*2.2. Tissue-Specific Expression Patterns of GmWRKYs*

To thoroughly study GmWRKY expression profiles under normal conditions, hierarchical clustering was conducted using expression levels of fifty-three (drought-responsive) and nine (salt-responsive) GmWRKY genes in young leaf, flower, one cm pod, pod shell 10 days after flowering (DAF), pod shell 14 DAF, seed 10 DAF, seed 14 DAF, seed 21 DAF, seed 25 DAF, seed 28 DAF, seed 35 DAF, seed 42 DAF, root and nodule (Figures 1 and 2). Approximately 28% of GmWRKYs from different tissues were expressed at low levels or unexpressed (*GmWRKY3*, *5*, *6*, *21*, *22*, *25*, *29*, *30*, *31*, *47*, *50*, *55*, *63*, *70* and *72*); by contrast, 45% of GmWRKYs were highly expressed in different tissues

(*GmWRKY4*, *9*, *11*, *14*, *16*, *17*, *19*, *28*, *32*, *34*, *35*, *36*, *37*, *39*, *41*, *42*, *46*, *48*, *49*, *52*, *56*, *57*, *60* and *61*). Among these GmWRKYs, *GmWRKY11* and *GmWRKY17* had the highest expression in four different tissues. *GmWRKY28*, *35*, *37*, *48* and *57* are highly expressed in nodule, seed 10 DAF, seed 42 DAF, root and flower. Within the nine GmWRKYs related to salt response, *GmWRKY3* and *GmWRKY21* had low expression and *GmWRKY14*, *28*, *35*, *49* and *59* were highly expressed in at least four different tissues. The analysis data are available in Tables S1 and S2. *Int. J. Mol. Sci.* **2018**, *19*, x FOR PEER REVIEW 5 of 20 *GmWRKY21* had low expression and *GmWRKY14*, *28*, *35*, *49* and *59* were highly expressed in at least four different tissues. The analysis data are available in Tables S1 and S2.

**Figure 1.** Expression pattern of fifty-three GmWRKYs in six different tissues (young leaf, flower, pod shell, seed, root and nodule). The fifty-three GmWRKYs were selected from drought treatment RNA-Seq data based on the rule that log2 (GH\_treat/CK1\_treat) >1. The tissue expression is from SoyDB (http://soykb.org/). The color legend refers to the different expression level under normal condition. "DAF" in the tissue label indicates days after flowering. **Figure 1.** Expression pattern of fifty-three GmWRKYs in six different tissues (young leaf, flower, pod shell, seed, root and nodule). The fifty-three GmWRKYs were selected from drought treatment RNA-Seq data based on the rule that log<sup>2</sup> (GH\_treat/CK1\_treat) >1. The tissue expression is from SoyDB (http://soykb.org/). The color legend refers to the different expression level under normal condition. "DAF" in the tissue label indicates days after flowering. **Figure 1.** Expression pattern of fifty-three GmWRKYs in six different tissues (young leaf, flower, pod shell, seed, root and nodule). The fifty-three GmWRKYs were selected from drought treatment RNA-Seq data based on the rule that log2 (GH\_treat/CK1\_treat) >1. The tissue expression is from SoyDB (http://soykb.org/). The color legend refers to the different expression level under normal condition. "DAF" in the tissue label indicates days after flowering.

**Figure 2.** Expression pattern of nine GmWRKYs in six different tissues (young leaf, flower, pod shell, seed, root and nodule). The nine GmWRKYs were selected from salt treatment RNA-Seq data based on the rule that log2 (NaCl\_treat/CK2\_treat) >1. The tissue expression is from SoyDB (http://soykb.org/). The color legend refers to the different expression level under normal condition. **Figure 2.** Expression pattern of nine GmWRKYs in six different tissues (young leaf, flower, pod shell, seed, root and nodule). The nine GmWRKYs were selected from salt treatment RNA-Seq data based on the rule that log2 (NaCl\_treat/CK2\_treat) >1. The tissue expression is from SoyDB (http://soykb.org/). The color legend refers to the different expression level under normal condition. "DAF" in the tissue label indicates days after flowering. **Figure 2.** Expression pattern of nine GmWRKYs in six different tissues (young leaf, flower, pod shell, seed, root and nodule). The nine GmWRKYs were selected from salt treatment RNA-Seq data based on the rule that log<sup>2</sup> (NaCl\_treat/CK2\_treat) >1. The tissue expression is from SoyDB (http://soykb.org/). The color legend refers to the different expression level under normal condition. "DAF" in the tissue label indicates days after flowering.

"DAF" in the tissue label indicates days after flowering.

