Understanding salt tolerance mechanism using transcriptome profiling and de novo assembly of wild tomato Solanum chilense

Soil salinity affects the plant growth and productivity detrimentally, but Solanum chilense, a wild relative of cultivated tomato (Solanum lycopersicum L.), is known to have exceptional salt tolerance. It has precise adaptations against direct exposure to salt stress conditions. Hence, a better understanding of the mechanism to salinity stress tolerance by S. chilense can be accomplished by comprehensive gene expression studies. In this study 1-month-old seedlings of S. chilense and S. lycopersicum were subjected to salinity stress through application of sodium chloride (NaCl) solution. Through RNA-sequencing here we have studied the differences in the gene expression patterns. A total of 386 million clean reads were obtained through RNAseq analysis using the Illumina HiSeq 2000 platform. Clean reads were further assembled de novo into a transcriptome dataset comprising of 514,747 unigenes with N50 length of 578 bp and were further aligned to the public databases. Genebank non-redundant (Nr), Viridiplantae, Gene Ontology (GO), KOG, and KEGG databases classification suggested enrichment of these unigenes in 30 GO categories, 26 KOG, and 127 pathways, respectively. Out of 265,158 genes that were differentially expressed in response to salt treatment, 134,566 and 130,592 genes were significantly up and down-regulated, respectively. Upon placing all the differentially expressed genes (DEG) in known signaling pathways, it was evident that most of the DEGs involved in cytokinin, ethylene, auxin, abscisic acid, gibberellin, and Ca2+ mediated signaling pathways were up-regulated. Furthermore, GO enrichment analysis was performed using REVIGO and up-regulation of multiple genes involved in various biological processes in chilense under salinity were identified. Through pathway analysis of DEGs, “Wnt signaling pathway” was identified as a novel pathway for the response to the salinity stress. Moreover, key genes for salinity tolerance, such as genes encoding proline and arginine metabolism, ROS scavenging system, transporters, osmotic regulation, defense and stress response, homeostasis and transcription factors were not only salt-induced but also showed higher expression in S. chilense as compared to S. lycopersicum. Thus indicating that these genes may have an important role in salinity tolerance in S. chilense. Overall, the results of this study improve our understanding on possible molecular mechanisms underlying salt tolerance in plants in general and tomato in particular.

Function annotation of assembled transcripts based on Gene Ontology (GO) analysis. According to their sequence homology, a total of 1,08,355 unigenes were assigned into 3 main GO categories viz., cellular component (CC), biological process (BP) and molecular function (MF).
Pathway assignment based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. A total of 7,923 unigenes were mapped into the reference canonical pathways and assigned to 127 KEGG pathways, and further divided into five different KEGG functional groups viz., cellular processes, environmental information processing, genetic information processing, metabolism and organismal systems.
Classification of assembled transcripts based on the euKaryotic Ortholog Groups (KOG) database. A total of 1,26,937 unigenes were assigned into 26 diverse functional categories.
Distribution of SSRs motif based on SSR unit size. A total of 1,06,239 potential SSRs were classified based on the SSR unit size.
Volcano plot of the differentially expressed genes shows the estimated log2 (fold change) (xaxis) against its statistical significance (y-axis) between control and treated groups. (a-c) DEGs between salt treated chilense and chilense (Chilense_Treated vs Control, a), salt treated DVRT 1 and DVRT 1 (DVRT-1_Treated vs Control, b), salt treated chilense and salt treated DVRT-1 (Chilense_Treated vs DVRT-1_Treated, c) displayed by volcano plots. The abscissa displays the fold change difference in the expression of genes in different comparison groups, and the vertical coordinates indicate the adjusted P-values for the differences in expression. Genes without significant differences are indicated by black dots. The up-regulated genes are represented by red dots, and the down-regulated genes are represented by green dots. Heatmap and clustering of differentially expressed genes (DEGs) between the salt-treated and untreated groups. Each column represents a different sample. Colour bar indicates the relative expression level from up-regulated (red) to down-regulated (green). Heatmap was created using Heatmapper online tool (http://heatmapper.ca/).
Gene ontology (GO) annotations of differentially expressed genes (DEGs) using Blast2GO program. DEGs were grouped into three main GO categories; biological process, cellular component and molecular function as well as into 27 subcategories.
Pathway analysis of differentially expressed genes (DEGs). PANTHER14.1 tool was used for identification of pathway of DEGs. A total of 30 pathways were identified and among these pathways, the "Wnt signaling pathway" was over-represented and has not been reported previously with response to the salinity function.
Pathway analysis of assembled unigenes (Chilense_Control). A total of 115 pathways were identified, top 20 pathways were taken Chilense_Control sample for Pie chart representation.
Pathway analysis of assembled unigenes (Chilense_Treated). A total of 131 pathways were identified, top 20 pathways were taken Chilense_Treated sample for Pie chart representation.
Pathway analysis of assembled unigenes (DVRT-1 Control). A total of 137 pathways were identified, top 20 pathways were taken DVRT-1 Control sample for Pie chart representation.
Pathway analysis of assembled unigenes (DVRT-1 Treated). A total of 123 pathways were identified, top 20 pathways were taken DVRT-1 Treated sample for Pie chart representation.
Expression patterns of selected candidate genes related with transporters, ROS scavenging and signaling transduction in salt treated and untreated Chilense and DVRT-1 determined by RNAseq and qPCR. The RNA-seq values represent the ratio of the expression level in chilense to the expression level in DVRT-1. Bars with distinct letters are significantly different at P ≤ 0.05 applying the DMRT test.
Expression patterns of selected candidate genes related with osmotic regulation, defence-stress, homeostasis, transporters and transcription factor in salt treated and untreated Chilense and DVRT-1 determined by RNA-seq and qPCR. The RNA-seq values represent the ratio of the expression level in chilense to the expression level in DVRT-1. Bars with distinct letters are significantly different at P ≤ 0.05 applying the DMRT test.
Picture depicting the salt sensitivity of the S. lycopersicum cv. DVRT-1 and salt tolerance of S. chilense; the salt stress was imposed by adding NaCl for 21 days. A non-stress or control treatment (0 days) was also carried out without NaCl with EC of 3.8 dSm -1 whereas; saline treatments received 500 mmol of NaCl for 21 days with EC of 26.8 dSm -1 , respectively.

Supplemental Table S1
Summary of transcriptome sequencing data  Supplemental Table S11: List of differentially expressed genes (DEGs) in DVRT-1_Treated vs Control.
Supplemental Table S13: Gene Ontology (GO) enrichment analysis of up-regulated differentially expressed genes in different treatment and control groups using REVIGO based on the lowest p values.
Supplemental Table S14: Primer sequences used for qRT-PCR analysis and NormFinder stability check value of reference genes. The primer names with sequences are shown. The unigene sequences obtained by transcriptome analysis were used to design primers with the help of http://www.ncbi.nlm.nih.gov/tools/primerblast/ online tool.