*2.4. Multiple Sequence Alignment and Phylogenetic Analysis of GmWRKY12*

Although WRKYGQK sequence is a conservative motif of WRKY proteins, WRKY variant domains, such as WRKYGEK, WRKYGKK, WQKYGQK, WSKYGQK and WRKYGM have been found in the genomes of *Arabidopsis* [28], rice [63], grape [64] and tomato [65]. This difference may be a variation of WRKY TFs developed over long-term evolution. The domain of these variations is unique and may represent a new type. Therefore, to identify conservation of *GmWRKY12*, *WRKY12* from 20 different species were selected for multiple sequence alignment (Figure 4A). Results showed that 20 species only harbored one WRKY variant WRKYGQK, with amino acid sequence similarity of 75%, which illustrated that *GmWRKY12* was highly conserved. To further evaluate the evolutionary relationship between *GmWRKY12* and *WRKY12* of 32 different species, a phylogenetic tree was constructed with the neighbor-joining method [66]. Phylogenetic results showed that the relationship between *GmWRKY12* and *VrWRKY12* (XP\_014515898.1) was the closest (Figure 4B). *Int. J. Mol. Sci.* **2018**, *19*, x FOR PEER REVIEW 7 of 20 Although WRKYGQK sequence is a conservative motif of WRKY proteins, WRKY variant domains, such as WRKYGEK, WRKYGKK, WQKYGQK, WSKYGQK and WRKYGM have been found in the genomes of *Arabidopsis* [28], rice [63], grape [64] and tomato [65]. This difference may be a variation of WRKY TFs developed over long-term evolution. The domain of these variations is unique and may represent a new type. Therefore, to identify conservation of *GmWRKY12*, *WRKY12*  from 20 different species were selected for multiple sequence alignment (Figure 4A). Results showed that 20 species only harbored one WRKY variant WRKYGQK, with amino acid sequence similarity of 75%, which illustrated that *GmWRKY12* was highly conserved. To further evaluate the evolutionary relationship between *GmWRKY12* and *WRKY12* of 32 different species, a phylogenetic tree was constructed with the neighbor-joining method [66]. Phylogenetic results showed that the relationship between *GmWRKY12* and *VrWRKY12* (XP\_014515898.1) was the closest (Figure 4B).

**Figure 4.** Multiple alignment and phylogenetic relationship of GmWRKY12 with different species. (**A**) Multiple alignment of GmWRKY12 with other WRKY12 proteins from other species. (**B**) Phylogenetic relationship of *GmWRKY12* in different species. The red dot in (**B**) means GmWRKY12. The number of nodes is the bootstrap value and the number on the branch is the evolutionary distance. Bootstrap replications are 1000. **Figure 4.** Multiple alignment and phylogenetic relationship of GmWRKY12 with different species. (**A**) Multiple alignment of GmWRKY12 with other WRKY12 proteins from other species. (**B**) Phylogenetic relationship of *GmWRKY12* in different species. The red dot in (**B**) means GmWRKY12. The number of nodes is the bootstrap value and the number on the branch is the evolutionary distance. Bootstrap replications are 1000.

#### *2.5. Expression Patterns of GmWRKY12 under Different Treatments 2.5. Expression Patterns of GmWRKY12 under Different Treatments*

*GmWRKY12* was responsive to drought and salt treatments (Figure 3). WRKY proteins are reported to be involved in signal transductions of plant hormones [39]. In order to identify whether *GmWRKY12* was responsive to other abiotic stresses, expression patterns were identified using qRT-PCR. Results indicated that *GmWRKY12* not only participated in drought and salt response but was also responsive to ABA and SA. Under low concentrations of SA, the expression profile of *GmWRKY12* was increased about 50-fold (Figure 5). *GmWRKY12* was responsive to drought and salt treatments (Figure 3). WRKY proteins are reported to be involved in signal transductions of plant hormones [39]. In order to identify whether *GmWRKY12* was responsive to other abiotic stresses, expression patterns were identified using qRT-PCR. Results indicated that *GmWRKY12* not only participated in drought and salt response but was also responsive to ABA and SA. Under low concentrations of SA, the expression profile of *GmWRKY12* was increased about 50-fold (Figure 5).

