Genome-wide identification and gene expression analysis of SOS family genes in tuber mustard (Brassica juncea var. tumida)

The Salt Overly Sensitive (SOS) pathway in Arabidopsis thaliana plays important roles in maintaining appropriate ion homeostasis in the cytoplasm and regulating plant tolerance to salinity. However, little is known about the details regarding SOS family genes in the tuber mustard crop (Brassica juncea var. tumida). Here, 12 BjSOS family genes were identified in the B. juncea var. tumida genome including two homologous genes of SOS1, one and three homologs of SOS2 and SOS3, two homologs of SOS4, two homologs of SOS5 and two homologs of SOS6, respectively. The results of conserved motif analysis showed that these SOS homologs contained similar protein structures. By analyzing the cis-elements in the promoters of those BjSOS genes, several hormone- and stress-related cis-elements were found. The results of gene expression analysis showed that the homologous genes were induced by abiotic stress and pathogen. These findings indicate that BjSOS genes play crucial roles in the plant response to biotic and abiotic stresses. This study provides valuable information for further investigations of BjSOS genes in tuber mustard.


Introduction
Tuber mustard, Brassica juncea var. tumida (AABB, 2n = 36), which belongs to Brassicaceae, is an allotetraploid species that was produced from a natural cross between B. rapa (AA, 2n = 20) and B. nigra (BB, 2n = 16), followed by chromosome doubling [1]. It is an important vegetable in China and some Southeast Asian countries, as it serves as the raw material for Fuling mustard and is also famous for its special flavor and nutritional value. However, during growth and development, tuber mustard frequently suffers from abiotic and biotic stresses, such as salinity and pathogens, resulting in the inhibition of plant growth and huge economic loss. Therefore, determining the mechanisms underlying its resistance to salt stress will be helpful for improving the production of this vegetable.
The Salt Overly Sensitive (SOS) signaling pathway plays important roles in the plant response to salt stress and has three components: SOS1, SOS2 and SOS3. SOS1 encodes a Na + / H + anti-transport protein in the cell membrane, which transports excess Na + from the PLOS  cytoplasm to the extracellular region [2]; SOS2 encodes a serine/threonine protein kinase; and SOS3 encodes a Ca 2+ binding protein [3,4]. Under salt stress, the concentration of Ca 2+ in the cytoplasm immediately increases and is perceived by SOS3. SOS3 further activates SOS2 protein kinase by combining with Ca 2+ . SOS2 can interact with SOS3 and forms the SOS2-SOS3 complex to regulate the expression of SOS1 by phosphorylation. The transport activity of SOS1 is activated by SOS2-SOS3, and excess Na + is discharged to alleviate the toxic effects of Na + on cells [4][5][6]. Besides that, AtSOS4, AtSOS5 and AtSOS6 were also identified using a root-bending assay for the regulation of ion homeostasis and cell expansion under salt stress [7][8][9][10]. AtSOS4 encodes a pyridoxal kinase which is involved in the biosynthesis of vitamin B6 and regulates Na + and K + homeostasis [7,8]. AtSOS5 encodes a putative cell surface adhesion protein and is required for normal cell expansion [9]. The root tips of sos5 mutant swell and root growth is arrested [9]. AtSOS6 encodes a cellulose synthase-like protein, AtCSLD5 [10].
The sos6-1 mutant shows hypersensitive to salt stress and osmotic stress, and accumulates high level of reactive oxygen species (ROS) [10].
The Arabidopsis mutants sos1, sos2 and sos3 are hypersensitive to Na + and Li + , indicating that SOS proteins are involved in the regulation of plant tolerance to salinity [11]. Transgenic Arabidopsis seedlings overexpressing SOS1 show enhanced tolerance to salt stress and less Na + content compared to wild-type plants under NaCl treatment [12]. The lateral root development of the sos3-1 mutant shows increased sensitivity even at low salt concentration, confirming that the SOS signaling pathway also modulates organ development in response to salt stress [13]. Overexpression of B. juncea SOS3 (BjSOS3) in the Arabidopsis mutant sos3 complements the sos3 mutant phenotype and transgenic plants exhibit enhanced tolerance to salinity, indicating that BjSOS3 has conserved function with AtSOS3 in regulating plant resistance to salt stress [13,14]. However, the role of the SOS gene family in tuber mustard remains mostly unknown. Therefore, we identified the SOSs genes and elucidated their putative role for better understanding of SOS signaling pathway in tuber mustard (Brassica juncea var. tumida).
In this study, we identified 12 SOS family genes in the B. juncea var. tumida genome. Based on the analysis of phylogenic relationship, gene structures, protein motifs, and promoter ciselements, similar gene characteristics were found between BjSOS and AtSOS. In addition, we analyzed the transcript levels of BjSOS family genes under biotic and abiotic stresses, including NaCl, ABA, low temperature, and the pathogen Plasmodiophora Brassicae. The results showed that BjSOS genes were induced by abiotic stresses and pathogen in tuber mustard. The findings indicate that SOS family genes play crucial roles in the plant response to biotic and abiotic stresses. The findings not only are helpful for further understanding of the SOS signaling pathway but also provide clues about the defense responses of tuber mustard against different stresses.

