*2.8. ZmWRKY106 Regulated the Expression of Stress-Related Genes 2.8. ZmWRKY106 Regulated the Expression of Stress-Related Genes*

To understand the molecular mechanisms of *ZmWRKY106* in stress responses, expression of stress-responsive genes, including *RD29A*, *HSP90*, *DREB2A*, *CuZnSOD*, *NCED3*, and *NCED6*, was examined using qRT-PCR under normal and drought conditions. The results showed that the expression levels of *HSP90* and *NCED3* were low in both WT and OE lines under normal conditions, while the expression levels in OE lines remained higher than WT plants after treatment (Figure 7B, E). Meanwhile, *CuZnSOD* and *NCED6* in OE lines were up-regulated after 4 h of stress treatment, and sharply increased to the maximum (Figure 7D, F). The expression levels of *RD29A* and *DREB2A* in OE lines were remarkably higher following all treatments (Figure 7A, C). Because the expressions of ABA and stress-related genes were altered in transgenic lines, we conjectured that *ZmWRKY106* may play a role in the abiotic stress response by regulating stress-related genes through the ABAsignaling pathway. To understand the molecular mechanisms of *ZmWRKY106* in stress responses, expression of stress-responsive genes, including *RD29A*, *HSP90*, *DREB2A*, *CuZnSOD*, *NCED3*, and *NCED6*, was examined using qRT-PCR under normal and drought conditions. The results showed that the expression levels of *HSP90* and *NCED3* were low in both WT and OE lines under normal conditions, while the expression levels in OE lines remained higher than WT plants after treatment (Figure 7B,E). Meanwhile, *CuZnSOD* and *NCED6* in OE lines were up-regulated after 4 h of stress treatment, and sharply increased to the maximum (Figure 7D,F). The expression levels of *RD29A* and *DREB2A* in OE lines were remarkably higher following all treatments (Figure 7A,C). Because the expressions of ABA and stress-related genes were altered in transgenic lines, we conjectured that *ZmWRKY106* may play a role in the abiotic stress response by regulating stress-related genes through the ABA-signaling pathway.

**Figure 7.** *Cont*.

**Figure 7.** The relative expression of stress-related genes. (**A**) *RD29A*, (**B**) *HSP90*, (**C**) *DREB2A*, (**D**) *CuZnSOD*, (**E**) *NCED3*, and (**F**) *NCED6* were examined under control and drought conditions for various time points (4, 12, 24 and 36 h). Values are means ± SDs of three replicates, and asterisks (\* or \*\*) represent the significant differences at *p* < 0.05 or *p* < 0.01, respectively (Student's *t*-test).

*2.9. Overexpression of ZmWRKY106 Reduced Reactive Oxygen Species (ROS) Content and Enhanced the Activities of Superoxide Dismutase (SOD), Peroxide Dismutase (POD), and Catalase (CAT) under Drought Treatment*

The ROS content and the enzyme activities were assessed in transgenic lines and WT plants at 0, 4, 12 and 24 h after drought treatment (Figure 8). As shown in Figure 8A, the ROS accumulation in transgenic lines was less than that in WT plants at all times, while the ROS content was increased in WT plants and remained at a higher level during the whole experiment. The activities of SOD, POD and CAT were increased in OE lines compared to WT lines (Figure 8B–D). The activity of SOD was almost unchanged in WT before and after drought treatment, whereas in OE lines the activity of SOD was greater, and reached a maximum at 24 h after drought treatment (Figure 8B). Increases of POD activity were observed in both WT and transgenic lines, but the increases in WT were smaller, and there was consistently higher POD activity in transgenic lines than in WT lines (Figure 8C). In the case of CAT, the CAT activity of WT lines remained consistent at 0.016 U and had almost no significant change during the stress treatment; however, the CAT concentration in the OE lines remained significantly higher compared to that in WT lines under drought stress (Figure 8D). In a word, overexpression of *ZmWRKY106* reduced ROS content by enhancing the activities of SOD, POD and CAT to resist drought stress. *Int. J. Mol. Sci.* **2018**, *19*, x FOR PEER REVIEW 9 of 15

**Figure 8.** *Cont*.

Biotic and abiotic stresses seriously affect plant growth and development. Under adverse environments, transcriptome changes are the earliest responses, and transcriptional regulation plays a crucial role in plant defense responses [15]. Thus far, many TFs have been identified as participating in plant defense responses, including MYB, bZIP, and WRKY proteins. There are many more biotic stress-related genes in WRKYs than in other TFs, and an increasing number of studies have revealed that WRKY TFs play positive or negative roles in plants' disease prevention [24]. For example, *AtWRKY46*, coordinated with *AtWRKY70* and *AtWRKY53*, positively regulated basal resistance to *Pseudomonas syringae* [25]; *OsWRKY6* played a positive role in plant defense response by activating the expression of defense-related genes [26]; *GhWRKY44* was induced by pathogen injection, and overexpression of *GhWRKY44* led to enhanced resistance against bacterial and fungal pathogens [27]. These results all suggest that the WRKY family plays an important role in responding to biotic

However, knowledge about the role of WRKYs in abiotic stresses is limited [29,30]. Maize is a major food and economic crop and plays an important role in basic and applied biological research. So far, known research about WRKYs has been mostly related to defense response in dicotyledon plants such as *Arabidopsis*, tomato, and tobacco, but little information about the role of maize WRKYs has been reported [31–33]. It is rather crucial to elucidate the functional maize WRKY protein in abiotic stress response. In maize, Wei et al. [22] have identified 136 WRKY proteins encoded by 119 WRKY genes, numbered them, and performed a phylogenetic tree analysis of the maize WRKYs with orthologs in *Arabidopsis*, rice, and barley, which improved knowledge of WRKYs in maize. In addition, Zhang et al. [23] identified three new additional *ZmWRKY* genes, analyzed the gene expression profiles of *ZmWRKYs* using data from various studies, and found that ten genes, including *ZmWRKY9*, *ZmWRKY25*, *ZmWRKY47*, *ZmWRKY97*, *ZmWRKY80*, *ZmWRKY39*, *ZmWRKY106*, *ZmWRKY53*, *ZmWRKY36* and *ZmWRKY113*, were responsive under drought treatment in at least in three studies, which provided the basis for cloning functional *ZmWRKY* genes. In this study, we

**Figure 8.** (**A**) The reactive oxygen species (ROS) content and the activities of (**B**) superoxide dismutase

represent the significant differences at *p* < 0.05 or *p* < 0.01, respectively (Student's *t*-test).

**3. Discussion**

stresses [28].

**Figure 8.** (**A**) The reactive oxygen species (ROS) content and the activities of (**B**) superoxide dismutase (SOD), (**C**) peroxide dismutase (POD), and (**D**) catalase (CAT) under different conditions at different time points (0, 4, 12, and 24 h). Values are means ± SDs of three replicates, and asterisks (\* or \*\*) represent the significant differences at *p* < 0.05 or *p* < 0.01, respectively (Student's *t*-test). **Figure 8.** (**A**) The reactive oxygen species (ROS) content and the activities of (**B**) superoxide dismutase (SOD), (**C**) peroxide dismutase (POD), and (**D**) catalase (CAT) under different conditions at different time points (0, 4, 12, and 24 h). Values are means ± SDs of three replicates, and asterisks (\* or \*\*) represent the significant differences at *p* < 0.05 or *p* < 0.01, respectively (Student's *t*-test).