*Int. J. Mol. Sci.* **2018**, *19*, x FOR PEER REVIEW 8 of 20

**Figure 5.** Expression patterns of *GmWRKY12* under drought, NaCl, exogenous ABA and SA. The ordinates are the relative expression level (fold) of *GmWRKY12* compared to non-stressed control. The horizontal ordinate is treatment time for 0, 0.5, 1, 2, 5, 8, 12 and 24 h. The expression level of *GmActin* as a loading control. All experiments were repeated three times. Error bars represent standard deviations (SDs). All data represent the means ± SDs of three independent biological **Figure 5.** Expression patterns of *GmWRKY12* under drought, NaCl, exogenous ABA and SA. The ordinates are the relative expression level (fold) of *GmWRKY12* compared to non-stressed control. The horizontal ordinate is treatment time for 0, 0.5, 1, 2, 5, 8, 12 and 24 h. The expression level of *GmActin* as a loading control. All experiments were repeated three times. Error bars represent standard deviations (SDs). All data represent the means ± SDs of three independent biological replicates.

#### replicates. *2.6. Cis-Acting Elements in Promoter*

*2.6. Cis-Acting Elements in Promoter*  To further understand the regulatory mechanism of *GmWRKY12*, we isolated its promoter region. *Cis*-elements correlated to stress were present in the promoter region, including the ABA and wound responsive elements ABER4 and MYC, drought responsive element MYB, salt stress responsive element GT-1 and wound responsive element W-box. In addition, there was another element that participated in heat and GA response in the promoter region of *GmWRKY12* (Table 3). To further understand the regulatory mechanism of *GmWRKY12*, we isolated its promoter region. *Cis*-elements correlated to stress were present in the promoter region, including the ABA and wound responsive elements ABER4 and MYC, drought responsive element MYB, salt stress responsive element GT-1 and wound responsive element W-box. In addition, there was another element that participated in heat and GA response in the promoter region of *GmWRKY12* (Table 3). This analysis suggested that *GmWRKY12* may function in abiotic stress response.


This analysis suggested that *GmWRKY12* may function in abiotic stress response. **Table 3.** *Cis*-elements analysis of *GmWRKY12* promotor.

GT-1 7 GAAAAA Salt stress responsive element "Numbers" corresponds to the number of *cis*-elements of each type present in the promoter.

DPBF 6 ACACNNG Dehydration-responsive element GARE 2 TAACAAR GA-responsive element "Numbers" corresponds to the number of *cis*-elements of each type present in the promoter.

#### *2.7. GmWRKY12 was Located in the Nucleus 2.7. GmWRKY12 was Located in the Nucleus*

To investigate GmWRKY12 subcellular localization, GmWRKY12 were fused to the N-terminus of the humanized green fluorescent protein (hGFP) and co-transformed into wheat mesophyll protoplasts with the nucleus marker AT2G03340 (AtWRKY3)-mCherry [67,68]. The 35S::GFP vector was transformed as the control. Fluorescence of GmWRKY12 was specifically detected in the nucleus, whereas GFP fluorescence was distributed throughout the cell (Figure 6). To investigate GmWRKY12 subcellular localization, GmWRKY12 were fused to the N-terminus of the humanized green fluorescent protein (hGFP) and co-transformed into wheat mesophyll protoplasts with the nucleus marker AT2G03340 (AtWRKY3)-mCherry [67,68]. The 35S::GFP vector was transformed as the control. Fluorescence of GmWRKY12 was specifically detected in the nucleus, whereas GFP fluorescence was distributed throughout the cell (Figure 6).

