*2.1. De Novo Transcriptome Sequencing Analysis*

To find maize stress-responsive genes under drought stress, three-leaf seedlings were dehydrated on filter paper for 4 h, and then were collected for transcriptome sequencing analysis. The results showed that the transcription levels of many genes had changed after drought treatment (Figure 1A). Gene ontology (GO) analyses were used to classify the differentially expressed genes (DEGs) into functional groups. Almost 30 functionally enriched GO terms were identified for DEGs, and the results are shown in Supplementary Figure S1A. Among the predominantly enriched GO terms, signaling process was the most enriched term related to biological process. To further understand which pathways the stress-responsive genes may be involved in, the DEGs were analyzed against the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database. The top 20 enriched pathways were identified, and the "plant hormone signal transduction" pathway enriched the most DEGs under drought treatment (Supplementary Figure S1B). DEGs including many transcription factors that play vital roles in plant growth, development, morphogenesis, and abiotic stress responses through regulating the expression of downstream genes [1,4,18]. Among these transcription factors, WRKYs play important roles in response to biotic and abiotic stresses [10,19]. We searched for *ZmWRKYs* among the DEGs, and found 14 *ZmWRKYs* induced by drought treatment (Figure 1B). We chose the gene GRMZM2G013391 named *ZmWRKY106* for further study.

*Int. J. Mol. Sci.* **2018**, *19*, x FOR PEER REVIEW 3 of 15

*Int. J. Mol. Sci.* **2018**, *19*, x FOR PEER REVIEW 3 of 15

**Figure 1.** *De novo* transcriptome sequencing analysis of maize under drought stress. (**A**) Cluster analysis of the differentially expressed genes (DEGs) under drought treatment. (**B**) Transcription levels of the 14 differentially expressed *ZmWRKYs* under drought treatment. Error bar represent standard deviations (SD). The data represent means ± SD of three biological replications. **Figure 1.** *De novo* transcriptome sequencing analysis of maize under drought stress. (**A**) Cluster analysis of the differentially expressed genes (DEGs) under drought treatment. (**B**) Transcription levels of the 14 differentially expressed *ZmWRKYs* under drought treatment. Error bar represent standard deviations (SD). The data represent means ± SD of three biological replications. analysis of the differentially expressed genes (DEGs) under drought treatment. (**B**) Transcription levels of the 14 differentially expressed *ZmWRKYs* under drought treatment. Error bar represent standard deviations (SD). The data represent means ± SD of three biological replications.

#### *2.2. Phylogenetic Analysis of Maize ZmWRKY106 2.2. Phylogenetic Analysis of Maize ZmWRKY106 2.2. Phylogenetic Analysis of Maize ZmWRKY106*

After selecting from the drought-treated maize *de novo* transcriptome data, we got a putative WRKY gene *ZmWRKY106* encoding 277 amino acids. The BLASTp online tool was used to search for the homologous amino acid sequences of *ZmWRKY106* in rice and *Arabidopsis*. The amino acid sequence alignment and phylogeny analysis of ZmWRKY106 orthologs are shown in Figure 2. ZmWRKY106 shared a mean identity of 28.47% with its rice, *Arabidopsis*, and barley orthologs and had a conserved signature WRKYGQK at the N-terminus followed by a C2H2 zinc-finger motif (C-X5-C-X23-H-X1-H), which characterized group II (Figure 2A). The sequences outside the conserved domain/motif were very different. The results of phylogenesis showed that ZmWRKY106 was closer to OsWRKY13, with a 61% bootstrap rate, followed by HvWRKY39, with a frequency of 100% (Figure 2B). However, the identity of ZmWRKY106 with other orthologs was lower than that with OsWRKY13, which indicated that ZmWRKY106 may have an extensive difference from other members. After selecting from the drought-treated maize *de novo* transcriptome data, we got a putative WRKY gene *ZmWRKY106* encoding 277 amino acids. The BLASTp online tool was used to search for the homologous amino acid sequences of *ZmWRKY106* in rice and *Arabidopsis*. The amino acid sequence alignment and phylogeny analysis of ZmWRKY106 orthologs are shown in Figure 2. ZmWRKY106 shared a mean identity of 28.47% with its rice, *Arabidopsis*, and barley orthologs and had a conserved signature WRKYGQK at the N-terminus followed by a C2H2 zinc-finger motif (C-X5-C-X23-H-X1-H), which characterized group II (Figure 2A). The sequences outside the conserved domain/motif were very different. The results of phylogenesis showed that ZmWRKY106 was closer to OsWRKY13, with a 61% bootstrap rate, followed by HvWRKY39, with a frequency of 100% (Figure 2B). However, the identity of ZmWRKY106 with other orthologs was lower than that with OsWRKY13, which indicated that ZmWRKY106 may have an extensive difference from other members. After selecting from the drought-treated maize *de novo* transcriptome data, we got a putative WRKY gene *ZmWRKY106* encoding 277 amino acids. The BLASTp online tool was used to search for the homologous amino acid sequences of *ZmWRKY106* in rice and *Arabidopsis*. The amino acid sequence alignment and phylogeny analysis of ZmWRKY106 orthologs are shown in Figure 2. ZmWRKY106 shared a mean identity of 28.47% with its rice, *Arabidopsis*, and barley orthologs and had a conserved signature WRKYGQK at the N-terminus followed by a C2H2 zinc-finger motif (C-X5-C-X23-H-X1-H), which characterized group II (Figure 2A). The sequences outside the conserved domain/motif were very different. The results of phylogenesis showed that ZmWRKY106 was closer to OsWRKY13, with a 61% bootstrap rate, followed by HvWRKY39, with a frequency of 100% (Figure 2B). However, the identity of ZmWRKY106 with other orthologs was lower than that with OsWRKY13, which indicated that ZmWRKY106 may have an extensive difference from other members.