Materials and growth conditions
The tuber mustard cultivar Yong'an was used in this study. The seeds were surface sterilized and plated on MS medium (Sigma-Aldrich, St. Louis, MO, USA) with 1% sucrose and 8 g/L agar (Sigma-Aldrich, St. Louis, MO, USA) and then cultivated in a growth room at 22˚C and 6000 lx under long-day conditions (16 h light/8 h dark). To analyze the gene expression patterns of BjSOS genes under abiotic stresses, 1-week-old seedlings grown on MS medium were treated with 50 μM ABA, 200 mM NaCl and low temperature (4˚C) for the indicated time points. To analyze the gene expression patterns of BjSOS genes under biotic stress, 2-week-old seedlings were irrigated with P. brassicae suspension liquid (OD 600 = 0.07) for 0, 0.25, 0.5, 1, 3, 5, 7, and 9 days.

Gene expression analysis
Total RNA was extracted from tuber mustard seedlings that had been subjected to NaCl, ABA, low temperature, and pathogen treatment using TRIzol reagent (Invitrogen, Carlsbad, CA, USA; Catalog No. 15596026). The RNA samples were used for cDNA synthesis using the cDNA synthesis Supermix with gDNA remover kit (Transgen Biotech, China; Catalog No. AT301) following the manufacturer's instructions. qRT-PCR was carried out using SYBR Green qPCR Supermix (Invitrogen, Carlsbad, CA, USA; Catalog No. 4309155). The transcript abundance was calculated by the comparative C T (cycle threshold) method, and BjActin3 was used as the internal control. The qRT-PCR experiments were carried out three times with three replicates each. The primers used in this study were listed in S1 Table.

Statistical analysis
All data were analyzed using SigmaPlot 10.0 (Systat Software, Inc., Chicago, IL) and SPSS 16.0 software. The averages and standard deviations of all results were calculated, and for multiple groups of samples, the one-way ANOVA followed by the Dunnett test was used. The statically significant treatments were marked with ' ��� ' (P<0.001), ' �� ' (P<0.01) and ' � ' (P<0.05).

Genome-wide identification and characterization of SOS homologs in B. juncea var. tumida
Twelve genes as homologs of SOS genes were identified in B. juncea var. tumida genome through BLASTP in Brassica database using six AtSOS protein sequences as references ( Table 1). The gene lengths ranged from 1191 bp to 5965 bp with 1-23 exons in each sequence. The protein lengths of these twelve SOS homologs ranged from 180 (BjSOS3-3) to 1192 (BjSOS6-2) amino acid (aa) residues. The relative molecular weights of those proteins varied from 20.59 kD (BjSOS3-3) to 133.61 kD (BjSOS6-2), and the isoelectric point (PI) ranged from 4.90 to 9.25 ( Table 1). The twelve BjSOS genes were distributed in 9 of the 18 chromosomes of B. juncea var. tumida. Each of the chromosomes A02, A04, A06, A10, B01, and B04 contained one gene, and each of the chromosomes A09, B02 and B03 contained two genes (Fig 1).

