*2.4. ZmWRKY106 Promoter Domain Contained Various Stress-Related Cis-Elements*

To further understand the regulation mechanism of *ZmWRKY106*, we isolated the promoter region upstream of the *ZmWRKY106* ATG start codon. Types of *cis*-elements correlated to stress were present in the promoter region, including the C-repeat/DRE element referred to cold and dehydration response, low-temperature responsive element LTR and the drought-induced element MBS. In addition, there was another TCA-element that participated in salicylic acid (SA) response in the promoter region of *ZmWRKY106* (Table 1). This analysis suggested that *ZmWRKY106* may function in abiotic stress response. *Int. J. Mol. Sci.* **2018**, *19*, x FOR PEER REVIEW 5 of 15 *2.4. ZmWRKY106 Promoter Domain Contained Various Stress-Related Cis-Elements* To further understand the regulation mechanism of *ZmWRKY106*, we isolated the promoter region upstream of the *ZmWRKY106* ATG start codon. Types of *cis*-elements correlated to stress were



DRE—dehydration responsive element; LTR—low-temperature responsive; SA—salicylic acid. LTR CCGAAA involved in low-temperature responsiveness

MBS TAACTG MYB binding site involved in drought-inducibility

#### *2.5. ZmWRKY106 Was Involved in Abiotic Stress Responses* TCA-element TCAGAAGAGG involved in SA responsiveness *DRE—dehydration responsive element; LTR—low-temperature responsive; SA—salicylic acid*

To explore the possible signal pathways which *ZmWRKY106* may be involved in, we performed qRT-PCR to investigate the expression patterns of *ZmWRKY106* in maize treated with drought, high-salt, high-temperature, and ABA treatments. *ZmWRKY106* was remarkably induced by drought, high temperature and ABA, but was weakly induced by salt (Figure 4). For dehydration treatment, the transcript of *ZmWRKY106* was rapidly up-regulated more than 10-fold after 1 h of dehydration stress (Figure 4A). *ZmWRKY106* was slightly induced by salt at a maximum level of about 1.5-fold (Figure 4B). High temperature also significantly affected the expression of *ZmWRKY106*. Under high-temperature stress, the transcription level of *ZmWRKY106* increased gradually, peaked at 7.6-fold after 2 h of stress, and then rapidly declined to a constitutive level. With exogenous ABA treatment, the transcription level of *ZmWRKY106* was increased more than three-fold at 6 h after treatment. *2.5. ZmWRKY106 Was Involved in Abiotic Stress Responses* To explore the possible signal pathways which *ZmWRKY106* may be involved in, we performed qRT-PCR to investigate the expression patterns of *ZmWRKY106* in maize treated with drought, highsalt, high-temperature, and ABA treatments. *ZmWRKY106* was remarkably induced by drought, high temperature and ABA, but was weakly induced by salt (Figure 4). For dehydration treatment, the transcript of *ZmWRKY106* was rapidly up-regulated more than 10-fold after 1 h of dehydration stress (Figure 4A). *ZmWRKY106* was slightly induced by salt at a maximum level of about 1.5-fold (Figure 4B). High temperature also significantly affected the expression of *ZmWRKY106*. Under hightemperature stress, the transcription level of *ZmWRKY106* increased gradually, peaked at 7.6-fold after 2 h of stress, and then rapidly declined to a constitutive level. With exogenous ABA treatment, the transcription level of *ZmWRKY106* was increased more than three-fold at 6 h after treatment.

**Figure 4.** Expression patterns of *ZmWRKY106* under (**A**) drought, (**B**) high-salt, (**C**) high-temperature, and (**D**) exogenous abscisic acid (ABA) stresses. The ordinates are the relative expression level (fold) **Figure 4.** Expression patterns of *ZmWRKY106* under (**A**) drought, (**B**) high-salt, (**C**) high-temperature, and (**D**) exogenous abscisic acid (ABA) stresses. The ordinates are the relative expression level (fold) of *ZmWRKY106* compared to the non-stressed control. The horizontal ordinate is treatment time for 0, 0.5, 1, 2, 4, 6, 12 and 24 h. All experiments were repeated three times. Error bars represent standard deviations (SDs). All the data represent the means ± SDs of three independent biological replicates. The different letters in the bar graphs indicate significant differences at *p* < 0.05.

