**5. Homo- and hetero-dimerization of TFs**

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

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

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

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

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 [1, 11].

oxidative damage in plant cells [111].

ABA-independent pathway [7].

Homo- and hetero-dimerization of TFs plays an important role in certain cases and is consid‐ ered as a pre-requisite for binding of DNA *cis-*elements. Formation of homo- and hetero-dimers plays a further function in modulating the DNA-binding specificity of TFs. Inability to form a dimeric complex may absolutely abolish the DNA binding ability of certain classes of TFs. The high complexity in the selection of hetero-dimerization partners and inability of some TFs to homo-dimerize but hetero-dimerize suggests that homo- and hetero-dimerization of TFs are not random processes, but that specific interactions between monomeric TFs forms are preferred. Hence, dimerization is likely to fulfill specific functions in gene regulation.

The dimerization ability of NAC proteins has been localized to the NAC domain [112, 113]. The residues in the highly conserved NAC domain are involved in the dimer contact and consist of hydrophobic interactions, a twisted anti parallel β-sheet sandwiched between two helices and two prominent salt bridges formed by the conserved arginine and glutamate [114, 115]. Experimental data suggest that NAC TFs are capable of forming both homo- and heterodimers. The NAC domain of NAC1 [116] and ANACO19 [113, 114] were shown to form homodimers. The NAC domains of OsNAC5 were shown to interact with the NAC domains of OsNAC5, OsNAC6, and SNAC1, generating both homo- and hetero-dimeric complexes. BnNAC14, a *Brassica napus* NAC protein, was shown to form hetero-dimers with BnNAC5-8, BnNAC485, and BnNAC3, but not homo-dimers. Mutational and deletion studies suggested that conserved NAC domains, in particular, the amino acids in close proximity to both the amino and carboxy-terminals, are necessary for mediating the formation of homo- or heterodimers [106, 112, 117].

Dimerization of the bZIP class of TFs is mediated by leucine zipper motifs, i.e., non-canonical repeats of leucine or other hydrophobic amino acid residues creating an amphipathic α-helix. The electrostatic attraction and repulsion of the polar residues situated next to the hydrophobic residues enables the formation and stabilization of dimers [118, 119]. Homo-dimeric rice OsbZIP71 is capable of exchanging its subunit to form hetero-dimers with members of the Group-C, in particular, with OsbZIP15, OsbZIP20, OsbZIP33, and OsbZIP88, suggesting a possible role of hetero-dimerization in efficient binding to *cis*-elements on promoters of target genes [25]. A member of Group-A, the G-box-binding factor AtGBF4, interacts with the Group-G AtGBF1 and the Group-H AtGBF2. Similarly, the members of *Arabidopsis* Group-E, bZIP34 and bZIP61, form hetero-dimers with bZIP51 of Group-I and bZIP43 of Group-S, but none of these TFs belonging to Groups E and I can form homo-dimers due to electrostatic violations in the leucine zipper regions [15, 120, 121]. These data suggest that dimerization between members of within and between groups of bZIP TFs is highly specific and acts as a crucial mechanism to modulate the affinity for *cis*-elements and function of TFs.

In HD-Zips, the leucine zipper that is immediately downstream of the helical domain enables dimerization of HD-Zip TFs, which is a pre-requisite for DNA binding. The HD-Zip leucine zipper is a canonical repeat of leucine amino acid at every seventh residue creating an amphipathic α-helix, which forms a coiled coil structure during dimerization. Formation of hydrophobic interface and complementary charge interactions by the residues present in the coiled coil structure permit or inhibit the formation of dimers from monomeric HD-Zip TFs [105]. *In-vitro* studies have shown that dimerization of HD-Zip is a pre-requisite for DNA binding, and it is assumed that members of HD-Zip Class I and Class II families form heterodimers exclusively with other members of their own family [105, 122–124].

Though there is clear evidence for homo- and hetero-dimerization of WRKY proteins, the extent to which they form a functional dimer is unknown and yet to be determined. Of the seven WRKY subclasses, interaction between members of four WRKY class TFs have been experimentally demonstrated. In Group IIa WRKY TFs, dimerization is mediated by a canonical leucine zipper sequence, whereas in members belonging to other Group II and Group III subclasses, presence of leucine/isoleucine/valine residues at approximate sevenresidue intervals at their N-termini form an amphipathic alpha helices similar to the secondary structure of a basic leucine zipper and mediate dimerization [125]. It is suggested that these potential leucine zipper sequences might mediate the formation of homo- and hetero-dimers within and between members of different subclasses of WRKY TFs. For example, *Arabidopsis* WRKY TFs belonging to the Group IIa, AtWRKY 18, AtWRKY40, and AtWRKY60, form homoand hetero-dimers [126]. Similarly AtWRKY30 interacted with AtWRKY53, AtWRKY54, and AtWRKY70 and formed hetero-dimers through leucine zipper motifs present at the N-termini of the subclass of WRKY TFs [127]. Interaction between different subclasses was observed in rice. OsWRKY71, a Group IIa WRKY TF, interacted not only with itself, but also with a Group IId WRKY protein, OsWRKY51 [128]. Formation of homo- and hetero-dimer complexes between different WRKY TFs can have positive or negative effects on their DNA binding activities.

Formation of homo- and hetero-dimers offers an additional large combinatorial flexibility in the regulation of transcription. Performing an accurate analysis and developing a deeper understanding of roles of TFs in various biological processes will require the knowledge of other interacting partners, downstream genes, and location of expression in plant organs along with mechanism of homo- and hetero-dimerization of particular TFs. Thus, it may prove difficult to attempt to make informative conclusions about the roles of specific TFs on the basis of their singular overexpression without this level of knowledge.