Phylogenic analysis and gene structures of SOS family genes
To analyze the evolutionary relationships between BjSOSs and AtSOSs, a phylogenetic tree was constructed using MEGA5 software with the neighbor-joining method. According to the phylogenic tree, twelve BjSOS genes with six AtSOS genes were identified and clustered into six clades. The first clade was AtSOS1 and two homologs BjSOS1-1 and BjSOS1-2; the second clade was AtSOS4 and two homologs BjSOS4-1 and BjSOS4-2; the third clade was AtSOS5 and its two homologs BjSOS5-1 and BjSOS5-2; the fourth clade was AtSOS6 and its two homologs BjSOS6-1 and BjSOS6-2; the fifth clade was AtSOS2 and its homolog BjSOS2; and the last clade was AtSOS3 and its homologs BjSOS3-1, BjSOS3-2 and BjSOS3-3 (Fig 2). SOS family genes in the same subfamilies may have similar functions. To understand their gene structures, we analyzed the gene exon-introns using the GSDS2.0 online server. According to the results, AtSOS1, BjSOS1-1 and BjSOS1-2 all had 23 exons and 22 introns; AtSOS2 and BjSOS2 contained 13 and 12 exons, respectively; AtSOS3, BjSOS3-1 and BjSOS3-2 all contained eight exons with the exception of BjSOS3-3 (7 exons); AtSOS4, BjSOS4-1 and BjSOS4-2 contained 13 and 12 exons, respectively; AtSOS5, BjSOS5-1 and BjSOS5-2 all contained one exon; AtSOS6, BjSOS6-1 and BjSOS6-2 all contained 3 exons (Fig 2). These results indicated that the homologs clustered into the same subfamily had similar gene structures and might have conserved functions.

Promoter cis-acting regulatory elements prediction of SOS homologs
To further understand the potential roles of SOS homologs and how their gene expression is regulated, we chose the 2000 bp DNA fragment upstream of the ATG start codon as the promoter sequences and performed promoter cis-element analysis using PlantCARE and PLACE online software. According to the results, the promoters of all BjSOS genes, except BjSOS2, contained at least one hormone-related elements such as the ABRE (ACGTG, responsive to abscisic acid stress) [17], p-box (CCTTTTG, responsive to gibberellin) [18], and CGTCA motif (CGTCA, responsive to MeJA) [19] (Fig 5 and S2 Table). In addition, the promoters of the SOS homologs contained at least eight stressed-related elements such as the DRE (GCCGAC, responsive to dehydration) [20], MBS (CAACTG, responsive to dehydration stress) [21], TCrich repeats (GTTTTCTTAC, responsive to defense and stress) [22], LTR (CCGAAA, responsive to low temperature) [23], and GT1GMSCAM4 motif (GAAAAA, responsive to pathogen and salt stress) [24] (Fig 5 and S2 Table). Together, the promoters of SOS homologs contained diverse cis-elements responsive to ABA, auxin, GA, SA, and abiotic stresses, indicating that the genes expression of SOS homologous were regulated by hormone and abiotic stresses, and they might play a role in regulating tuber mustard response to hormone and stresses.

Tissue specific expression pattern analysis of SOS homologs genes
To investigate the tissue specific expression patterns of SOS homologs, we analyzed the gene expression levels at different growth stages and tissues (root, stem, swollen stem, leaf, pod and inflorescence) using qRT-PCR. The results showed that the SOS1 homolog BjSOS1-2 was highly expressed in the root, leaf, and inflorescence. In contrast, the expression level of another homolog BjSOS1-1 was very low, with nearly no expression in pod; BjSOS2 was highly expressed in the stem; the SOS3 homologs BjSOS3-1 and BjSOS3-2 were highly expressed in the leaf, pod, and inflorescence, whereas there was very low BjSOS3-3 expression in B. juncea var. tumida (Fig 6). BjSOS4-1 and BjSOS5-1 was highly expressed in stem; BjSOS5-2 was highly expressed in leaf and flower; and the expression level of BjSOS6-1 and BjSOS6-2 were high in almost all the tissues (Fig 6). According to the results, the expression levels of the SOS homologs varied in different tissues and organs, indicating that they may play different roles in different tissues. In addition, the different expression patterns of the same gene in different tissues and organs suggest that the expression pattern of the genes was existence of space-time specificity.