#### *2.6. ZmWRKY106 Enhanced Drought Tolerance in Transgenic Arabidopsis 2.6. ZmWRKY106 Enhanced Drought Tolerance in Transgenic Arabidopsis*

To investigate the function of *ZmWRKY106*, the pBI121-*ZmWRKY106* recombinant was transformed into wild-type (WT) *Arabidopsis* (Columbia-0). T<sup>3</sup> generation transgenic lines with relatively high expressions were selected by qRT-PCR for further analysis. The expression levels of transgenic lines are exhibited in Supplementary Figure S2. On MS medium, no significant differences in seed germination rates were observed between transgenic and WT plants. In the presence of 4% PEG6000, the germination rate of transgenic seeds was nearly 9% higher than WT after four days. Moreover, the germination was suppressed under 8% PEG6000, but transgenic seeds showed a higher germination rate than WT seeds (Figure 5A). For root growth assays, as shown in Figure 5B, *ZmWRKY106* transgenic lines had similar phenotypes to WT on MS medium. When supplemented with PEG6000, the growth of all transgenic and WT plants was repressed; however, transgenic plants showed clear differences compared to WT ones, with significantly longer total root lengths than those of WT under both PEG treatments. These results showed that *ZmWRKY106* transgenic lines had a stronger capacity to resist drought. To investigate the function of *ZmWRKY106*, the pBI121-*ZmWRKY106* recombinant was transformed into wild-type (WT) *Arabidopsis* (Columbia-0). T<sup>3</sup> generation transgenic lines with relatively high expressions were selected by qRT-PCR for further analysis. The expression levels of transgenic lines are exhibited in supplementary Figure S2. On MS medium, no significant differences in seed germination rates were observed between transgenic and WT plants. In the presence of 4% PEG6000, the germination rate of transgenic seeds was nearly 9% higher than WT after four days. Moreover, the germination was suppressed under 8% PEG6000, but transgenic seeds showed a higher germination rate than WT seeds (Figure 5A). For root growth assays, as shown in Figure 5B, *ZmWRKY106* transgenic lines had similar phenotypes to WT on MS medium. When supplemented with PEG6000, the growth of all transgenic and WT plants was repressed; however, transgenic plants showed clear differences compared to WT ones, with significantly longer total root lengths than those of WT under both PEG treatments. These results showed that *ZmWRKY106* transgenic lines had a stronger capacity to resist drought.

*Int. J. Mol. Sci.* **2018**, *19*, x FOR PEER REVIEW 6 of 15

of *ZmWRKY106* compared to the non-stressed control. The horizontal ordinate is treatment time for 0, 0.5, 1, 2, 4, 6, 12 and 24 h. All experiments were repeated three times. Error bars represent standard

**Figure 5.** Phenotypes of *ZmWRKY106* transgenic *Arabidopsis* under drought treatment. (**A**) Seed germinations of wild-type (WT) and *ZmWRKY106*-overexpressing lines. (**B**) Root lengths of WT and *ZmWRKY106* transgenic plants. Five-day-old seedlings were transferred to MS medium supplemented with or without PEG6000 for seven days, and then root lengths were measured. All the data represent the means ± SDs of three independent biological replicates and asterisks (\*\*) **Figure 5.** Phenotypes of *ZmWRKY106* transgenic *Arabidopsis* under drought treatment. (**A**) Seed germinations of wild-type (WT) and *ZmWRKY106*-overexpressing lines. (**B**) Root lengths of WT and *ZmWRKY106* transgenic plants. Five-day-old seedlings were transferred to MS medium supplemented with or without PEG6000 for seven days, and then root lengths were measured. All the data represent the means ± SDs of three independent biological replicates and asterisks (\*\*) represent the significant differences at *p* < 0.01 (Student's *t*-test).
