**3. Discussion**

**3. Discussion** 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 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 stresses [28].

stresses [28]. 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 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 revealed the function of *ZmWRKY106* in abiotic stress responses. Our study showed that ZmWRKY106 belongs to group II, shares a mean identity with its rice, *Arabidopsis* and barley orthologs, and is closer to *OsWRKY13* (Figure 2).

Increasing evidence has indicated that WRKYs play an important role in abiotic stress response, for example, *GmWRKY21* improved freezing tolerance in transgenic *Arabidopsis*, and *GmWRKY54* played a positive role in response to salt and drought stresses, whereas *GmWRKY13* markedly increased sensitivity to salt and mannitol [34]. Overexpression of *AtWRKY25* and *AtWRKY33* in *Arabidopsis* led to enhanced resistance to salt and hypersensitivity to ABA [35]. In rice, *OsWRKY11* enhanced heat and drought tolerance [29]. In barley, *Hv-WRKY38* played key roles in the response to cold and drought stresses, and enhanced drought tolerance in turf and forage grass [36,37]. In this study, expression

profiles analysis revealed that *ZmWRKY106* was induced significantly by drought, high temperature and ABA (Figure 4), possibly related to various stress-related cis-elements in its promoter region (Table 1). Under drought treatment, the transgenic seeds of *ZmWRKY106* germinated faster than WT seeds, and roots of OE lines were remarkably longer than those of WT lines (Figure 5). Meanwhile, overexpression of *ZmWRKY106* reduced ROS content and enhanced the activities of SOD, POD and CAT under drought treatment (Figure 8). Furthermore, the survival rates of OE lines were higher than those of WT lines (Figure 6). These results all showed that *ZmWRKY106* exhibited drought tolerance and thermotolerance.

ABA is a major phytohormone referred to plant response under drought stress, and there exist ABA-dependent and ABA-independent pathways in drought stress response. In our study, the expression levels of six stress-related genes were assessed under normal and drought conditions (Figure 7). *DREB2A* is a well-known marker gene in ABA-independent stress responses [38]. ABRE and DRE/CRT motifs were found in the promoters of many stress-inducible genes, such as *RD29A*, which contained several DREs and one ABRE in the promoter domain, and was strongly induced by cold, drought and salt stresses [39–41]. *HSP90* played a major role in stress signal transduction, and overexpression of HSP90 affected the phenotype of transgenic plants [42–45]. In our study, the expressions of *RD29A*, *HSP90*, and *DREB2A* genes were all up-regulated in *ZmWRKY106* transgenic lines (Figure 7A–C), suggesting that *ZmWRKY106* may play a positive role in drought and heat response. *CmWRKY10* acted as a positive factor in response to drought stress by regulating the expression of *DREB1A*, *DREB2A*, *CuZnSOD*, *NCED3A*, and *NCED3B*, which proved that *CmWRKY10* enhanced the drought tolerance through the ABA-dependent pathway [19]. These genes could play key roles in the physiological process of abiotic stress response [46,47]. We found that the expressions of ABA-related genes were higher in transgenic lines, which indicated that overexpression of *ZmWRKY106* led to enhanced tolerance of drought stress through the ABA-dependent pathway (Figure 7D–F). These results all indicated that *ZmWRKY106* may play a role in the abiotic stress response by regulating stress-related genes through the ABA-signaling pathway (Figure 7). Nevertheless, the role and regulation mechanisms of *ZmWRKY106* in maize still need further research.
