity of sos1 mutants [36]. In addition, overexpression of cotton GhSOS1, soybean GmSOS1 or S. portulacastrum SpSOS1 in Arabidopsis could improve salt tolerance in transgenic plants [37][38][39]. In this study, through overexpression of the RsSOS1 gene in Arabidopsis, it was found that transgenic seeds had higher germination rates, fresh weights and longer roots under salt stress compared with WT Arabidopsis, which was consistent with the results in several other crops including cotton and soybean. In the Salt Overly Sensitive pathway, the calcium-ion binding protein SOS3 senses and binds to the calcium signal caused by salt stress, and activates the Ser/threonine protein kinase SOS2, which phosphorylates the Ser residue in the DSPS unit at the C-terminal of SOS1 protein, then the functional active region of SOS1 protein is released, so that the Na + can be excreted from cells [11,35]. In this study, overexpression of RsSOS1 improved salt tolerance of transgenic Arabidopsis, which may be due to the fact that radish RsSOS1 protein has a DSPS structure being similar to that of AtSOS1 protein. When transgenic Arabidopsis is subjected to salt stress, AtSOS2 was activated by AtSOS3, and then phosphorylated the DSPS region of exogenous RsSOS1 to activate its ion transport activity. The function of RsSOS1 is very similar to that of the SOS1 gene in other plants, suggesting that the contribution of SOS1 to plant salt tolerance is conserved. RsSOS1 mainly participates in the enhancement of Na + extrusion and the regulation of Na + and K + homeostasis in plant roots under salt stress, thus improving root vigor, reducing cell membrane damage and maintaining normal plant growth [40]. Plant salt tolerance is a quantitative trait controlled by multiple genes, involving a complex regulatory network of many signaling pathways. The salt stress response mechanism of plants is more complicated than in the single-celled organism yeast, which needs to be further studied.
More evidence has demonstrated that SOS1 with a short cut in the autoinhibitory C-terminal domain can confer yeast mutant AXT3 with strong salt tolerance [11]. It is speculated that radish RsSOS1 also has a similar mechanism. In this study, homologous comparison was conducted between the amino acid sequence of radish RsSOS1 and AtSOS1, and it was inferred that the autoinhibitory region of RsSOS1 was located behind the 999th amino acid according to the known autoinhibitory domain of AtSOS1. Through the yeast functional complement analysis, it was found that the transferred RsSOS1 could not compensate the defective function of Na + efflux of the yeast mutant AXT3, which was consistent with the results in Bruguiera gymnorrhiza [41]. While the SOS1 of Arabidopsis, rice and wheat could partially compensate the defective function of yeast mutant AXT3 [9,11,42]. After removing the autoinhibitory region in SOS1 of the above species, AXT3 yeast could recover growth with lower Na + content, higher K + content and a higher K + /Na + ratio under severe salt stress conditions. Similar results were obtained in this study. In conclusion, the Na + /H + antiporter domain exists before the 999th amino acid of RsSOS1, which performs the function of sodium ion exportation and transport in cells under salt stress, laying a foundation for the further investigation of the functional domain of RsSOS1. When exogenous RsSOS1 was transferred into AXT3, it was speculated that due to the lack of homologous genes to activate RsSOS1 in yeast, the ion transport functional domain could not be released. However, the RsSOS1 999 was in a hyperactive state without auto inhibitory region, so it could effectively compensate the defective function of AXT3 yeast. The RsSOS1 protein without the autoinhibitory region acts as a super-active ion transporter, and could be used as a new salt-tolerance gene in the genetic improvement of salt tolerance traits in crops. This result would facilitate clarifying the mechanisms underlying salt tolerance, and the breeding of elite salt-tolerant cultivars of radish.