*Int. J. Mol. Sci.* **2018**, *19*, x FOR PEER REVIEW 9 of 20

**Figure 6.** Co-localization of GmWRKY12. The recombinant plasmid of GmWRKY12-GFP and At2G03340-mCherry were co-transformed into wheat mesophyll protoplasts under the control of the CaMV 35S promoter. GmWRKY12 was localized in the nucleus of wheat mesophyll protoplasts protoplasts. Results were observed by a confocal laser scanning microscope (LSM700; CarlZeiss, Oberkochen Germany) after incubating in darkness at 22 °C for 18–20 h. Scale bars = 10 μm. **Figure 6.** Co-localization of GmWRKY12. The recombinant plasmid of GmWRKY12-GFP and At2G03340-mCherry were co-transformed into wheat mesophyll protoplasts under the control of the CaMV 35S promoter. GmWRKY12 was localized in the nucleus of wheat mesophyll protoplasts protoplasts. Results were observed by a confocal laser scanning microscope (LSM700; CarlZeiss, Oberkochen Germany) after incubating in darkness at 22 ◦C for 18–20 h. Scale bars = 10 µm.

#### *2.8. GmWRKY12 Improved Drought and Salt Tolerance of Soybean 2.8. GmWRKY12 Improved Drought and Salt Tolerance of Soybean*

confers stress tolerance in transgenic hairy roots.

We further used transgenic hairy root assays to investigate the roles of *GmWRKY12* in abiotic stress responses. Amplified cDNA sequence of *GmWRKY12* was constructed into pCAMBIA3301 to create an overexpression transgenic line and the control was pCAMBIA3301 plant expression vector with CaMV35S promoter. Two constructs were transferred into *Agrobacterium rhizogenes* strain K599 (NCPPB2659) [69] then transformed into soybean hairy roots as previously described [70,71]. After drought treatment for 20 days, both control and over-expression soybean seedlings had leaf shedding to different degrees, especially the old leaves of the plants (Figure 7A). However, compared with transgenic soybean seedlings, the control seedlings were severely wilted and almost 99% of the leaves had serious dehydration and drying. By contrast, there was slight shedding of the old leaves of transgenic soybean seedlings but the new leaves were still growing vigorously. Results of proline and malondialdehyde (MDA) content determination showed that overexpression of *GmWRKY12* increased proline content in transgenic lines, while the MDA content was decreased due to drought stress (Figure 7B,C). Fresh weight and main length of transgenic soybean hair roots under drought treatment were measured (Figure 8E,F), results showed overexpressed *GmWRKY12*  in soybean roots enhanced drought tolerance of soybean by increasing the length of transgenic hair roots and the number of transgenic hair roots. We further used transgenic hairy root assays to investigate the roles of *GmWRKY12* in abiotic stress responses. Amplified cDNA sequence of *GmWRKY12* was constructed into pCAMBIA3301 to create an overexpression transgenic line and the control was pCAMBIA3301 plant expression vector with CaMV35S promoter. Two constructs were transferred into *Agrobacterium rhizogenes* strain K599 (NCPPB2659) [69] then transformed into soybean hairy roots as previously described [70,71]. After drought treatment for 20 days, both control and over-expression soybean seedlings had leaf shedding to different degrees, especially the old leaves of the plants (Figure 7A). However, compared with transgenic soybean seedlings, the control seedlings were severely wilted and almost 99% of the leaves had serious dehydration and drying. By contrast, there was slight shedding of the old leaves of transgenic soybean seedlings but the new leaves were still growing vigorously. Results of proline and malondialdehyde (MDA) content determination showed that overexpression of *GmWRKY12* increased proline content in transgenic lines, while the MDA content was decreased due to drought stress (Figure 7B,C). Fresh weight and main length of transgenic soybean hair roots under drought treatment were measured (Figure 8E,F), results showed overexpressed *GmWRKY12* in soybean roots enhanced drought tolerance of soybean by increasing the length of transgenic hair roots and the number of transgenic hair roots.

