*4.3. RNA-Seq Data Analysis*

A total of 72 RNA-Seq data, including 30, 24, 6 and 12 data from salt, drought, waterlogging and osmotic stresses, respectively, were used in this study. The raw data were first processed with FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) to filter out adapters and low-quality sequences. The raw data was submitted to NCBI SRA (PRJNA552167). Then, the clean reads were mapped to the sesame genome v.1.0 (https://www.ncbi.nlm.nih.gov/genome/?term=sesamum) [95] using HISAT [96]. The RSEM package v1.3.0 [97] was used to calculate gene expression level for each sample expressed as fragments per kilobase of transcript per million fragments mapped (FPKM). For each treatment, the gene expression levels in the stressed samples were compared with those in the control samples in order to identify the differentially expressed genes (DEG). The DEGs were detected as described by Tarazona et al. [98] based on the parameters: Fold change ≥ 2 and Probability ≥ 0.8, with a significant false discovery rate-adjusted *p* value (FDR) < 0.05 based on the three biological replicates. Gene Ontology enrichment analysis for the DEGs was performed using the clusterProfiler version 3.8.

#### *4.4. Analysis of Co-Expression Modules Based on WGCNA*

Weighted Gene Co-Expression Network Analysis (WGCNA) package version: 1.61 [39] in the R software (http://www.r-project.org/) was used to construct the gene co-expression networks from the normalized log2-transformed FPKM matrix as described by Wan et al. [99] and Yang et al. [100]. This analysis was based on the core conserved DEGs between the four abiotic stress treatments. The gradient method was used to test the independence and the average connectivity degree of different modules with different power value (the power value ranging from 1 to 20). The appropriate power value was determined when the degree of independence was 0.8. Then, the adjacency was transformed into a topological overlap matrix (TOM), which could measure the network connectivity of a gene defined as the sum of its adjacency with all other genes for network generation, and the corresponding dissimilarity (1-TOM) was calculated. To classify genes with similar expression profiles into gene modules, average linkage hierarchical clustering was conducted according to the TOM-based dissimilarity measure with a minimum size (gene group) of 50 for the genes' dendrogram.

Module-trait associations were estimated using the correlation between the module eigengene and the stress treatments. Network visualization for each module was performed using the Cytoscape software version 3.6.1 [101] with a cut off of the weight parameter (obtained from the WGCNA) set at 0.30.

## *4.5. Enrichment Analysis of Cis-Regulatory Motifs*

To detect the enriched *cis*-regulatory motifs within the promoters of the genes belonging to each module detected by WGCNA, first, all the sesame transcription factor binding motifs were downloaded from the JASPAR website (http://jaspar.genereg.net/, [102]. Then, the sequences of 1 kb upstream from the transcription start sites of the genes belonging to each module detected by WGCNA were retrieved from the sesame genome v1.0 [95]. The promoter regions were scanned for presence/absence of the DNA binding motifs using the FIMO tool v5.0.3 [103] with a threshold (*p* < 0.01). Finally, we performed an enrichment analysis of each motif within the promoters of the genes belonging to each module using the cumulative hypergeometric distribution (*p* ≤ 0.05).

#### *4.6. Vector Construction and Arabidopsis Genetic Transformation*

We selected two genes, including a hub gene (*SiERF5*, *SIN\_1005239*) and a no-hub gene (*SiNAC104*, *SIN\_1026079*) to demonstrate their involvement in abiotic stress responses in *Arabidopsis thaliana* following descriptions of Dossa et al. [38]. Briefly, the protein coding region were cloned by PCR

