**4. TFs target gene network activated in response to drought**

binding motif [86] in maize, rice, and barely. The overexpression of several cereal bZIP TFs was identified to be in response to drought stress. In barley, 11 HvbZIP members were identified to be down- or up-regulated by drought [86]. In maize, ZmbZIP37, -17, and -112 showed high expression levels in drought stress conditions [88, 89] and the overexpression of ZmbZIP72 enhanced the drought tolerance in transgenic *Arabidopsis* [90]. In rice, OsbZIP12 [34], -16 [91], -23, -45 [92], -71 [25], and -72 [93] play a positive role in drought tolerance through ABA signal, while OsbZIP52 [24] and -46 [94] were suggested to be a negative regulator in water deficiency. In wheat, the gene encoding *TabZIP60* was highly induced by salt, cold, and ABA, and the overexpression of *TabZIP60* enhanced the drought and frost tolerance in

12 Abiotic and Biotic Stress in Plants - Recent Advances and Future Perspectives

The homeodomain leucine zipper (HD-Zip) family is a group of proteins that are unique to the plant kingdom. All members of the HD-Zip family contain the combination of homeodo‐ main (HD) [6] with a following leucine zipper (Zip or LZ). HD is a conserved protein domain containing a 60 amino acids sequence, which is present in all eukaryotic species [95]. HD is a folded structure with three helices, which are responsible for the specific protein-DNA interactions [95]. HD-Zip family proteins have been classified into HD-Zip I, HD-Zip II, HD-Zip III, and HD-Zip IV [96–100] according to different domain structure and functions. According to Chan et al. [101], HD-Zip I TFs have less conserved motifs than HD-Zip II, and the sequences of HD-Zip I outside the HD-Zip domain are quite different, whereas HD-Zip II TFs have several common sequences outside the HD-Zip domain. The HD-Zip III TFs have four additional amino acids on the conjunction of HD and LZ compared with other three subfamilies [102]. HD-Zip I TFs contain no lipid/sterol-binding domain, designated StARrelated lipid transfer (START) domain, which was found in HD-Zip III and HD-Zip IV TFs [102]. Hence, the special structural feature of HD-Zip I TFs is the presence of HD and leucine

zipper and the absence of common sequences outside the HD-Zip domain [103].

In rice, the *HD-Zip I* subfamily has 14 members: *Oshox4-6*, *Oshox*8, *Oshox*12-14, *Oshox*16, and *Oshox*20-25 [104]. Three of them*, OsHOX6*, *OsHOX22* and *OsHOX24*, are homologs of the ABA and abiotic stress-inducible genes *AtHB7* and *AtHB12 in Arabidopsis* [105]. *OsHOX22* and *OsHOX24* have been identified to be involved in drought, cold, and ABA response. *OsH‐ OX22* is strongly activated by high salinity and ABA, but it is weakly induced by frost [27]. Zhang et al. [27] found that the insertion of T-DNA into the *OsHOX22* promoter region led to a decreased gene expression level of *OsHOX22* and reduced ABA content, but improved drought and salt endurance of rice seedlings. The authors believe that *OsHOX22* is a negative regulator for stress response by regulating an ABA-mediated signal transduction pathway and ABA biosynthesis [27]. The *OsHOX24* promoter has shown strong activation by water deficiency and high salinity [106]. According to the results of Agalou et al. [104], *OsHOX22* and *OsHOX24* can be induced by drought in drought-sensitive and drought-resistant cultivars, whereas *OsHOX6* can only be induced in drought sensitive cultivars*.* Although the role of OsHOX6 and OsHOX24 TFs is still not clear, the homologs of these TFs, AtHB7 and AtHB12, have been found to be involved in ABA modulation by regulating the protein phosphatase 2C

transgenic *Arabidopsis* [84].

**3.6. HD-Zip family**

TFs are involved in target gene network regulation through their DBD interaction with different gene promoter *cis*-elements mediated by ABA-dependent or ABA-independent signal transduction pathway (see Figure 3).

**Figure 3.** Cereal TFs target gene networks in response to drought through ABA-dependent and -independent path‐ ways. The green arrows show the ABA-independent pathway and the purple arrows show the ABA-dependent path‐ way.