Meanwhile, under NaCl (200 mM) treatment, control and overexpression soybean seedlings had different degrees of leaf shedding (Figure 7D). Compared with the control, transgenic soybean seedlings were slightly wilted and slowly drying out, while the control seedlings were almost dry due to the osmotic stress. Results of Pro and MDA content in transgenic lines (Figure 7E,F) fresh weight and main length of transgenic soybean hair roots (Figure 8H,I) also showed that *GmWRKY12* improved salt tolerance of soybean. These results demonstrated that *GmWRKY12*

*Int. J. Mol. Sci.* **2018**, *19*, x FOR PEER REVIEW 10 of 20

**Figure 7.** Phenotype identification of *GmWRKY12* under drought and salt treatments. (**A**) Images of drought stress resistance phenotypes of CK and *35S::GmWRKY12* soybean seedlings after drought treatment for 20 days. (**B**) Proline contents in CK and *35S::GmWRKY12* soybean seedlings under normal growth conditions and drought treatment. (**C**) MDA contents in in CK and *35S::GmWRKY12* soybean seedlings under normal growth conditions and drought treatment. (**D**) Images of salt stress resistance phenotypes of CK and *35S::GmWRKY12* soybean seedlings after 200 mM NaCl treatment for 7 days. (**E**) Proline contents in CK and *35S::GmWRKY12* soybean seedlings under normal growth conditions and salt treatment. (**F**) MDA contents in CK and *35S::GmWRKY12* soybean seedlings under normal growth conditions and salt treatment. All data represent the means ± SDs of three independent biological replicates. ANOVA tests demonstrated that there were significant differences (\* *p* < 0.05, \*\* *p* < 0.01). **Figure 7.** Phenotype identification of *GmWRKY12* under drought and salt treatments. (**A**) Images ofdrought stress resistance phenotypes of CK and *35S::GmWRKY12* soybean seedlings after droughttreatment for 20 days. (**B**) Proline contents in CK and *35S::GmWRKY12* soybean seedlings under normal growth conditions and drought treatment. (**C**) MDA contents in in CK and *35S::GmWRKY12* soybean seedlings under normal growth conditions and drought treatment. (**D**) Images of salt stress resistance phenotypes of CK and *35S::GmWRKY12* soybean seedlings after 200 mM NaCl treatment for 7 days. (**E**) Proline contents in CK and *35S::GmWRKY12* soybean seedlings under normal growth conditions and salt treatment. (**F**) MDA contents in CK and *35S::GmWRKY12* soybean seedlings under normal growth conditions and salt treatment. All data represent the means ± SDs of three independent biological replicates. ANOVA tests demonstrated that there were significant differences (\* *p* < 0.05, \*\* *p* < 0.01).

Meanwhile, under NaCl (200 mM) treatment, control and overexpression soybean seedlings had different degrees of leaf shedding (Figure 7D). Compared with the control, transgenic soybean seedlings were slightly wilted and slowly drying out, while the control seedlings were almost dry due to the osmotic stress. Results of Pro and MDA content in transgenic lines (Figure 7E,F) fresh weight and main length of transgenic soybean hair roots (Figure 8H,I) also showed that *GmWRKY12* improved salt tolerance of soybean. These results demonstrated that *GmWRKY12* confers stress tolerance in transgenic hairy roots.

