Overview on the main cereal transcription factor family members involved in drought.
Drought is the major abiotic stress in many wheat environments, decreasing grain yields and farmer’s income. Finding ways to improve drought tolerance in wheat is therefore a global effort. Transcription factors (TFs) play important roles in drought tolerance by stimulating plant’s protective genome activities in response to heat and water limitation. TFs are specialized proteins which can bind to specific DNA elements in gene promoters and modulate gene expression in response to various external and internal stimuli. Thus TFs is a crucial part of plant signal transduction pathway mediated by signal receptors, phytohormones and other regulatory compounds. The activities of TFs are closely related to their structure, and their binding specificity is determined by the homo-/hetero-dimerization of TFs. The expression of downstream genes may produce a subset of TFs or regulate other functional proteins involved in physiological drought adaptation. Thus, the hierarchic regulations of TF activities, downstream gene expression and protein–protein interaction comprise a complex regulatory network, which participates in drought response and adaptation in cereal crops. Basic mechanisms of this regulatory network have been described, but more insight is needed to find new tools for enhancing cereals’ adaptation to drought stress.
- Abiotic stress
- regulatory networks
- transcription factors
Drought is the major environmental factor that limits crop growth and yield globally. Improving crop performance under water limiting conditions is, therefore, an important research focus of plant scientists around the world. Limited water availability evokes adaptive physiological responses regulated by changes in expression of numerous stress-responsible genes. Transcription factors (TFs) are groups of proteins that bind to specific regulatory DNA elements located in gene promoters upstream of transcription initiation sites, repressing or activating target gene expression. Intensive research in recent years has shown that temporal and spatial modulation of stress-related TFs provides an efficient way for plants to deal with unfavourable growth conditions. TFs involved in drought response were identified and characterized in various cereal species, including all major food crops such as in rice [1–4], wheat [5–8], barley [9, 10], and maize [11, 12]. Overexpression of several target genes encoding stress-responsive TFs led to improved survival rate under water limitation in transgenic plants [4, 10, 12–14].
The activities of TFs are closely related to their structure. TFs usually contain a DNA-binding domain (DBD) and a transcriptional activation domain (TAD) . The DBD enables TFs to bind with specific promoter elements of target genes, and TAD mediates regulation of the downstream gene either directly or in cooperation with other proteins. TAD usually represents a low-complexity sequence that prevents protein self-folding and facilitates protein–protein interactions (PPI). The transcriptional response to drought in cereals is controlled by a large number of TFs, which have been grouped into several different families based on their structure and binding specificity. The main TFs discussed in this article belong to the following families: the DRE-binding protein/C-repeat binding factors (DREB/CBF) [1, 13, 16, 17], the NAM/ATAF1/CUC2 (NAC) factors [18, 19], the MYB family [20, 21], the WRKY family [9, 22], the basic leucine zipper family (bZIP) [14, 23–25], and the homeodomain-leucine-zipper (HD-Zip) family [26–28].
When plants suffer water deficiency, receptors from the cell membrane/cell wall sense the extracellular stress signals and convert them into intracellular secondary messengers such as Ca2+ and inositol phosphate . How exactly the signal is transmitted toward gene activation is still poorly understood and is a subject of intensive multidisciplinary investigations. However, it is well agreed that plant hormones, especially abscisic acid (ABA), play significant role in drought stress–related transcription, in many cases through modulating phosphorylation status of transcription factors and other regulatory proteins.
The objective of this article is to review the involvement of TFs in drought response and adaptation in cereals and to illuminate the complexity of the factors and processes involved. The article is subdivided into four sections, which will (1) give examples of drought-related hierarchy in TF interactions regulated by plant hormones, (2) provide an overview of major families of cereal TFs involved in drought response, (3) overview existing data on TF target gene networks activated in response to drought, and (4) describe the homo- and hetero-dimerization in relation to TF’s activities.
2. Plant hormone crosstalk in drought relevant regulatory pathways
Phytohormones play critical roles in linking the stress-responsive signaling cascades. ABA is a key plant hormone that functions as a link between environmental stress reception and adaptive transcriptional programs such as the regulation of cellular mechanisms, carbohydrate and lipid metabolism. Similar to ABA, gibberellic acid (GA) and jasmonic acid (JA) play important functions in cellular stress network signaling. Different receptors have been reported to recognize and bind with these plant growth regulators in order to activate or modulate downstream responses .
Drought and ABA-mediated signals are perceived through three main pathways by different receptors (see Figure 1). The first receptor PYR/PYL/ RCARs (PYRABACTIN RESISTANCE / PYRABACTIN RESISTANCE-LIKE / REGULATORY COMPONENT OF ABA RECEPTORSPYR) binds ABA and inactivates the type 2C protein phosphatases (PP2Cs), which leads to the accumulation of SNF1-RELATED PROTEIN KINASES (SnRK2s) . SnRK2s activate ABA-responsive TFs such as
It was also demonstrated that ABA can increase the transcription level of reactive oxygen species (ROS) network genes . ROS are reactive oxygen–based molecules such as superoxide (O2–), hydrogen peroxide (H2O2), and hydroxyl radical (OH–), which not only toxically damage cells through membrane peroxidation and de-esterification under environmental stresses but also trigger stress endurance in plants . For example, ABA has been shown to trigger the activity of cytosolic aldehyde oxidase and xanthine dehydrogenase, which separately produce H2O2 and O2– in drought .