Most DREB family members such as OsDREB1A [1] and TaDREB1 [41] were found to be activated in ABA-independent pathways. They can enhance the stress tolerance by activating the expression of downstream genes such as late embryogenesis abundant (*LEA*) genes driven by DRE/CRT *cis-*element. *LEA* genes, such as *COR15A* and *rd29A* (*Cor78*), are also designated as dehydrins (DHNs) or cold-responsive (COR) genes. Their expression products participate in cells protection from stresses by enhancing the membrane stability and correction of protein folding [13]. However, recent studies showed that some members of DREB also participate in ABA-dependent pathways. Wang et al. [3] found that the overexpression of *OsDREB1F* in transgenic rice results in the expression of ABA-induced genes *rd29B* and *RAB18*, whose promoters only contain an AREB element that cannot be recognized by DREB TFs. So far, there is no explanation on illuminating the way for OsDREB1F activating the expression of these two genes. The hypothesis is that OsDREB1F might interact with bZIP family members that can bind with an AREB element and modulate the transcription of *rd29B* and *RAB18* [3]. The same is true for ZmDREB2A [110] and the mechanism is still not clear. Besides, ZmDREB2A was identified to activate some downstream genes encoding detoxification enzymes that can protect cells from ROS. However, these genes do not contain a DRE/CRT element. Hence, it is possible that ZmDREB2A indirectly affects these genes' expression [110]. Besides, DREBs in different species might have different preference in regulating the expression of downstream genes with different core elements in their promoters. The rice OsDREB1A, e.g., prefers to interact with the CRT/DRE core element GCCGAC of genes such as *cor15A, rd29A,* and *rd17* instead of core element ACCGAC*,* while the *Arabidopsis* DREB1A and maize ZmDREB1A have equal competition for recognizing core element GCCGAC and core element ACCGAC in the downstream genes [1, 11].

In the WRKY family, more than 10 TFs isolated from rice were found to co-express under drought and cold stresses, but the downstream genes were still not determined [81]. TaWR‐ KY44 from wheat was identified to recognize the core element (TTGACC/TTAACC) in the promoter region of downstream genes and up-regulate genes encoding antioxidant enzymes such as NtSOD, stress-defensive proteins such as NtERD10C, and lipid-transfer proteins such as NtLTP1 to increase plants survival rate in drought. Thus, TF TaWRKY44 participates in regulating antioxidant enzyme activity and decreasing the ROS levels in order to prevent oxidative damage in plant cells [111].

Members of the MYB family regulate the expression level of different target genes involved in the ABA-dependent and independent pathways. The overexpression of OsMYB48-1 was found to regulate genes such as *OsPP2C68*, *RAB21,* and *OsNCED4,* respectively, involving in ABA early signaling, late response, and the ABA synthesis pathway, contributing to increased drought tolerance under water deficiency [72]. In transgenic *Arabidopsis*, the overexpression of *OsMYB3R-2* increased the expression level of the downstream genes *DREB2A*, *COR15A,* and *RCI2A* and enhanced the plants adaptation to abiotic stresses [20]. Besides, TaMYB30-B was found to induce the expression of stress inducible genes *rd29A* and *ERD1*, involved in the ABA-independent pathway [7].

Members of the bZIP family were also found to regulate downstream gene transcription through the ABA-dependent and independent pathway. OsbZIP52 was suggested to bind to G-box *cis-*elements and down-regulate genes such as *OsLEA3* and *OsTPP* that can improve drought or cold tolerance in rice via the ABA-independent pathway [24]. The expression of downstream genes *LEA3* and *Rab16* was activated by the transcription factor OsbZIP12 under water deficiency mediated by the ABA synthesis pathway [34].

Most TFs in the NAC family participate in the ABA synthesis pathway. OsNAC5 and OsNAC6 can recognize a core sequence (CACG) of the downstream gene *OsLEA3* and regulate the gene expression that changed root structure and resulted in higher drought tolerance through the ABA signaling pathway [35]. OsNAC6 also participates in up-regulating the transcription of genes encoding peroxidase, which can catalyze a series of oxidative reactions [33]. Some TFs even participate in both ABA-dependent and independent pathways. For example, TaNAC67 was found to up-regulate 10 abiotic stress responsive genes such as *rd29A* and *rd29B*, which were separately related to ABA-independent and -dependent pathways and four ABA synthesis/responsive genes such as *ABI1* [54]*,* thereby improving stress tolerance in plants.