Discussion
The SOS signaling pathway plays important roles in the plant response to salt stress; however, the identity and expression patterns of B. juncea var. tumida SOS genes are unknown. Here, a total of twelve BjSOS genes were identified and located in 9 of 18 chromosomes (Fig 1). There was high sequence identity between BjSOSs and their relative homologs in A. thaliana, and they shared the same protein motifs (Fig 4). The expression patterns of BjSOS genes indicated that they might play specific roles in different tissues and stress conditions. These results shed light on the roles of BjSOS genes in regulating plant growth and response to abiotic and biotic stresses, which will be helpful for improving the production of tuber mustard.
In A. thaliana, six SOS genes SOS1, SOS2, SOS3, SOS4, SOS5 and SOS6 were identified [2][3][4][5][7][8][9][10]. According to our results, twelve SOS homologs were found in the genome of B. juncea var. tumida. There were more members of the SOS genes in the B. juncea var. tumida genome than in A. thaliana, most likely because B. juncea var. tumida is an allotetraploid species that resulted from the hybridization between B. rapa and B. nigra following with genome duplication [1]. The comparable homologous gene number in the A sub-genome and B sub-genome indicated the B. juncea var. tumida genome experienced co-linearity gene duplication. However, the homologous genes of AtSOS2 were lost or not duplicated in B. juncea var. tumida, suggesting that these homologous genes may have had functional redundancy or divarication during the evolutionary process. The loss of genes during the genome duplication event has also frequently occurred in other Brassica species, such as chitinase gene family in B. rapa [25].
The cis-elements and functional characterization of the promoters of SOS genes have been identified in many species such as Salicornia brachiate, B. juncea, and A. thaliana [26][27][28]. In    this study, we analyzed the promoter cis-elements of SOS homologs in B. juncea var. tumida, and found that all of the promoters contained diverse cis-elements responsive to plant hormones (ABA, auxin, GA, and SA) and abiotic stresses (drought, cold, and salt stresses) (Fig 5). These promoter cis-elements of SOS genes in B. juncea var. tumida are in accordance with previous studies in other species [26][27][28], indicating that the expression patterns of SOS homologs were regulated by hormone and abiotic stresses, and that these homologs might play roles in regulating the tuber mustard response to hormone and abiotic stresses.
To date, although the expression patterns of SOS family genes have been determined in other species, such as wheat, Arabidopsis and Brassica [12,14,[27][28][29][30], no detailed studies on the expression of BjSOS genes were found. Here, tissue specific expression pattern analysis results revealed that majority of BjSOS genes expressed in various tissues, such as root, stem, pod, leaf and flower (Fig 6). Notably, some genes highly expressed in root, stem and leaf, pointing to the important roles of BjSOS genes in those tissues and the diverse biological functions of different BjSOS genes in B. juncea var. tumida. In Arabidopsis, AtSOS1 promoter-driven GUS expression was mainly found in root, inflorescence and leaf [31], similar tissue specific expression patterns were also found for BjSOS1 genes, suggesting that SOS1 genes played conserved functions in Arabisopsis and tuber mustard (Fig 6). In Arabidopsis, AtSOS4 expressed ubiquitously in all organs, in contrast, BjSOS4-1 mainly expressed in stem, while almost no expression of BjSOS4-2 could be detected in stem, leaf and flower (Fig 6), indicating the different roles of BjSOS4 genes in the regulation of tuber mustard growth and development [7,8]. The expression patterns of BjSOS genes under different stress treatments were examined by qPCR and found that they were differentially expressed after different stress treatments. According to the results of gene expression pattern analysis, all of the SOS homologs were significantly response to salt stress; BjSOS3-1 was induced by ABA and low temperature; BjSOS4-1, BjSOS4-2, BjSOS5-1, BjSOS5-2, BjSOS6-1 and BjSOS6-2 were induced by low temperature, BjSOS2, BjSOS3-1, BjSOS4-2, BjSOS5-2, BjSOS6-1 and BjSOS6-2 were significantly induced by treatment with the pathogen P. brassicae, indicating that BjSOS genes might regulate the tuber mustard response to abiotic and biotic stresses (Figs 7-10). In rice, OsSOS1 was highly induced in root after 15 h salt stress treatment comparing with non-treated plant [32], in accordance with this, both the expression levels of BjSOS1-1 and BjSOS1-2 were significantly induced in root by salt treatment at 12 h (Fig 7), and the expression induction was also found for PabSOS1 Populus [33]. Besides that, AtSOS1 was significantly induced by NaCl but not ABA and cold [2], similar expression patterns were also found for BjSOS1-1 and BjSOS1-2 (Figs 7-9), those results suggested that the functions of SOS1 genes are conserved in the regulation of plant response to salt stress in different species. Although abiotic and biotic stresses responsive ciselements could be found in the promoters of some BjSOS genes, the expressions of those genes were not induced by ABA, low temperature or pathogen. The event of gene expression level not in agreement with the promoter analysis of cis-element also frequently exists in other species, such as BnPYLs in B. napus and BjuTIR1/AFBs in B. juncea var. tumida [34][35].
In conclusion, our study identified twelve BjSOS genes in tuber mustard and analyzed their transcript levels under the biotic and abiotic stresses. The results suggest that SOS family genes might potentially be utilized for improving the tolerance of B. juncea var. tumida to biotic and abiotic stresses.
Supporting information S1