The GA receptor GA INSENSITIVE DWARF1 (GID1) was reported from rice and is a homolog of the
The regulation network of TFs plays an important role in stress-relevant hierarchic regulatory pathways. OsNAC10, a NAC TF, can up-regulate the downstream genes encoding AP2 and WRKY TFs involved in ROS detoxification and scavenging for drought response through the ABA synthesis pathway. The mechanisms of plant response to drought include cell wall development and cuticle formation . The promoter region of the gene
3. Major families of cereal TFs involved in drought response
Transcription factors are classified into several family groups mainly based on characteristic amino acid sequences of its conserved DBDs [36, 37]. Of these, the families DREB/CBF, NAC, MYB, WRKY, bZIP, and HD-Zip are the main TFs involved in drought. Their structural features, classification, and representative family members in cereals are summarized in Table 1 and Figure 2.
3.1. DREB/CBF family
The DREB/CBF family is a member of the AP2/EREBF superfamily of TFs, responsive to several stresses including drought [3, 8]. A cDNA encoding the first identified DREB/CBF family member CBF1 was isolated from
Many reported drought-inducible cereal DREBs were shown to be regulators improving stress endurance. In wheat, the gene
3.2. NAC family
The NAM/ATAF/CUC (NAC) TFs contain a unique feature, a conserved N-terminus DBD and a dissimilar C-terminus regulatory domain, and they are spread across the plant kingdom . The name NAC is an abbreviation of three genes designated as no apical meristem (
3.3. MYBs family
MYB is a group of ancient TFs found in viruses  and eukaryotes such as plants, animals, and fungi . The first gene (c1) identified to encode MYB in plants was from
3.4. WRKY family
WRKY proteins belong to the superfamily WRKY-GCM1 of zinc finger TFs . They exist in numerous plant species  and were reported to be involved in several biotic and abiotic stress responses and developmental processes such as embryogenesis and leaf senescence . WRKY family members contain a highly conserved WRKY domain with 60 amino acids comprised of two motifs. One is the conserved WRKYGQK motif on the N-terminus and the other one is a zinc-finger-like motif on the C-terminus . There are three main groups in the WRKY family according to the different number of WRKY domains and the variable structure of the zinc-finger-like motif . Group I has two WRKY domains, whereas groups II and III have one WRKY domain. The zinc finger motifs of the WRKY domain in groups I and II are the same, but different in group III . The group II has been divided into five subgroups by Eulgem et al. , designated as IIa, IIb, IIc, IId, and IIe, according to the conserved motifs outside the WRKY domain. Within these five subgroups, Zhang et al.  distinguished another three new groups (2_a+2_b, 2_c, 2_d+2_e). Several drought-related WRKY TFs were found in rice, wheat, and barley [78–80]. In rice,
3.5. bZIP family
The basic leucine zipper (bZIP) family is another big group of TFs involved in diverse functions such as hormone and sugar signaling and organ development . bZIP proteins commonly have a basic region for DNA binding and a conserved leucine zipper motif . These TFs specifically bind to a DNA sequence with a core
3.6. HD-Zip family
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 homeodomain (HD)  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 . HD is a folded structure with three helices, which are responsible for the specific protein-DNA interactions . 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. , 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 . HD-Zip I TFs contain no lipid/sterol-binding domain, designated StAR-related lipid transfer (START) domain, which was found in HD-Zip III and HD-Zip IV TFs . 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 .
In rice, the
In maize, the HD-Zip TF Zmhdz10 was found to play an important role in drought response , and in wheat, only two HD-Zip TFs, TaHDZipI-1 and TaHDZipI-2, were reported so far . However, there is no information about the function of wheat HD-Zip proteins in drought response.
4. TFs target gene network activated in response to drought
TFs are involved in target gene network regulation through their DBD interaction with different gene promoter
Most DREB family members such as OsDREB1A  and TaDREB1  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 (
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 . TaWRKY44 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 .
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
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
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
5. Homo- and hetero-dimerization of TFs
Homo- and hetero-dimerization of TFs plays an important role in certain cases and is considered as a pre-requisite for binding of DNA
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 hetero-dimers. The NAC domain of NAC1  and ANACO19 [113, 114] were shown to form homo-dimers. 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
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
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 .
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 seven-residue intervals at their N-termini form an amphipathic alpha helices similar to the secondary structure of a basic leucine zipper and mediate dimerization . 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,
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.
TFs play a vital role in regulating gene transcription through different signal pathways to enable plants to adapt to harsh environments and abiotic stresses such as drought. Those TFs can recognize and interact with specific