**Figure 2.** *Cont*.

*Int. J. Mol. Sci.* **2018**, *19*, x FOR PEER REVIEW 4 of 15

**Figure 2.** Multiple alignment and phylogenetic relationships of ZmWRKY106 with other orthologs in rice, *Arabidopsis*, and barley. The phylogenetic tree was produced using the aligned file with 1000 bootstraps in the MEGA 5.0 program. (**A**) Multiple alignment of ZmWRKY106 homologous proteins in rice, *Arabidopsis*, and barley. The different background colors represent the similar degree of amino acid sequences. (**B**) Phylogenetic relationship of ZmWRKY106 and other orthologs in different species. The first red box indicates the WRKYGQK motif, and the second indicates the conserved C2H2 zinc-finger motif. **Figure 2.** Multiple alignment and phylogenetic relationships of ZmWRKY106 with other orthologs in rice, *Arabidopsis*, and barley. The phylogenetic tree was produced using the aligned file with 1000 bootstraps in the MEGA 5.0 program. (**A**) Multiple alignment of ZmWRKY106 homologous proteins in rice, *Arabidopsis*, and barley. The different background colors represent the similar degree of amino acid sequences. (**B**) Phylogenetic relationship of ZmWRKY106 and other orthologs in different species. The first red box indicates the WRKYGQK motif, and the second indicates the conserved C2H2 zinc-finger motif. (**B**) **Figure 2.** Multiple alignment and phylogenetic relationships of ZmWRKY106 with other orthologs in rice, *Arabidopsis*, and barley. The phylogenetic tree was produced using the aligned file with 1000 bootstraps in the MEGA 5.0 program. (**A**) Multiple alignment of ZmWRKY106 homologous proteins in rice, *Arabidopsis*, and barley. The different background colors represent the similar degree of amino acid sequences. (**B**) Phylogenetic relationship of ZmWRKY106 and other orthologs in different species. The first red box indicates the WRKYGQK motif, and the second indicates the conserved C2H2 zinc-finger motif.

#### *2.3. ZmWRKY106 Was Localized in the Nucleus 2.3. ZmWRKY106 Was Localized in the Nucleus 2.3. ZmWRKY106 Was Localized in the Nucleus*

The transient expression vector p16318h-ZmWRKY106 was transformed to maize mesophyll protoplasts by the PEG-mediated method to determine the cell localization. After incubation in darkness for 18 h, the fluorescence signals were monitored by a confocal laser scanning microscope. As shown in Figure 3, relative to the control distributed throughout the cell, the p16318h-ZmWRKY106 fusion protein was specifically detected in the nucleus. The transient expression vector p16318h-ZmWRKY106 was transformed to maize mesophyll protoplasts by the PEG-mediated method to determine the cell localization. After incubation in darkness for 18 h, the fluorescence signals were monitored by a confocal laser scanning microscope. As shown in Figure 3, relative to the control distributed throughout the cell, the p16318h-ZmWRKY106 fusion protein was specifically detected in the nucleus. The transient expression vector p16318h-ZmWRKY106 was transformed to maize mesophyll protoplasts by the PEG-mediated method to determine the cell localization. After incubation in darkness for 18 h, the fluorescence signals were monitored by a confocal laser scanning microscope. As shown in Figure 3, relative to the control distributed throughout the cell, the p16318h-ZmWRKY106 fusion protein was specifically detected in the nucleus.

**Figure 3.** Subcellular localization of *ZmWRKY106*. The p16318hGFP and p16318hGFP-ZmWRKY106 constructs were transiently expressed in maize protoplasts. The green indicates green fluorescent, and the red indicates chloroplast autofluorescence. Results were observed after transformation for 18 h with confocal microscopy. Scale bars = 10 μm. **Figure 3.** Subcellular localization of *ZmWRKY106*. The p16318hGFP and p16318hGFP-ZmWRKY106 constructs were transiently expressed in maize protoplasts. The green indicates green fluorescent, and the red indicates chloroplast autofluorescence. Results were observed after transformation for 18 h with confocal microscopy. Scale bars = 10 μm. **Figure 3.** Subcellular localization of *ZmWRKY106*. The p16318hGFP and p16318hGFP-ZmWRKY106 constructs were transiently expressed in maize protoplasts. The green indicates green fluorescent, and the red indicates chloroplast autofluorescence. Results were observed after transformation for 18 h with confocal microscopy. Scale bars = 10 µm.