from sesame root cDNA (SiERF5-F-GCTTTCGCGAGCTCGGTACCATGAGAATGATTCTCAAGAA, SiERF5-R-CGACTCTAGAGGATCCTGTCAAGTGAGATGGTTTGA); (SiNAC104-F- GCTTTCGCG AGCTCGGTACCATGGCTGAAGGGAGGAAATG, SiNAC104-R- CGACTCTAGAGGATCCAGAT CAGCTTGCCTAACTAG) and inserted into a pCAMBIA 1301s vector (which is a modified form of the pCAMBIA1301 vector) between KPnI (50 -end) and BamHI (30 -end) sites, driven by the CaMV 35S promoter. The plasmids containing the 35S::SiERF5 and 35S::SiNAC104 constructs were transformed first into *Agrobacterium tumefaciens* strain LBA4404 and then into Arabidopsis ecotype Col-0 cv. Columbia by the floral dipping method [104]. Transgenic seeds were screened by sowing on MS medium containing 1% agar, and 1% sucrose and 50 <sup>µ</sup>g·ml−<sup>1</sup> hygromicin. All the putative T1 transgenic plants and vector control (VC) plants (containing an empty pCAMBIA1301 vector) were screened by PCR with genomic DNA from leaves. Furthermore, qRT-PCR was performed to confirm the expression of the transgene [105]. Three independent T3 transgenic homozygous lines were used for the stress treatments, gene expression assay and phenotypic analyses.

## *4.7. Evaluation of Transgenic Lines Exposed to Osmotic, Salt, Waterlogging and Drought Stresses*

First, to analyze the response of the transgenic plants to osmotic stress, seeds of VC and three T3 lines for each transgene, were surface sterilized and plated on solid MS medium. The seeds were stratified for 2 days in the dark at 4 ◦C and then transferred to a growth chamber under a 16-h light period (long-day condition) provided by fluorescent light at 120 <sup>µ</sup>M·m−<sup>2</sup> ·s <sup>−</sup><sup>1</sup> and day/night temperatures of 22/16 ◦C and 60/75% relative humidity. 10 days-old seedlings were transferred into solid MS medium supplemented with 0/250 mM Mannitol. Plates were placed vertically and after 10 days, seedling root length was recorded.

Next, 10-day-old seedlings (transgenic lines and VC plants) were transferred into pots (two plants per pot) containing organic potting mix and grown in normal conditions for 15 days. Then, 1/4 of the pots were subjected to dehydration stress by withholding watering for 20 days and subsequently, plants were allowed to recover for 1 week by supplementing water [38]. Another 1/4 of the pots were watered with 200 mM NaCl solution every three days for four times and subsequently, plants were allowed to recover for 1 week by supplementing water [38]. Another 1/4 of the pots were subjected to waterlogging stress for 18 days. Pots were placed inside plastic tanks and filled with tap water up to 5 mm above the ground [106]. After waterlogging stress, plants were allowed to recover for 1 week by drainage. The remaining plants (1/4) were kept under normal growth condition throughout the experiment. Leaf samples were collected at the end of each stress treatment and in the control condition for gene expression analysis. After recovery, the plant survival rate, the above-ground rosette biomass fresh weights were recorded and pictures were captured to show visible phenotypes. We estimated the relative rosette biomass as the ratio of the records under stress and control conditions. For each treatment, eight survived plants (four pots) were kept until maturation to evaluate the seed yield. The experiment was repeated twice with four replicates in each experiment for statistical analysis.

### *4.8. Sesame Materials and Stress Treatments*

Ten cultivars of sesame were obtained from the China National Genebank, Oil Crops Research Institute, Chinese Academy of Agricultural Sciences and used in this experiment. The genotypes G059, G079, G207, G208, G209, G210, G212, G213, G214 and G215, all originating in China, were used in this experiment. The sesame seeds were sterilized with 3% sodium hypochlorite for 7 min and washed three times using sterile water. For the drought experiment, the seeds were sown in pots containing loam soil mixed with 10% vermiculite and plants were regularly watered. After 6 weeks, seedlings were submitted to a water stress for 7 days [7]. For the waterlogging treatment, seedlings were flooded by standing in a plastic bucket filled with tap water to 3 cm above the soil surface and maintained for 9 h according to the experimental descriptions of Dossa et al. [105]. Concerning the salt, heat and osmotic stress treatments, seedlings were hydroponically grown in a box containing half-strength Hoagland solution for 2 weeks under ambient temperature of 35 ◦C. Then, they were

transferred to a new nutrient solution containing 200 mM NaCl for 48 h (salt stress treatment), in a nutrient solution containing 2% PEG6000 for 5 days (osmotic stress treatment) or under 45 ◦C for 48 h (heat stress treatment). Root samples of stressed and control plants were collected at the same periods.