*Int. J. Mol. Sci.* **2018**, *19*, x FOR PEER REVIEW 11 of 20

**Figure 8.** Different growth stage of transgenic soybean seedlings and phenotypes of transgenic soybean hair roots. (**A**) Images of different growth stage of transgenic soybean seedlings cultivated in flowerpot before any treatment. (**A1**) Soybean seedlings of 5-days-old without injected *A. rhizogenes* carrying *GmWRKY12*. (**A2**) Soybean seedlings which have injected *A. rhizogenes* carrying *GmWRKY12* for 7 days. (**A3**) Soybean seedlings which have injected *A. rhizogenes* carrying *GmWRKY12* for 14 days (The original main roots were removed by cutting from 1 cm below the infection site and the hairy roots of the seedlings were cultivated in nutritious soil with full water and grown with 16 h light (100 μM photons m<sup>−</sup>2·s−1)/8 h dark at 25 °C). (**B**) Images of different growth stage of signal transgenic soybean seedling before any treatment. (**B1**) Soybean seedling of 5-days-old without injected *A. rhizogenes* carrying *GmWRKY12* and the red circle shows the inject site of *A. rhizogenes.* (**B2**) Soybean seedling which have injected *A. rhizogenes* carrying *GmWRKY12* for 7 days and new hair roots have generated. (B**3**) Soybean seedling which have injected *A. rhizogenes* carrying *GmWRKY12* for 14 days. (B**4**) Soybean seedling that have salt treatment for 7days. (**C**) Relative expression of CK and *35S::GmWRKY12* transgenic soybean hair roots under normal growth conditions. (**D**) Images of drought stress resistance phenotypes of CK and *35S::GmWRKY12* transgenic soybean hair roots after drought treatment for 20 days. (**E**) Fresh weight in CK and *35S::GmWRKY12* transgenic soybean hair roots under normal growth conditions and drought treatment. (**F**) Length in CK and *35S::GmWRKY12* transgenic soybean hair roots under normal growth conditions and drought treatment. (**G**) Images of salt stress resistance phenotypes of CK and *35S::GmWRKY12* transgenic soybean hair roots after 200 mM NaCl treatment for 7 days. (**H**) Fresh weight in CK and *35S::GmWRKY12* transgenic soybean hair roots under normal growth conditions and salt treatment. (**I**) Length in CK and *35S::GmWRKY12* transgenic soybean hair roots under normal growth condition and salt treatment. All data represent the means ± SDs of three independent biological replicates. ANOVA tests demonstrated that there were significant differences (\* *p* < 0.05, \*\* *p* < 0.01). **Figure 8.** Different growth stage of transgenic soybean seedlings and phenotypes of transgenic soybean hair roots. (**A**) Images of different growth stage of transgenic soybean seedlings cultivated in flowerpot before any treatment. (**A1**) Soybean seedlings of 5-days-old without injected *A. rhizogenes* carrying *GmWRKY12*. (**A2**) Soybean seedlings which have injected *A. rhizogenes* carrying *GmWRKY12* for 7 days. (**A3**) Soybean seedlings which have injected *A. rhizogenes* carrying *GmWRKY12* for 14 days (The original main roots were removed by cutting from 1 cm below the infection site and the hairy roots of the seedlings were cultivated in nutritious soil with full water and grown with 16 h light (100 µM photons m−<sup>2</sup> ·s −1 )/8 h dark at 25 ◦C). (**B**) Images of different growth stage of signal transgenic soybean seedling before any treatment. (**B1**) Soybean seedling of 5-days-old without injected *A. rhizogenes* carrying *GmWRKY12* and the red circle shows the inject site of *A. rhizogenes.* (**B2**) Soybean seedling which have injected *A. rhizogenes* carrying *GmWRKY12* for 7 days and new hair roots have generated. (B**3**) Soybean seedling which have injected *A. rhizogenes* carrying *GmWRKY12* for 14 days. (B**4**) Soybean seedling that have salt treatment for 7days. (**C**) Relative expression of CK and *35S::GmWRKY12* transgenic soybean hair roots under normal growth conditions. (**D**) Images of drought stress resistance phenotypes of CK and *35S::GmWRKY12* transgenic soybean hair roots after drought treatment for 20 days. (**E**) Fresh weight in CK and *35S::GmWRKY12* transgenic soybean hair roots under normal growth conditions and drought treatment. (**F**) Length in CK and *35S::GmWRKY12* transgenic soybean hair roots under normal growth conditions and drought treatment. (**G**) Images of salt stress resistance phenotypes of CK and *35S::GmWRKY12* transgenic soybean hair roots after 200 mM NaCl treatment for 7 days. (**H**) Fresh weight in CK and *35S::GmWRKY12* transgenic soybean hair roots under normal growth conditions and salt treatment. (**I**) Length in CK and *35S::GmWRKY12* transgenic soybean hair roots under normal growth condition and salt treatment. All data represent the means ± SDs of three independent biological replicates. ANOVA tests demonstrated that there were significant differences (\* *p* < 0.05, \*\* *p* < 0.01).