#### *2.3. GmWRKYs Responsive to Both Drought and Salt Treatments* order to confirm whether the seven GmWRKY genes are responsive to drought and salt, 10-day-old soybean seedlings were subjected to stress treatments. For drought treatment, soybean seedlings

*2.3. GmWRKYs Responsive to Both Drought and Salt Treatments* 

Based on RNA-Seq data and result of Venn method [62], seven *GmWRKY* genes were found to respond to both drought and salt treatments (*GmWRKY3*, *12*, *14*, *21*, *35*, *4*3 and *49*) (Figure S1A). In order to confirm whether the seven GmWRKY genes are responsive to drought and salt, 10-day-old soybean seedlings were subjected to stress treatments. For drought treatment, soybean seedlings were put on filter paper to stimulate drought and then sampled 0.1 g of leaf on different periods (0, 0.5, 1, 2, 5, 8, 12 and 24 h); for salt treatment, the roots of soybean were soaked in 100 mM NaCl solution then sampled 0.1 g of leaf on different periods (0, 0.5, 1, 2, 5, 8, 12 and 24 h), all samples were submerged immediately in liquid nitrogen and stored at −80 ◦C for RNA extraction then quantitative real-time PCR (qRT-PCR) was conducted. Results confirmed that the seven *GmWRKY* genes were responsive to both treatments (Figure 3). Under drought treatment, the expression levels of *GmWRKY12* and *GmWRKY43* were gradually increased at 0, 0.5, 1, 2, 5, 8, 12 and 24 h. *GmWRKY12* was highly expressed after 12 h of drought treatment. While *GmWRKY14*, *GmWRKY21* and *GmWRKY35* had a tendency to rise first and then decrease, *GmWRKY49* was highly expressed at 2 h. Under drought conditions, the expression profiles of five *GmWRKY* genes were little changed at 0 to 5 h and then increased significantly at 12 h. were put on filter paper to stimulate drought and then sampled 0.1 g of leaf on different periods (0, 0.5, 1, 2, 5, 8, 12 and 24 h); for salt treatment, the roots of soybean were soaked in 100 mM NaCl solution then sampled 0.1 g of leaf on different periods (0, 0.5, 1, 2, 5, 8, 12 and 24 h), all samples were submerged immediately in liquid nitrogen and stored at −80 °C for RNA extraction then quantitative real-time PCR (qRT-PCR) was conducted. Results confirmed that the seven *GmWRKY* genes were responsive to both treatments (Figure 3). Under drought treatment, the expression levels of *GmWRKY12* and *GmWRKY43* were gradually increased at 0, 0.5, 1, 2, 5, 8, 12 and 24 h. *GmWRKY12* was highly expressed after 12 h of drought treatment. While *GmWRKY14*, *GmWRKY21* and *GmWRKY35* had a tendency to rise first and then decrease, *GmWRKY49* was highly expressed at 2 h. Under drought conditions, the expression profiles of five *GmWRKY* genes were little changed at 0 to 5 h and then increased significantly at 12 h. Under salt treatment, the expression profile increased first and then decreased, meanwhile, there was a notable change at 0 to 0.5 h and *GmWRKY3*, *12*, *14* and *35* were highly expressed. *GmWRKY12*, which was 714 bp in length, encoded 237 amino acids and had low expression in different tissues under normal conditions but was highly expressed under drought and salt treatments was selected for further investigation (Figure S1B).

*Int. J. Mol. Sci.* **2018**, *19*, x FOR PEER REVIEW 6 of 20

respond to both drought and salt treatments (*GmWRKY3*, *12*, *14*, *21*, *35*, *4*3 and *49*) (Figure S1A). In

**Figure 3.** Quantitative RT-PCR of seven GmWRKYs under drought and salt treatment. (**A**) qRT-PCR of seven GmWRKYs under drought treatment. (**B**) qRT-PCR of seven GmWRKYs under salt treatment. The expression level of *GmActin* as a loading control. The data represent means ± SD of three biological replications. **Figure 3.** Quantitative RT-PCR of seven GmWRKYs under drought and salt treatment. (**A**) qRT-PCR of seven GmWRKYs under drought treatment. (**B**) qRT-PCR of seven GmWRKYs under salt treatment. The expression level of *GmActin* as a loading control. The data represent means ± SD of three biological replications.

*2.4. Multiple Sequence Alignment and Phylogenetic Analysis of GmWRKY12*  Under salt treatment, the expression profile increased first and then decreased, meanwhile, there was a notable change at 0 to 0.5 h and *GmWRKY3*, *12*, *14* and *35* were highly expressed. *GmWRKY12*, which was 714 bp in length, encoded 237 amino acids and had low expression in different tissues under normal conditions but was highly expressed under drought and salt treatments was selected for further investigation (Figure S1B).
